**Plasticity of Dendritic Spines. Not Only for Cognitive**

## **Processes**

Ignacio González-Burgos,

Dulce A. Velázquez-Zamora, David González-Tapia,

Nallely Vázquez-Hernández and

Néstor I. Martínez-Torres

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67127

#### **Abstract**

Excitatory synaptic transmission is associated with the input of "new" information at synaptic junctions established by dendritic spines. The role that each type of spine plays in the transmission of the synaptic impulses is different. Indeed, there is a close relationship between the shape of spines and the differential processing of the excitatory synaptic information that is relayed to them, influencing in turn the transmission of synaptic information related to several psychoneural processes.

The vast majority of the experimental evidence shows that specific plastic interchanges of dendritic spines' shapes are related to specific functional effects in the postsynapse, i.e., the acquisition or learning of new information (thin spines), or to the storage of information in memory (mushroom spines).

Several brain regions are involved in other functions different than those of a cognitive nature, and all projection neurons in these areas possess dendritic spines. However, the functional significance of the changes that the spines of these neurons express has not been studied. Thus, in this Chapter we will discuss experimental evidence supporting the claim that dendritic spines express plastic changes in some brain regions that are not directly related to cognition, and we will also preliminarily approach their possible functional meaning.

**Keywords:** plasticity, dendritic spines, synapse, cognition

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **1. Cognition and noncognition: basic concepts**

Person A walks down the sidewalk alongside a fence when suddenly, a dog leaps out behind the fence and barks at Person A. Person A, caught off guard, jerks back and then kicks the fence. Coming to his sense, he realizes that the dog is a harmless little Chihuahua, still stuck behind the fence. Now conscious of the situation, he looks around and sees that various passers-by have been watching him and are laughing at his situation. Feeling exposed, Person A continues walking, now blushing furiously.

Looking at this situation, we might ask, if Person A was going to be embarrassed by his actions, why he reacted the way he did in the first place. Why he did not avoid the scare, and with it, the situation? Why did he kick an object without first working through what it was? This type of event—and Person A's type of reaction—is unrelated to cognition, which is the processing of information that permits the abstraction and manipulation of the environment by way of symbols (language and thought, for example) [1]. In this example, Person A could not place the origin of the stimulus nor relate it to as a previous experience, so his reaction had no cognitive component.

Certain *noncognitive* processes express themselves simultaneously with cognitive processes [2]. These processes arise consciously or unconsciously [3], automatically or implicitly [4]. These noncognitive processes do not require attention, perception, learning, memory, language, or thought for their integration; be that as it may, cognitive processes may be present for the regulation or modulation of those noncognitive ones [5].

Izdar [2] explores the role of noncognitive processes in emotional processing. He mentions that there exists a neural basis for the expression of the emotions that escape cognition and which precede the conscious experience of the stimulus. A series of experiments by LeDoux [6] provide experimental evidence for the two-way configuration for the expression of fear: one of them, the faster, permits the organism to generate a reaction to the stimulus without being fully aware of the situation. Person A's situation illustrates this case clearly.

Noncognitive processing has been the object of conceptual debate, since at one time it was thought that all nerve activity was aimed at the expression of processes related to cognition. Lazarus [7] writes that "cognition is the end of all cortical or subcortical activity." Likewise, Lazarus [8] defends that position by arguing that even simple perceptual phenomena (a type of which can be seen in Person A's case) depend on and create meanings or evaluations with respect to the stimulus. Frijda [9] argues that the nervous system is capable of generating emotions, but only when those emotions had been previously acquired through cognition. Frijda [10] argues that "emotions are the result of meaning, and that meaning is the result of inferences about causes and consequences." However, Zajonc [11] responds to Lazarus with experimental evidence wherein he illustrated the primacy of some noncognitive processes that do not themselves attribute meanings to stimuli. Zajonc mentions that even the integration of information from the retinohypothalamic tract is sufficient for the organism to produce a response, "leaving the attribution of the meaning of the stimulus a synapse away" [11].

Izdar [2] talks about the existing predisposition to argue that cognition is anything that goes hand in hand with learning, memory, and, in general, with "mental" life, leaving aside all those instinctive behaviors or those which present without cognitive acquisition or previous experience. An example of this type of behavior is the sucking reflex, the response to aversive stimuli that presents in infants even without prior cognitive processing to give it meaning. Likewise, the expression of circadian rhythms like the sleep-wake cycle. Some of these behaviors come from ancestral information accumulated in the course of a species' evolution, which shapes the brain architecture in the absence of experience with the environment [12] and that establishes itself in the absence of cognitive processing, serving the latter as a potentiator and moderator in later stages of life. It is worth mentioning that the processing of information can take place even in infants without previous experience or learning, that is, without cognitive processing [13]. The above leads to the argument that the processing of information has as much a noncognitive component as a cognitive one.

One can make a distinction between cognitive and noncognitive processes: a cognitive process depends on experience, learning, and memory, whose neural basis is the function of the areas of association of the cerebral cortex, changing stimuli into abstractions, meanings, and manageable symbols [14]. On the other hand, noncognitive processes have as their neural basis the function of subcortical structures such as the hypothalamus [15], the amygdala [6], and the functioning of primary areas of the cerebral cortex [14] that do not depend on learning, on memory, or on previous experience for the expression of certain behaviors, like sexual ones, those based on the emotional fast track [6], and the execution of voluntary movement; among others.

Noncognitive processing generates controversy within psychological epistemology. It creates a heuristic conflict that requires a solution not only from psychology but also from those sciences that provide evidence about the determinants of behavior.

This chapter presents experimental evidence about: the expression of behaviors that do not depend on cognition, as is the case in sexual behavior, which is expressed by virtue of neurophysiological changes in hypothalamic nuclei as well as in the ventromedial nucleus; about the formation of biological rhythms, like the sleep-wake cycle, which depend predominantly on the function of the preoptical area and the suprachiasmatic nucleus of the hypothalamus; about the execution of voluntary motor activity that depends on the function of the primary motor area; and about the expression of emotions through a fast track that is integrated in the nuclei of the amygdala even in the absence of the participation of the cerebral cortex and, therefore, of conscious experience.

## **2. Neuronal plasticity**

**1. Cognition and noncognition: basic concepts**

for the regulation or modulation of those noncognitive ones [5].

continues walking, now blushing furiously.

154 Synaptic Plasticity

Person A walks down the sidewalk alongside a fence when suddenly, a dog leaps out behind the fence and barks at Person A. Person A, caught off guard, jerks back and then kicks the fence. Coming to his sense, he realizes that the dog is a harmless little Chihuahua, still stuck behind the fence. Now conscious of the situation, he looks around and sees that various passers-by have been watching him and are laughing at his situation. Feeling exposed, Person A

Looking at this situation, we might ask, if Person A was going to be embarrassed by his actions, why he reacted the way he did in the first place. Why he did not avoid the scare, and with it, the situation? Why did he kick an object without first working through what it was? This type of event—and Person A's type of reaction—is unrelated to cognition, which is the processing of information that permits the abstraction and manipulation of the environment by way of symbols (language and thought, for example) [1]. In this example, Person A could not place the origin of the stimulus nor relate it to as a previous experience, so his reaction had no cognitive component. Certain *noncognitive* processes express themselves simultaneously with cognitive processes [2]. These processes arise consciously or unconsciously [3], automatically or implicitly [4]. These noncognitive processes do not require attention, perception, learning, memory, language, or thought for their integration; be that as it may, cognitive processes may be present

Izdar [2] explores the role of noncognitive processes in emotional processing. He mentions that there exists a neural basis for the expression of the emotions that escape cognition and which precede the conscious experience of the stimulus. A series of experiments by LeDoux [6] provide experimental evidence for the two-way configuration for the expression of fear: one of them, the faster, permits the organism to generate a reaction to the stimulus without

Noncognitive processing has been the object of conceptual debate, since at one time it was thought that all nerve activity was aimed at the expression of processes related to cognition. Lazarus [7] writes that "cognition is the end of all cortical or subcortical activity." Likewise, Lazarus [8] defends that position by arguing that even simple perceptual phenomena (a type of which can be seen in Person A's case) depend on and create meanings or evaluations with respect to the stimulus. Frijda [9] argues that the nervous system is capable of generating emotions, but only when those emotions had been previously acquired through cognition. Frijda [10] argues that "emotions are the result of meaning, and that meaning is the result of inferences about causes and consequences." However, Zajonc [11] responds to Lazarus with experimental evidence wherein he illustrated the primacy of some noncognitive processes that do not themselves attribute meanings to stimuli. Zajonc mentions that even the integration of information from the retinohypothalamic tract is sufficient for the organism to produce a response, "leaving the attribution of the meaning of the stimulus a synapse away" [11].

Izdar [2] talks about the existing predisposition to argue that cognition is anything that goes hand in hand with learning, memory, and, in general, with "mental" life, leaving aside all

being fully aware of the situation. Person A's situation illustrates this case clearly.

The plastic capacity of the structures related to the expression of some of the previously mentioned behaviors that are usually related to cognition. However, as the experimental evidence presented in this chapter will show, the neural structures involved in those cognitive processes also form a part of the repertoire involved in the variable expression "noncognitive" processes through their plastic capacity.

Transmission of excitatory information between neurons is mediated by the activation of receptors located on dendritic spines. These neural substructures exist in all projection neurons, and by virtue of participating in the functional integration of the afferent information by those neurons, they "add" a psychoneural attribute to the conformation of functions integrated into the corresponding neural circuits.

The dendritic spines are cytoplasmic protrusions that cover varying portions of the tubular surface of the dendrites. Depending on the excitatory afferents, the spines show varying densities and distributions along the length of the dendrites. Although the spines generally translate the excitatory information, the way in which they process it depends on their geometric structure. According to their shape, six types of spines have been described: (1) thin; (2) mushroom; (3) stubby; (4) wide; (5) branched; and (6) double [16, 17].

The primary characteristic of these structural distinctions lies in the presence or absence of a neck and a head. The thin spines have a long, narrow neck that results in a bulbous structure, or "head," whose length is shorter than the neck. The mushroom spines have a short, narrow neck that leads to a head whose diameter and length are greater than those of its neck. Stubby spines are protoplasmic protrusions that show no difference between head and neck and whose length is less than their diameter. Similarly, wide spines show neither neck nor head, but their length is greater than their diameter. Branched spines, for their part, display a narrow neck that emerges from the dendrite and which divides into two similar necks before terminating in two similar heads. Double spines have a neck that emerges from the dendrite and forms a head, which then forms another neck, and which finally terminates in a second head.

Typically, the different types of spines show variable amounts between the neurons that host them, but their proportional density remains relatively similar in all neurons: thin > mushroom > fat > wide > branched > double.

Bioelectrically, thin spines have been linked to the rapid transmission of afferent information and are functionally related to the acquisition of new information (learning). Meanwhile, mushroom spines have been related to the slow transmission of afferent synaptic information and with the storage of the same (memory). The other types of spines have not been much studied. There is, however, evidence that suggests that stubby and wide spines could be related to the regulation of the excitability of postsynaptic neurons, while branched spines could be a transformation of other, larger spines (presumably mushroom spines) into two new (branched) spines—hypothetically thin ones. Finally, no functional evidence of the activity of the double spines has been uncovered, although their geometric structure suggests that they represent two independent sites of synaptic contact.

From this chapter's perspective, thin and mushroom spines are particularly relevant. According to the evidence, both are related to the processing of cognitive information: learning from thin spines and memory from mushrooms. However, it is clear that certain lines of projection neurons—like those located in the primary motor cortex, some nuclei of the amygdala, and certain hypothalamic nuclei, like the ventromedial, the preoptic, and the suprachiasmatic—possess dendritic spines that have shown plastic changes, which have been induced experimentally.

## **3. Neuronal plasticity in cognitive and noncognitive processes**

Transmission of excitatory information between neurons is mediated by the activation of receptors located on dendritic spines. These neural substructures exist in all projection neurons, and by virtue of participating in the functional integration of the afferent information by those neurons, they "add" a psychoneural attribute to the conformation of functions inte-

The dendritic spines are cytoplasmic protrusions that cover varying portions of the tubular surface of the dendrites. Depending on the excitatory afferents, the spines show varying densities and distributions along the length of the dendrites. Although the spines generally translate the excitatory information, the way in which they process it depends on their geometric structure. According to their shape, six types of spines have been described: (1) thin; (2)

The primary characteristic of these structural distinctions lies in the presence or absence of a neck and a head. The thin spines have a long, narrow neck that results in a bulbous structure, or "head," whose length is shorter than the neck. The mushroom spines have a short, narrow neck that leads to a head whose diameter and length are greater than those of its neck. Stubby spines are protoplasmic protrusions that show no difference between head and neck and whose length is less than their diameter. Similarly, wide spines show neither neck nor head, but their length is greater than their diameter. Branched spines, for their part, display a narrow neck that emerges from the dendrite and which divides into two similar necks before terminating in two similar heads. Double spines have a neck that emerges from the dendrite and forms a head, which then forms another neck, and which finally terminates in

Typically, the different types of spines show variable amounts between the neurons that host them, but their proportional density remains relatively similar in all neurons: thin > mush-

Bioelectrically, thin spines have been linked to the rapid transmission of afferent information and are functionally related to the acquisition of new information (learning). Meanwhile, mushroom spines have been related to the slow transmission of afferent synaptic information and with the storage of the same (memory). The other types of spines have not been much studied. There is, however, evidence that suggests that stubby and wide spines could be related to the regulation of the excitability of postsynaptic neurons, while branched spines could be a transformation of other, larger spines (presumably mushroom spines) into two new (branched) spines—hypothetically thin ones. Finally, no functional evidence of the activity of the double spines has been uncovered, although their geometric structure suggests that

From this chapter's perspective, thin and mushroom spines are particularly relevant. According to the evidence, both are related to the processing of cognitive information: learning from thin spines and memory from mushrooms. However, it is clear that certain lines of projection neurons—like those located in the primary motor cortex, some nuclei of the amygdala, and certain hypothalamic nuclei, like the ventromedial, the preoptic, and the suprachiasmatic—possess dendritic spines that have shown plastic changes, which have been induced experimentally.

mushroom; (3) stubby; (4) wide; (5) branched; and (6) double [16, 17].

grated into the corresponding neural circuits.

a second head.

156 Synaptic Plasticity

room > fat > wide > branched > double.

they represent two independent sites of synaptic contact.

The hypothalamus is a structure that predominantly participates in the regulation of emotion/ affective behavior, in the control of visceral functions, and in the maintenance of the body's homeostasis [18]. The hypothalamus is a structure associated with noncognitive functions. It is neuroanatomically divided into several nuclei, among which is the ventromedial nucleus, which participates in the regulation of sexual behavior in females [18–21]. This portion of the hypothalamus receives information primarily from the medial amygdala and passes the information along to other structures, like the periaqueductal gray and the medulla oblongata, provoking the display of female sexual behavior [21].

Research conducted on the ventromedial nucleus of the hypothalamus has shown the presence of adaptive plastic changes in the neurons of this structure. There is evidence that estrogenic activity reduces the density of dendritic spines on the projection neurons of the ventromedial hypothalamus, thereby facilitating lordosis behavior in female rats [22]. Studies in our laboratory [15] have shown plastic changes in projection neurons in the ventrolateral area of the ventromedial nucleus during different stages of the estrous cycle. Among other plastic changes in the neurons, we observed changes in the densities of spines and in the proportional densities of thin and mushroom spines. The density of spines was greater in diestrus, proestrus, and estrus with respect to metestrus, which was reflected in the greater proportional density of thin and mushroom spines in those stages in which the circulating levels of estradiol are higher. We suggest that these changes are associated with neuroendocrine mechanisms, and that they do not respond to any kind of activity related to cognition. Furthermore, these findings evidence that the functional role of the types of spines that have been classically linked to learning (thin spines) and memory (mushroom spines) would also be linked to other neuropsychophysiological events, beyond those related to the expression of cognitive functions.

The largest number of thin spines in stages like those above could be related to the rapid transmission of synaptic information that is, in fact, mediated by spines with the same geometric characteristics as the thin spines [23–25]. Thus, thin spines would then mediate those changes that in the short term influence female behavior in the shorter phases of the reproductive cycle. Meanwhile, the largest proportion of mushroom spines could be related to the changes that may occur slowly in the formation of patterns of sexual behavior by virtue of the fact that the transmission mediated by this type of spine triggers responses mediated by secondary messengers that, when transmitted to the nucleus, generate the synthesis of proteins [16, 17, 26] capable of modifying the psychophysiology of sexual behavior. Thus, the plasticity of dendritic spines in this region unrelated to cognitive activity might be more related to patterns of synaptic activity. Yes, like those related to cognition, but whose functional significance should be associated with the bioelectric activity that is most fundamental to synaptic activity. Consequently, the interpretation of the plastic changes mediated by dendritic spines should be attributed to the very psychophysiological activity of the noncognitive region in question.

Sensory input is of great importance in the deployment of sexual behavior. Olfactory information that has passed through regions such as the olfactory lobe, the amygdala, the stria terminalis, and the medial preoptic area or the hypothalamus has not yet been involved in nervous centers that give that olfactory information a cognitive character. In all of these structures, it has been seen that exposing rodents to the smell of the opposite sex results in an increase in *fos*protein, which is related to the corresponding increase in metabolic and cellular activity [27]. Thus, the brain generates a plastic response at the molecular level in the presence of relevant sexual stimuli. Studies of brain lesions have shown that massive damage to the medial preoptic area eliminates sexual behavior of male rates throughout the entire life of the individual [28]. In the case of females, the same occurs when the ventromedial hypothalamus is injured [29], a site that, as has already been mentioned, relates to the organization of female sexual behavior.

Despite those findings, there exists in the literature a vast quantity of work, which mentions the role of the prefrontal cortex—an area clearly associated with cognition—in relation to sexual behavior [30–32]. A study by Agmo et al. [33] reported that injuries to the prefrontal cortex and particularly to regions that receive information from the amygdala considerably delay the onset of sexual behavior. However, that same study reported that once male sexual behavior does begin, it then develops normally despite the injury. These data suggest that the prefrontal cortex could be playing an important role in the integration of the information necessary to initiate the approach during sexual arousal. Moreover, other structures participate in the emergence of sexual behavior as sensory receptors, as well as in the expression of copulatory behavior. It is important to highlight that there exists significant gender dimorphism in humans and rodents related to the structures involved in the expression of sexual behavior [27]. This might mark some tendencies about the way processes not directly associated with cognition could differ with the gender of the individuals.

There is experimental evidence that shows a direct relationship between good and bad performance in cognitive tasks and varying levels of hormones such as estradiol and progesterone during the menstrual cycle. It has been observed that in the execution of cognitive tests involving verbal fluency, perceptual speed, fine motor skills, verbal memory, and working memory, performance is higher during the follicular phase, when the greatest amount of estradiol is present in blood plasma. Likewise, when plasma progesterone levels reach their peak in the cycle—halfway through the luteal phase—performance improves on tests of visual memory, in comparison with the menstrual phase [34, 35]. This suggests that there is a differential modulation of cognitive processes by some ovarian hormones, depending on the variation of their concentrations throughout the menstrual cycle. Fernández et al. [36] conducted a longitudinal study that used fMRI to observe the brain activity of young women as they completed cognitive tests focused on language use during different phases of their menstrual cycle. They obtained data that suggested that the neural recruitment necessary to carry out such tasks is very sensitive to the hormonal fluctuations—progesterone and estradiol—of the menstrual cycle. The results likewise showed that the activity of the cortical areas associated with language varies through the different stages of the cycle, and that both progesterone and estradiol were capable of modulating neuronal plasticity of certain areas during the tests. The influence that hormones—which do not imply any cognitive process—have on tasks that are entirely associated with cognition is remarkable.

As in the case of the female reproductive cycle, other hypothalamic structures play a key role in the establishment of some biological rhythms. The suprachiasmatic nucleus and preoptic area of the hypothalamus are associated with the expression of the sleep-wake cycle. This cycle is regulated by the activity of a number of genes, such as the *per*, *clock*, and *tim* genes, which are transcribed cyclically [37, 38]. From the viewpoint of synaptic plasticity, a study from Girardet et al. [39] on rats showed an increase of afferent glutamatergic synapses toward the suprachiasmatic nucleus of the retina, a phenomenon regulated by the input of photic information [40]. That working group also associated with the synaptic arrangements and the configuration of the glia in the suprachiasmatic nucleus with the entrance of photic information that regulates the sleep-wake cycle.

centers that give that olfactory information a cognitive character. In all of these structures, it has been seen that exposing rodents to the smell of the opposite sex results in an increase in *fos*protein, which is related to the corresponding increase in metabolic and cellular activity [27]. Thus, the brain generates a plastic response at the molecular level in the presence of relevant sexual stimuli. Studies of brain lesions have shown that massive damage to the medial preoptic area eliminates sexual behavior of male rates throughout the entire life of the individual [28]. In the case of females, the same occurs when the ventromedial hypothalamus is injured [29], a site that, as has already been mentioned, relates to the organization of female sexual behavior. Despite those findings, there exists in the literature a vast quantity of work, which mentions the role of the prefrontal cortex—an area clearly associated with cognition—in relation to sexual behavior [30–32]. A study by Agmo et al. [33] reported that injuries to the prefrontal cortex and particularly to regions that receive information from the amygdala considerably delay the onset of sexual behavior. However, that same study reported that once male sexual behavior does begin, it then develops normally despite the injury. These data suggest that the prefrontal cortex could be playing an important role in the integration of the information necessary to initiate the approach during sexual arousal. Moreover, other structures participate in the emergence of sexual behavior as sensory receptors, as well as in the expression of copulatory behavior. It is important to highlight that there exists significant gender dimorphism in humans and rodents related to the structures involved in the expression of sexual behavior [27]. This might mark some tendencies about the way processes not directly associated with

There is experimental evidence that shows a direct relationship between good and bad performance in cognitive tasks and varying levels of hormones such as estradiol and progesterone during the menstrual cycle. It has been observed that in the execution of cognitive tests involving verbal fluency, perceptual speed, fine motor skills, verbal memory, and working memory, performance is higher during the follicular phase, when the greatest amount of estradiol is present in blood plasma. Likewise, when plasma progesterone levels reach their peak in the cycle—halfway through the luteal phase—performance improves on tests of visual memory, in comparison with the menstrual phase [34, 35]. This suggests that there is a differential modulation of cognitive processes by some ovarian hormones, depending on the variation of their concentrations throughout the menstrual cycle. Fernández et al. [36] conducted a longitudinal study that used fMRI to observe the brain activity of young women as they completed cognitive tests focused on language use during different phases of their menstrual cycle. They obtained data that suggested that the neural recruitment necessary to carry out such tasks is very sensitive to the hormonal fluctuations—progesterone and estradiol—of the menstrual cycle. The results likewise showed that the activity of the cortical areas associated with language varies through the different stages of the cycle, and that both progesterone and estradiol were capable of modulating neuronal plasticity of certain areas during the tests. The influence that hormones—which do not imply any cognitive process—have on tasks that

As in the case of the female reproductive cycle, other hypothalamic structures play a key role in the establishment of some biological rhythms. The suprachiasmatic nucleus and preoptic

cognition could differ with the gender of the individuals.

158 Synaptic Plasticity

are entirely associated with cognition is remarkable.

The suprachiasmatic nucleus is comprised of general bipolar small neurons whose dendrites may branch or not. Those dendrites display the presence of dendritic spines whose distribution is irregular [41] and which establish synaptic contact with 33% of all of the synapses in the said nucleus [42]. Despite the relevance of the eventual synaptic plasticity that could implicate the circadian regulation of the sleep-wake cycle, there are no studies that show variations in the synaptology of the suprachiasmatic nucleus, which strongly suggests that investigations ought to be performed.

Some studies discussed the relevance of the use of diverse techniques to establish which circuits are involved in insomnia in human adults [43, 44]. However, it seems clear that the participation of structures associated with cognition (like the prefrontal cortex) is also linked with the adequate establishment of those circuits that govern the sleep-wake cycle. Among these structures, it has been reported that the medial prefrontal cortex reduces its functional connectivity with the medial temporal cortex [45].

It has also been reported that the medial and inferior prefrontal cortex showed a decrease in activity when performing fMRI [46], and, particularly, it has been shown that pyramidal neurons from the infralimbic cortex layer III experience alternating plastic changes during both phases of the cycle: in the nocturnal phase, neurons show a pattern of dendritic arborization that is more profuse, and a greater density of spines in comparison with the diurnal phase, which could relate to the cyclical activity of the liberation of different neurotransmitters, growth factors, and corticosterone, in association with the afferent activities of the fibers of the suprachiasmatic nucleus [47]. Another study showed a decrease in the gray matter of the orbitofrontal cortex and in the parietal cortex [48]. In these studies, the results were associated with the presence of insomnia. Together, these investigations suggest that the sleep-wake cycle, although it is a biological rhythm, could also be regulated by structures associated with cognition.

Voluntary motor activity is the result of a series of mostly cognitive processes in which certain areas of the neocortex such as the prefrontal cortex, the premotor cortex, the parietal posterior cortex, and the primary motor cortex, as well as subcortical areas such as the basal ganglia, the thalamus, and the cerebellum participate [49, 50]. All these structures form different circuits for programming and establishing the commands necessary for the execution of movement. The information that is processed in them is sent through the spinal cord by the pyramidal tract from the Betz pyramidal neurons in the primary motor cortex. These last neurons perform the final step in circuits, integrating all the information that has been processed previously.

The pyramidal neurons in layer V of the primary motor cortex do not perform cognitive processing of the information they receive, but rather represent the final necessary filter for that information before motor function [49–51]. Little has been studied about the plastic capacity of these neurons. However, there is research that shows that after behaviors with a cognitive component, such as motor learning [52] and self-stimulation [53, 54], changes occur in the dendritic arborization of pyramidal neurons in layer V of the primary motor cortex. At the same time, preliminary studies conducted in our laboratory (submitted to publication) showed a greater density of dendritic spines (thin, mushroom, and branched spines) on neurons in the layer V of the motor cortex of mice subjected to forced motor activity over a week under differing levels of intensity using a treadmill device. The increase in branched spines corresponded directly to the increase in thin spines, a phenomenon that could be interpreted as a circumstantial demand for the integration of information coming to those neurons due to the increased demand for motor performance. For its part, the greater proportion of mushroom spines could be interpreted as the establishment of patterns of motor activity, which adjusted throughout the study to meet the increasing demand for physical effort on the part of the rats.

Another necessary component for the performance of voluntary movement is the adjustment of patterns of motor execution at the cerebellar level, a characteristic that, it has been suggested, is not associated with the processing of cognitive information. In this sense, it has been shown that Purkinje cells of the simple lobe of the cerebellum present plastic changes at the level of their dendritic spines during the performance of moderate motor activity. Such modifications consist of an increase in the stubby dendritic spines, which could be due to the input of excessive afferent synaptic information—inherent in the requirements of motor activity—which stimulates the postsynaptic components (the dendritic spines), thus causing the formation of the type of spines that would regulate the hypothetical hyperexcitability of the Purkinje neurons involved [55].

The role of the cerebellum during motor learning, a cognitive process, has also been approached. Those studies have investigated metabolic activity in the cerebellar cortex [56] and the plastic changes of the dendritic spines on the Purkinje neurons, particularly in the region that corresponds with the paramedian lobe [57]. In keeping with the role attributed to the spines in paradigms involving cognitive activity, the study found that there was a particular increase in thin spines (acquisition of new information, or learning) and in mushroom spines (consolidation of acquired information, or memory).

Overall, the results of research related to the functional activity of the cerebellum suggest that differential regions of this brain structure work in concert as much in cognitive processing—motor learning—as in noncognitive processing—motor adjustments. In both cases, there is evidence of plastic events at dendritic spines level underlying these processes.

As mentioned in a previous section, there exists a debate over the role of cognition in the emotions. There are certain brain structures involved in the neural circuits that lead to the expression of emotions, including the amygdala and the prefrontal cortex. In the noncognitive processing of emotion, the amygdala plays a key role. This structure participates in two distinct ways. In the more direct way, the amygdala receives thalamic sensory afferents that provide the information necessary to generate emotional responses and, given their connections with the hypothalamus, that induce autonomic activation. Moreover, the connections of the amygdala with the periaqueductal gray matter and the medulla give way to the responses of "freezing" or fight/flight, respectively. The slower way for the amygdala to participate is an indirect route in which the prefrontal cortex processes information associated with the emotional significance of experiences, providing the amygdaloid complex with the information necessary to trigger the appropriate response [6].

From the above, it can be concluded that the expression of emotions depends on structures that are related both to the organization of cognitive functions—the prefrontal cortex, in the indirect route—and with noncognitive functions—the amygdala, in the direct route. It has been reported that chronic stress induces an increase in dendrite length, in dendritic arborization, and in the density and length of the spines of the neurons in the nuclei of the basolateral amygdala. In turn, acute stress provokes an increase in the density of spines, which could be associated with an increase in elevated circulating levels of glucocorticoids [58], and it is clear that the neuronal cytoarchitecture of the amygdala related to noncognitive processes is also subject to plastic changes.

It is common to think of us processing emotions either "consciously" or "unconsciously" [2]. From a neuroscientific point of view, these two terms are what we have in this chapter referred to as "cognitive" and "noncognitive." A recent review of Lee et al. [59] discusses the importance of the noncognitive processing of information necessary for the expression of emotions, particularly in individuals diagnosed with anxiety disorders, schizophrenia, bipolar disorder, and stress. This paper makes a clear distinction between the perception of the emotions without the involvement of a cognitive process and the analysis of emotion that involves the assignation of meaning and emotional valence. It establishes that the perception of emotion that would involve structures such as the amygdala, the insular cortex, the anterior cingulate, and the primary visual cortex [60, 61] is a phenomenon in which information is processed about the stimulus that provoked an emotion without cognitive attribution [62] almost like an automatic processing of emotions without being aware of their meaning.

Although conflicting data regarding the structures that participate in this first step of the recognition of emotions [63] exists, it seems clear that the set of brain processes that involve emotional processing are not only limited to cognition, but that there exist other structures and previous, noncognitive processes that lead to emotional experience [2, 11, 13].

## **4. Conclusions**

The pyramidal neurons in layer V of the primary motor cortex do not perform cognitive processing of the information they receive, but rather represent the final necessary filter for that information before motor function [49–51]. Little has been studied about the plastic capacity of these neurons. However, there is research that shows that after behaviors with a cognitive component, such as motor learning [52] and self-stimulation [53, 54], changes occur in the dendritic arborization of pyramidal neurons in layer V of the primary motor cortex. At the same time, preliminary studies conducted in our laboratory (submitted to publication) showed a greater density of dendritic spines (thin, mushroom, and branched spines) on neurons in the layer V of the motor cortex of mice subjected to forced motor activity over a week under differing levels of intensity using a treadmill device. The increase in branched spines corresponded directly to the increase in thin spines, a phenomenon that could be interpreted as a circumstantial demand for the integration of information coming to those neurons due to the increased demand for motor performance. For its part, the greater proportion of mushroom spines could be interpreted as the establishment of patterns of motor activity, which adjusted throughout the study to meet the increasing demand for physical effort on the part

Another necessary component for the performance of voluntary movement is the adjustment of patterns of motor execution at the cerebellar level, a characteristic that, it has been suggested, is not associated with the processing of cognitive information. In this sense, it has been shown that Purkinje cells of the simple lobe of the cerebellum present plastic changes at the level of their dendritic spines during the performance of moderate motor activity. Such modifications consist of an increase in the stubby dendritic spines, which could be due to the input of excessive afferent synaptic information—inherent in the requirements of motor activity—which stimulates the postsynaptic components (the dendritic spines), thus causing the formation of the type of spines that would regulate the hypothetical hyperexcitability of the

The role of the cerebellum during motor learning, a cognitive process, has also been approached. Those studies have investigated metabolic activity in the cerebellar cortex [56] and the plastic changes of the dendritic spines on the Purkinje neurons, particularly in the region that corresponds with the paramedian lobe [57]. In keeping with the role attributed to the spines in paradigms involving cognitive activity, the study found that there was a particular increase in thin spines (acquisition of new information, or learning) and in mushroom

Overall, the results of research related to the functional activity of the cerebellum suggest that differential regions of this brain structure work in concert as much in cognitive processing—motor learning—as in noncognitive processing—motor adjustments. In both cases, there is evidence of plastic events at dendritic spines level underlying

As mentioned in a previous section, there exists a debate over the role of cognition in the emotions. There are certain brain structures involved in the neural circuits that lead to the expression of emotions, including the amygdala and the prefrontal cortex. In the noncognitive

of the rats.

160 Synaptic Plasticity

Purkinje neurons involved [55].

these processes.

spines (consolidation of acquired information, or memory).

It should be clear that the processing of synaptic information in distinct regions of the brain is independent of the conceptual aspects of the neuropsychological process in question. Thus, the plastic events that underlie the functional organization of "cognitive" and "noncognitive" processes appear to present common neurophysiological and neuromorphological bases. That is, they consist of adaptive changes in different levels of behavioral organization that, more than depending on the plastic events at a cellular level, depend on the structures involved and on the circuits that they establish among themselves in order to result in the expression of behavior.

Based on that, we would propose the following:

1. The intensification of experimental studies of neuronal plasticity related with "noncognitive" processes.

2. The broadening of the criteria of interpretation with regard to the functional significance of such plastic events.

## **Author details**

Ignacio González-Burgos1\* Dulce A. Velázquez-Zamora1,2 David González-Tapia1,2 Nallely Vázquez-Hernández<sup>1</sup> and Néstor I. Martínez-Torres<sup>1</sup>

\*Address all correspondence to: igonbur@hotmail.com

1 Laboratorio de Psicobiología, División de Neurociencias, Centro de Investigación Biomédica de Occidente, Instituto Mexicano del Seguro Social, Jalisco, México

2 Universidad Politécnica de la Zona Metropolitana de Guadalajara, Tlajomulco de Zúñiga, Jalisco, México

#### **References**


bases. That is, they consist of adaptive changes in different levels of behavioral organization that, more than depending on the plastic events at a cellular level, depend on the structures involved and on the circuits that they establish among themselves in order to result in the

1. The intensification of experimental studies of neuronal plasticity related with "noncogni-

2. The broadening of the criteria of interpretation with regard to the functional significance of

Ignacio González-Burgos1\* Dulce A. Velázquez-Zamora1,2 David González-Tapia1,2 Nallely

1 Laboratorio de Psicobiología, División de Neurociencias, Centro de Investigación Biomédica

2 Universidad Politécnica de la Zona Metropolitana de Guadalajara, Tlajomulco de Zúñiga,

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and Néstor I. Martínez-Torres<sup>1</sup>

de Occidente, Instituto Mexicano del Seguro Social, Jalisco, México

\*Address all correspondence to: igonbur@hotmail.com

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such plastic events.

**Author details**

Vázquez-Hernández<sup>1</sup>

Jalisco, México

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## **GABAergic Synapse Dysfunction and Repair in Temporal Lobe Epilepsy**

Meghan A. Van Zandt and Janice R. Naegele

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67218

#### **Abstract**

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166 Synaptic Plasticity

Severe medial temporal lobe epilepsy (mTLE) is often associated with pharmacoresistant seizures, impaired memory and mood disorders. In the hippocampus, GABAergic inhibitory interneuron dysfunction and other neural circuit abnormalities contribute to hyperexcitability, but the mechanisms are still not well understood. Experimental approaches aimed at correcting deficits in hippocampal circuits in mTLE include attempts to replace GABAergic interneurons through neural stem cell transplantation. Evidence from studies in rodent mTLE models indicates that transplanted GABAergic progenitor cells integrate into the hippocampus, form inhibitory synapses, reduce seizures and improve cognitive deficits. Here, we review current work in this field and describe potential molecular mechanisms underlying successful transplantation.

**Keywords:** GABA, temporal lobe, epilepsy, hippocampus, GABAergic, interneurons, neuroligin, neurexin, seizures, inhibition, gephyrin, collybistin, cognition, spatial memory, behaviour, transplantation, therapy, stem cells

## **1. Introduction**

Epilepsy is a brain disorder characterized by a predisposition to generate epileptic seizures that may have subsequent neurological, cognitive, psychological and social effects. Many patients with severe medial temporal lobe epilepsy (mTLE) experience intractable seizures and degenerative changes in the temporal lobes of the brain, particularly the hippocampus [1]. Pharmacological treatments for these patients may become ineffective [2–5], and chronic severe pharmacoresistant seizures in mTLE patients can lead to memory impairments, anxiety and depression. While removing epileptogenic foci provides better seizure control in these patients, surgery may not be feasible if the seizures are generated bilaterally or at

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

multiple foci. Moreover, one of the challenges of treating mTLE is that seizures trigger neuroplastic changes in the adult hippocampus, including axonal sprouting, rewiring and abnormal migration and growth of new dentate granule cells (GCs). Which of these changes are necessary and sufficient for generating recurrent seizures that can be corrected through cellreplacement therapies is not well known.

A major focus of current research is developing rigorous protocols for deriving human neural stem cells from pluripotent stem cells (PSCs) and directing their differentiation into the specific types of neurons, including subtypes of GABAergic interneurons. Additional studies are focusing on the circuit-level and molecular mechanisms that regulate incorporation of transplanted mouse or human GABAergic interneuron progenitors into host brains. Studies of the functional impact of transplanting GABAergic interneurons into the brain and spinal cord in models of different neurological disorders are still in their infancy [6], and relatively little is known about mechanisms guiding the survival, differentiation and synaptic integration of transplanted GABAergic interneurons in the adult brain. This review focuses on current work in this field of regenerative medicine and new directions for regenerating neural circuits.

## **2. Pathology of medial temporal lobe epilepsy**

Dentate gyrus (DG) reorganization has been extensively studied in rodent models of mTLE, particularly the chemoconvulsant models that employ kainic acid or pilocarpine injections to induce status epilepticus (SE). Prominent features of DG reorganization are the loss of glutamatergic mossy cells and subsets of GABAergic interneurons. Depletion of these neuronal populations results in loss of feed-forward inhibition of DG GCs [7–11]. Some of the principal cells of CA1 and CA3, as well as adult-generated GCs of the DG born around the time of SE, sprout excitatory axon collaterals, increasing recurrent excitatory drive between neighbouring neurons [12, 13]. Many somatostatin (SOM) -expressing GABAergic interneurons degenerate in the DG and CA1 of the hippocampus in mTLE, and some residual GABAergic interneurons form compensatory synaptic connections [10, 14–18]. Axonal sprouting by surviving hippocampal GABAergic interneurons increases the number of inhibitory synaptic puncta above control values in chronically epileptic rodents, although this response is insufficient to counter the development of spontaneous recurrent seizures typical of mTLE [15, 19]. Together, these studies suggest that replacing hippocampal GABAergic interneurons in pharmacoresistant mTLE may be a promising strategy for suppressing seizures (**Figure 1**).

GCs are a type of excitatory glutamatergic neuron in the granule cell layer (GCL) of the DG of the hippocampus, and studies suggest that they are relatively more resistant to seizureinduced injury than many other cell types in the hippocampus [32]. They form axons called the mossy fibres that project to CA3 pyramidal neurons and other cell types [1, 33–35]. GCs are generated throughout life [28, 36–41]. In rodent models of mTLE and human patients, many GCs, born around the time of SE, develop altered morphology, excitability and connectivity. These adult-generated GCs form recurrent axon collaterals in the inner molecular layer, a form of neuroplasticity termed mossy fibre sprouting (MFS) [26]. Overgrowth of mossy

multiple foci. Moreover, one of the challenges of treating mTLE is that seizures trigger neuroplastic changes in the adult hippocampus, including axonal sprouting, rewiring and abnormal migration and growth of new dentate granule cells (GCs). Which of these changes are necessary and sufficient for generating recurrent seizures that can be corrected through cell-

A major focus of current research is developing rigorous protocols for deriving human neural stem cells from pluripotent stem cells (PSCs) and directing their differentiation into the specific types of neurons, including subtypes of GABAergic interneurons. Additional studies are focusing on the circuit-level and molecular mechanisms that regulate incorporation of transplanted mouse or human GABAergic interneuron progenitors into host brains. Studies of the functional impact of transplanting GABAergic interneurons into the brain and spinal cord in models of different neurological disorders are still in their infancy [6], and relatively little is known about mechanisms guiding the survival, differentiation and synaptic integration of transplanted GABAergic interneurons in the adult brain. This review focuses on current work in this field of regenerative medicine and new directions for regenerating neural circuits.

Dentate gyrus (DG) reorganization has been extensively studied in rodent models of mTLE, particularly the chemoconvulsant models that employ kainic acid or pilocarpine injections to induce status epilepticus (SE). Prominent features of DG reorganization are the loss of glutamatergic mossy cells and subsets of GABAergic interneurons. Depletion of these neuronal populations results in loss of feed-forward inhibition of DG GCs [7–11]. Some of the principal cells of CA1 and CA3, as well as adult-generated GCs of the DG born around the time of SE, sprout excitatory axon collaterals, increasing recurrent excitatory drive between neighbouring neurons [12, 13]. Many somatostatin (SOM) -expressing GABAergic interneurons degenerate in the DG and CA1 of the hippocampus in mTLE, and some residual GABAergic interneurons form compensatory synaptic connections [10, 14–18]. Axonal sprouting by surviving hippocampal GABAergic interneurons increases the number of inhibitory synaptic puncta above control values in chronically epileptic rodents, although this response is insufficient to counter the development of spontaneous recurrent seizures typical of mTLE [15, 19]. Together, these studies suggest that replacing hippocampal GABAergic interneurons in pharmacoresistant mTLE may be a promising strategy for

GCs are a type of excitatory glutamatergic neuron in the granule cell layer (GCL) of the DG of the hippocampus, and studies suggest that they are relatively more resistant to seizureinduced injury than many other cell types in the hippocampus [32]. They form axons called the mossy fibres that project to CA3 pyramidal neurons and other cell types [1, 33–35]. GCs are generated throughout life [28, 36–41]. In rodent models of mTLE and human patients, many GCs, born around the time of SE, develop altered morphology, excitability and connectivity. These adult-generated GCs form recurrent axon collaterals in the inner molecular layer, a form of neuroplasticity termed mossy fibre sprouting (MFS) [26]. Overgrowth of mossy

replacement therapies is not well known.

168 Synaptic Plasticity

suppressing seizures (**Figure 1**).

**2. Pathology of medial temporal lobe epilepsy**

**Figure 1.** Studies of GABAergic interneuron transplantation are investigating whether it is feasible to replace populations of endogenous interneurons damaged by temporal lobe epilepsy. **(A)** In the non-epileptic mouse dentate gyrus (DG), self-renewing, quiescent Type 1 progenitor cells in the subgranular zone (SGZ) extend radial glial processes through the granule cell layer (GCL) (1). They divide asymmetrically to produce Type 2 progenitors (2) which further divide to generate pools of migratory Type 3 neuroblasts (3). As these neuroblasts mature, they differentiate into GCs, migrate into the GCL, extend their dendrites towards the molecular layer (ML)(4) and project axons to hilar interneurons and mossy cells and CA3 pyramidal cells (5). Inhibitory GABAergic interneurons (6) synapse with GCs and provide inhibition. GCs are shown in yellow, inhibitory interneurons in light blue and pyramidal cells in magenta. **(B)** High-resolution confocal image of retrovirally labelled GCs (green) and neuronal nuclei (NeuN, red) in the naïve mouse hippocampus. White arrow indicates a normal Type 1 progenitor cell in the SGZ. **(C)** In mTLE, some of the hilar GABAergic interneurons die, resulting in an overall loss of inhibition to GCs (1). Additionally, some adult-generated GCs undergo abnormal migration into the hilus, becoming ectopic. They typically form abnormal dendrites and sprout recurrent axonal collaterals, forming excitatory feedback projections onto other GCs (3) as well as abnormal excitatory projections to the CA3 pyramidal cells (4) [20–30]. GCs are shown in yellow; an abnormal, ectopic GC in orange; and pyramidal cells in magenta. **(D)** Highresolution confocal image of retrovirally labelled GCs (red) in the hippocampus of a mouse with mTLE. Nuclei are marked using a Nissl stain (blue). White arrows show ectopic GCs born after induction of epilepsy located in abnormal locations in the hilus. **(E)** Studies employing transplantation of GABAergic progenitors into either the normal or epileptic hippocampus show that they migrate away from the site of injection and form dense axonal arbors throughout the hilus, GCL and molecular layer. These interneurons appear to form functional synapses with hyperexcitable GCs, including those with aberrant morphologies (1), increasing synaptic inhibition in the epileptic circuit. GCs are shown in yellow; abnormal, ectopic GC in orange; transplanted GABAergic inhibitory interneurons in green; and pyramidal cells in magenta [31]. **(F)** High-resolution confocal image of a retrovirally labelled GC (red) receiving dense synaptic contacts from transplanted MGE-derived GABAergic progenitors (green). Nuclei are labelled using Nissl staining (blue) [31].

fibre recurrent collaterals onto GCs and pyramidal cells contributes to a hyperexcitable dentate environment [27, 29, 42–44].

As demonstrated by computer simulations, a few hubs of highly interconnected GCs are sufficient to create a hyperexcitable network [30, 45]. Additional epilepsy-induced neuroplastic changes to the DG include GC dispersion, formation of GC basal dendrites and ectopic migration of GCs into the hilus of the DG [23–25, 46–50]. Many adult-generated GCs born in epileptic rodents also have reduced dendritic spines and hypertrophic cell bodies [20, 21, 44, 51]. These cells often have higher baseline firing rates than normally positioned GCs in epileptic animals and non-epileptic controls and more depolarized resting membrane potentials, predisposing them to hyperexcitability [22]. Although increasing adult neurogenesis is not sufficient to cause seizures, it contributes to hyperexcitability [52, 53].

## **3. Cognitive changes in medial temporal lobe epilepsy**

Severe mTLE is linked to a number of comorbidities including cognitive deficits, heightened anxiety, increased aggression and depression [54, 55]. Rodents with mTLE show decreased social recognition, greater preference for closed arms in the elevated zero or plus maze test for anxiety and longer periods of immobility in the forced swim test for depression [56]. Additionally, a number of studies demonstrated severe learning deficits in the Morris water maze test of spatial memory [57, 58] and other spatial memory tasks [59]. Human mTLE patients exposed to virtual environments that test spatial memory also showed memory deficits [60, 61].

Changes in the properties of hippocampal and entorhinal cortex circuits may be responsible for cognitive changes in mTLE, but the nature of these changes is not well understood. Both human and rodent spatial memory formation and recall are dependent on place cells in the hippocampus and grid cells in the entorhinal cortex. These important cells exhibit distinct, spatially specific firing patterns, forming a topographical memory map of an area as an individual moves through space [62–65]. Hippocampal inhibitory interneurons, similar to place cells, show distinct, spatially specific discharges, implicating a role in the formation and fine tuning of spatial memories [66, 67]. Studies suggest that impairments in receptive field properties of the grid and place cells may occur in mTLE [68–71].

The regulation by inhibitory interneurons of various brain rhythms may also become altered, as rodents with mTLE show distinctly lower frequency and power of theta rhythms correlated with poor performance in spatial memory tasks [72–74]. Although the exact alterations occurring in the grid-place cell network are not yet clear, it is evident that the hyperexcitable firing of GCs and the overall disinhibition of the network by loss of inhibitory interneurons severely disrupts spatial memory formation [72, 75–79]. The disinhibition of the hippocampal networks following epileptogenesis and the subsequent development of spatial memory deficits suggest that the loss of inhibitory interneurons may disrupt place fields, providing a further rationale for cell-based therapies aimed at GABAergic interneuron transplantation.

## **4. Seizure suppression following transplantation of medial ganglionic eminence-derived neural progenitors**

fibre recurrent collaterals onto GCs and pyramidal cells contributes to a hyperexcitable den-

As demonstrated by computer simulations, a few hubs of highly interconnected GCs are sufficient to create a hyperexcitable network [30, 45]. Additional epilepsy-induced neuroplastic changes to the DG include GC dispersion, formation of GC basal dendrites and ectopic migration of GCs into the hilus of the DG [23–25, 46–50]. Many adult-generated GCs born in epileptic rodents also have reduced dendritic spines and hypertrophic cell bodies [20, 21, 44, 51]. These cells often have higher baseline firing rates than normally positioned GCs in epileptic animals and non-epileptic controls and more depolarized resting membrane potentials, predisposing them to hyperexcitability [22]. Although increasing adult neurogenesis is not suf-

Severe mTLE is linked to a number of comorbidities including cognitive deficits, heightened anxiety, increased aggression and depression [54, 55]. Rodents with mTLE show decreased social recognition, greater preference for closed arms in the elevated zero or plus maze test for anxiety and longer periods of immobility in the forced swim test for depression [56]. Additionally, a number of studies demonstrated severe learning deficits in the Morris water maze test of spatial memory [57, 58] and other spatial memory tasks [59]. Human mTLE patients exposed to virtual environments that test spatial memory also showed memory defi-

Changes in the properties of hippocampal and entorhinal cortex circuits may be responsible for cognitive changes in mTLE, but the nature of these changes is not well understood. Both human and rodent spatial memory formation and recall are dependent on place cells in the hippocampus and grid cells in the entorhinal cortex. These important cells exhibit distinct, spatially specific firing patterns, forming a topographical memory map of an area as an individual moves through space [62–65]. Hippocampal inhibitory interneurons, similar to place cells, show distinct, spatially specific discharges, implicating a role in the formation and fine tuning of spatial memories [66, 67]. Studies suggest that impairments in receptive field prop-

The regulation by inhibitory interneurons of various brain rhythms may also become altered, as rodents with mTLE show distinctly lower frequency and power of theta rhythms correlated with poor performance in spatial memory tasks [72–74]. Although the exact alterations occurring in the grid-place cell network are not yet clear, it is evident that the hyperexcitable firing of GCs and the overall disinhibition of the network by loss of inhibitory interneurons severely disrupts spatial memory formation [72, 75–79]. The disinhibition of the hippocampal networks following epileptogenesis and the subsequent development of spatial memory deficits suggest that the loss of inhibitory interneurons may disrupt place fields, providing a further rationale for cell-based therapies aimed at GABAergic interneuron transplantation.

ficient to cause seizures, it contributes to hyperexcitability [52, 53].

**3. Cognitive changes in medial temporal lobe epilepsy**

erties of the grid and place cells may occur in mTLE [68–71].

tate environment [27, 29, 42–44].

170 Synaptic Plasticity

cits [60, 61].

Initial studies established proof of concept for cell-based therapies for treating epilepsy by demonstrating that transplants of non-neural cells engineered to release GABA non-synaptically could increase seizure thresholds [80]. Increasingly, studies have aimed to identify the functional classes of interneurons that can migrate, integrate and suppress seizures in different models of epilepsy in rodents. The large variety of functional classes of cortical inhibitory interneurons and their sites of origin in the ventricular zones of the embryonic forebrain have been extensively studied. During development, forebrain GABAergic interneurons are born in the embryonic ventricular regions called the ganglionic eminences, including the medial, lateral and caudal ganglionic eminences (MGE, LGE, CGE). These transient proliferative zones lining the forebrain lateral ventricles generate different types of GABAergic interneuron progenitors, which then migrate to their final destinations in the forebrain, including the cerebral cortex, hippocampus and striatum [81–83]. Forebrain GABAergic progenitors expressing SOM or parvalbumin (PV) emerge from the MGE in early embryonic life and migrate tangentially into the cerebral cortex and hippocampus [84].

In naïve rodents and different epilepsy models, transplanted GABAergic interneuron progenitors from the embryonic MGE have been found to be highly migratory, a prerequisite for transplantation therapies aimed at repairing large brain areas. MGE-derived GABAergic progenitors transplanted into postnatal 3–4-day old mouse cerebral cortex differentiated into inhibitory interneurons expressing markers of mature GABAergic phenotypes, including PV, SOM, calretinin (CR) and neuropeptide Y (NPY), and displayed mature firing properties characteristic of inhibitory interneurons [85]. MGE-derived PV-positive interneurons transplanted into naïve postnatal 1–2-day old pups integrated into the endogenous circuitry and, upon maturation, displayed firing properties similar to endogenous PV-expressing interneurons and formed functional synapses onto pyramidal neurons [86]. In a mouse model of mTLE generated through neurotoxin-induced ablation of GABAergic interneurons, MGE transplantation significantly increased inhibitory postsynaptic currents (IPSCs) in CA1 pyramidal cells and reduced seizure frequency and severity [87]. These grafts contained high percentages of GABAergic interneurons that co-expressed PV, NPY or CR. Transplanting MGE cells into an epilepsy model caused by mutations of the Kv1.1 potassium channel also increased IPSCs in nearby endogenous pyramidal cells [88]. Additionally, MGE cell transplants into a cyclin D2 knockout model of hippocampal disinhibition restored lost inhibitory input and normalized hyperactivity and fear conditioning [89].

The efficacy of MGE cell transplantation for controlling seizures has also been studied in chemoconvulsant models of mTLE, including the kainic acid and pilocarpine (PILO) models. In an early ground-breaking study in the rat kainic acid model, Shetty and colleagues transplanted neurospheres derived from embryonic day-14 rat MGE progenitors and found that they reduced seizure duration, total time spent in seizures and seizure severity; however, these grafts failed to improve spatial memory deficits [90]. It is important to note that the degree of cognitive impairment may differ between kainic acid or pilocarpine models, different species and even different strains of mice [91, 92].

The mouse PILO model shows a pattern of loss of hippocampal interneurons that is similar to human mTLE, making this model highly appropriate for preclinical studies investigating GABAergic interneuron transplantation [93]. Work from our laboratory showed that MGE cells transplanted into the hilus of the DG led to significant reductions in seizure frequency, duration and severity in the mouse PILO model [31]. The transplanted neurons matured into GABAergic interneurons that expressed CB, SOM or PV and formed dense networks of inhibitory synapses onto dentate GCs. Optogenetic experiments in hippocampal slices from these mice showed that light-induced depolarization of MGE transplants expressing channelrhodopsin (ChR2) triggered strong postsynaptic inhibitory currents in GCs, indicating that the transplanted neurons had integrated synaptically. These findings suggest that seizure suppression can be achieved with focal transplants into the DG. In this study, which employed continuous video-EEG recording for periods of up to 3 months, some of the mice show a reoccurrence of seizures several months after transplantation, suggesting that achieving enduring seizure suppression may require more widespread dispersion of the transplanted interneurons throughout different subfields of the hippocampus. Determining the optimal sites and cell types for permanent seizure suppression will be important for moving into clinical applications.

## **5. Transplantation therapy using human embryonic stem cell-derived progenitors**

For treating patients with severe mTLE, sources of human interneurons are required. Previous work showed that differentiating human embryonic stem cells (hESCs) into GABAergic inhibitory interneuron progenitors can be achieved using specific combinations of signalling molecules and growth factors [94–100]. Carpentino et al. (2008) found that maturation of transplanted mouse or hESCs is highly dependent on the environment into which the cells are transplanted. For instance, in the mouse systemic kainic acid model, it was shown that ESCderived neural progenitors transplanted in the CA3 area tended to migrate into the DG and differentiate into GCs, whereas those implanted into the fimbria tended to mature into astrocytes [101]. Lee et al. also transplanted undifferentiated hESCs into the CA3 region of the hippocampus in epileptic rats. Some of these differentiated into GABAergic interneurons (~21% of engrafted cells) and at 8 weeks post transplantation displayed immature morphology. Even with low numbers of GABAergic neurons, the animals with transplants showed reduced seizure frequency and seizure duration for 2–3 months. EEG recordings in these animals were limited to 60 hours per week, with a 2-week recording period, during daylight hours only. Additionally, when tested 3 months after transplantation, improvements in Morris water maze performance were not found [102]. More recent studies have focused on purified, fate-determined populations of hESCs for transplantation. Treating hESCs with the signalling molecule sonic hedgehog (SHH) or a sonic hedgehog agonist (SAG), in combination with modulating the WNT and FGF signalling pathways, can be used in vitro to induce ventral forebrain neural fates and MGE-like cell types [97–100, 103–106]. Ventralized hESC-derived progenitors have identities similar to that of mouse MGE-derived GABAergic interneuron progenitors, potentially allowing the large-scale *in vitro* production of human cells for therapies to treat clinical disorders [94]. However, undifferentiated hESCs can cause teratomas, making it important to develop protocols for eliminating them prior to transplantation [107].

degree of cognitive impairment may differ between kainic acid or pilocarpine models, differ-

The mouse PILO model shows a pattern of loss of hippocampal interneurons that is similar to human mTLE, making this model highly appropriate for preclinical studies investigating GABAergic interneuron transplantation [93]. Work from our laboratory showed that MGE cells transplanted into the hilus of the DG led to significant reductions in seizure frequency, duration and severity in the mouse PILO model [31]. The transplanted neurons matured into GABAergic interneurons that expressed CB, SOM or PV and formed dense networks of inhibitory synapses onto dentate GCs. Optogenetic experiments in hippocampal slices from these mice showed that light-induced depolarization of MGE transplants expressing channelrhodopsin (ChR2) triggered strong postsynaptic inhibitory currents in GCs, indicating that the transplanted neurons had integrated synaptically. These findings suggest that seizure suppression can be achieved with focal transplants into the DG. In this study, which employed continuous video-EEG recording for periods of up to 3 months, some of the mice show a reoccurrence of seizures several months after transplantation, suggesting that achieving enduring seizure suppression may require more widespread dispersion of the transplanted interneurons throughout different subfields of the hippocampus. Determining the optimal sites and cell types for

permanent seizure suppression will be important for moving into clinical applications.

**5. Transplantation therapy using human embryonic stem cell-derived** 

For treating patients with severe mTLE, sources of human interneurons are required. Previous work showed that differentiating human embryonic stem cells (hESCs) into GABAergic inhibitory interneuron progenitors can be achieved using specific combinations of signalling molecules and growth factors [94–100]. Carpentino et al. (2008) found that maturation of transplanted mouse or hESCs is highly dependent on the environment into which the cells are transplanted. For instance, in the mouse systemic kainic acid model, it was shown that ESCderived neural progenitors transplanted in the CA3 area tended to migrate into the DG and differentiate into GCs, whereas those implanted into the fimbria tended to mature into astrocytes [101]. Lee et al. also transplanted undifferentiated hESCs into the CA3 region of the hippocampus in epileptic rats. Some of these differentiated into GABAergic interneurons (~21% of engrafted cells) and at 8 weeks post transplantation displayed immature morphology. Even with low numbers of GABAergic neurons, the animals with transplants showed reduced seizure frequency and seizure duration for 2–3 months. EEG recordings in these animals were limited to 60 hours per week, with a 2-week recording period, during daylight hours only. Additionally, when tested 3 months after transplantation, improvements in Morris water maze performance were not found [102]. More recent studies have focused on purified, fate-determined populations of hESCs for transplantation. Treating hESCs with the signalling molecule sonic hedgehog (SHH) or a sonic hedgehog agonist (SAG), in combination with modulating the WNT and FGF signalling pathways, can be used in vitro to induce ventral forebrain neural fates and MGE-like cell types [97–100, 103–106]. Ventralized hESC-derived progenitors have

ent species and even different strains of mice [91, 92].

**progenitors**

172 Synaptic Plasticity

Evidence that fate-directed human GABAergic interneuron progenitors integrate into the epileptic circuitry of the hippocampus following transplantation into the hilus has emerged in several recent studies. To reduce immune rejection of cell grafts, human and mouse ESC transplantation studies generally use immunodeficient host animals. The nonobese diabetic (NOD)-severe combined immunodeficiency (SCID) mice are an immunodeficient mouse strain lacking mature T and B cells and with reduced natural killer (NK) cell activity. Another mouse strain, the Nod-scid-gamma (NSG) triple mutant, has a mutation at the interleukin-2 receptor (IL-2R) γ-chain locus. This strain shows the highest impairment in T-cell, B-cell and NK-cell development, resulting in low graft rejection [108]. Both strains have been used to study differentiation of ESC-derived GABAergic interneurons [109].

In a recent study in which hESC-derived progenitors were differentiated in vitro into MGElike progenitors and transplanted into NSG mice, the transplanted cells differentiated into GABAergic neurons expressing SOM, PV, CB, CR or NPY after approximately 4 months. Additionally, optogenetic stimulation of the transplanted cells produced action potentials and resulted in IPSCs in endogenous hippocampal neurons, suggesting successful synaptic integration into the existing circuitry of the hippocampus. Video-EEG monitoring of these animals 3 months post-transplant showed reduced numbers of seizures in engrafted animals [110]. However, the EEG monitoring was only for short durations of 5–10 days, which is likely too brief a period to reliably evaluate seizures in rodent chemoconvulsant models, due to the clustered and periodic nature of the spontaneous recurrent seizures.

## **6. Ameliorating cognitive and behavioural abnormalities in epilepsy by transplantation of GABAergic interneurons**

Inconsistent results regarding spatial memory improvement have been reported following GABAergic interneuron transplantation. In the Morris water maze test of spatial memory, C57BL/6 mice with PILO induced mTLE and received mouse MGE cell transplants showed significantly reduced escape latencies in training, significantly more platform crossings in the probe trial; improved path efficiency; and a greater amount of time spent in the target quadrant than epileptic controls [111]. In another study, rats with mTLE that MGE-derived stem cell grafts showed no improvements in the Morris water maze task 8 weeks post-engraftment relative to non-engrafted mTLE controls [90]. However, transplantation took place approximately 3 months following induction of epilepsy, a longer time interval than other studies. The lack of cognitive improvement at this later transplantation time point suggests a potentially limited time window in which transplanted GABAergic interneurons must integrate to confer cognitive improvements. In a third study in NSG mice with mTLE, engrafted hESCderived GABAergic interneuron progenitors appeared to improve performance in the Y-maze test of spatial memory and memory in the novel recognition test [110].

Behavioural tests also suggested that mTLE mice receiving interneuron grafts were less hyperactive and aggressive, compared to mTLE controls with only intrahippocampal injections of media. In the handling test of aggression, in which mice are scored for aggressive reactions to a series of increasingly uncomfortable stimuli, TLE mice with hESC or foetal mouse MGE interneuron transplants scored significantly lower in aggression ratings than controls [109, 110]. These transplants also reduced hyperactive behaviour [110]. Taken together, these results suggest that both rodent and human GABAergic interneuron transplants may ameliorate some of the psychological comorbidities in rodents with mTLE. While the Morris water maze is currently one of the standard tests in the industry for spatial memory, rodents with mTLE often exhibit a phenomenon known as thigmotaxis, in which animals will locomote or swim adjacent to the walls of an apparatus or make repeated circles [91, 112]. In such animals, it is uncertain whether the data reflect poor spatial memory or an anxiety phenotype. Therefore, alternative spatial memory tests should be used to gain a more complete understanding of how GABAergic interneuron transplantation affects cognition. An alternative test of hippocampal-dependent spatial memory is a modification of the novel object recognition test in which animals must learn to recognize that a previously familiar object has changed location. This test, called novel object location task, takes advantage of the rodent preference for novelty and desire to explore changes in its environment [113, 114]. Another test of spatial memory is the Barnes maze, consisting of an elevated platform with closed holes around the circumference. One hole is available for escape into a dark box. Remaining on the platform is unpleasant to the rodent, due to bright lights, fans and/or loud ambient noise, encouraging a swift escape to the box. As this test measures a very natural desire to escape an unpleasant environment, it is considered an effective test of normal rodent behaviour and spatial memory [115]. The Barnes maze also has no walls, eliminating thigmotaxis, although care must be taken to prevent animals from falling from the raised platform. Additional tests of spatial memory include the Y-maze, T-maze and the radial arm maze, all of which measure the ability of a rodent to remember previously travelled areas [116–121]. This extensive array of spatial memory tests can provide a more complete picture of the behavioural improvements following GABAergic interneuron transplantation in rodents with mTLE.

Currently, most testing of aggression has been done using the handling test, which, while effective, is an unnatural stimulus to the rodent [110, 111]. In addition to the handling test, the resident-intruder test can be used to analyse the response of a rodent to more natural stimuli. Male rodents are territorial, and the resident-intruder test measures the aggressive reactions to a male rival within their space. Although care must be taken to avoid injury to animals, this test measures an innate animal response and can be an effective measure of aggression in mTLE animals with transplants [122].

Although heightened anxiety is a common and well-characterized comorbidity in rodent mTLE models and human patients, surprisingly little work has been done to examine the effects of GABAergic transplants on correcting anxiety phenotypes. As rodents with mTLE have a tendency to exhibit thigmotaxis, which skews results in tests such as the open field test or the Morris water maze [91, 112], paradigms such as the elevated plus maze, elevated zero maze or the light-dark box can be used to provide more accurate measures of anxiety in rodents with mTLE [123–128].

## **7. GABAergic synapse formation and stability: potential mechanisms of transplanted cell integration**

Behavioural tests also suggested that mTLE mice receiving interneuron grafts were less hyperactive and aggressive, compared to mTLE controls with only intrahippocampal injections of media. In the handling test of aggression, in which mice are scored for aggressive reactions to a series of increasingly uncomfortable stimuli, TLE mice with hESC or foetal mouse MGE interneuron transplants scored significantly lower in aggression ratings than controls [109, 110]. These transplants also reduced hyperactive behaviour [110]. Taken together, these results suggest that both rodent and human GABAergic interneuron transplants may ameliorate some of the psychological comorbidities in rodents with mTLE. While the Morris water maze is currently one of the standard tests in the industry for spatial memory, rodents with mTLE often exhibit a phenomenon known as thigmotaxis, in which animals will locomote or swim adjacent to the walls of an apparatus or make repeated circles [91, 112]. In such animals, it is uncertain whether the data reflect poor spatial memory or an anxiety phenotype. Therefore, alternative spatial memory tests should be used to gain a more complete understanding of how GABAergic interneuron transplantation affects cognition. An alternative test of hippocampal-dependent spatial memory is a modification of the novel object recognition test in which animals must learn to recognize that a previously familiar object has changed location. This test, called novel object location task, takes advantage of the rodent preference for novelty and desire to explore changes in its environment [113, 114]. Another test of spatial memory is the Barnes maze, consisting of an elevated platform with closed holes around the circumference. One hole is available for escape into a dark box. Remaining on the platform is unpleasant to the rodent, due to bright lights, fans and/or loud ambient noise, encouraging a swift escape to the box. As this test measures a very natural desire to escape an unpleasant environment, it is considered an effective test of normal rodent behaviour and spatial memory [115]. The Barnes maze also has no walls, eliminating thigmotaxis, although care must be taken to prevent animals from falling from the raised platform. Additional tests of spatial memory include the Y-maze, T-maze and the radial arm maze, all of which measure the ability of a rodent to remember previously travelled areas [116–121]. This extensive array of spatial memory tests can provide a more complete picture of the behavioural improve-

ments following GABAergic interneuron transplantation in rodents with mTLE.

mTLE animals with transplants [122].

174 Synaptic Plasticity

rodents with mTLE [123–128].

Currently, most testing of aggression has been done using the handling test, which, while effective, is an unnatural stimulus to the rodent [110, 111]. In addition to the handling test, the resident-intruder test can be used to analyse the response of a rodent to more natural stimuli. Male rodents are territorial, and the resident-intruder test measures the aggressive reactions to a male rival within their space. Although care must be taken to avoid injury to animals, this test measures an innate animal response and can be an effective measure of aggression in

Although heightened anxiety is a common and well-characterized comorbidity in rodent mTLE models and human patients, surprisingly little work has been done to examine the effects of GABAergic transplants on correcting anxiety phenotypes. As rodents with mTLE have a tendency to exhibit thigmotaxis, which skews results in tests such as the open field test or the Morris water maze [91, 112], paradigms such as the elevated plus maze, elevated zero maze or the light-dark box can be used to provide more accurate measures of anxiety in Relatively few studies have examined the molecular mechanisms responsible for guiding synaptic integration of transplanted cells into mature neural circuits. Previous findings suggest that cell-cell interactions mediate the formation and stabilization of both excitatory and inhibitory synapses [129–131]. The synaptic scaffolding complex between GABAergic interneurons and their postsynaptic targets in the developing brain may also guide recruitment and stabilization of the new synaptic connections formed by transplanted interneurons (**Figure 2**).

**Figure 2.** Interactions between cell surface molecules that are binding partners provide a potential mechanism for forming or stabilizing new synapses between transplanted GABAergic interneurons and endogenous neurons in the hippocampus. GABAergic synapse formation is coordinated by multiple molecules in the pre- and postsynaptic sites. Binding between presynaptic neurexin molecules and postsynaptic neuroligin2 (NLGN2) molecules may be important for initial formation or maintenance of GABAergic synapses. NLGN2 is associated with a postsynaptic complex containing collybistin, gephyrin and GABAARs, which are necessary in the formation of functional inhibitory circuitry. Collybistin, gephyrin, NLGN2 or GABAAR subunit γ2 deficiency results in impaired inhibitory synapses [132–141].

The synaptic scaffolding protein gephyrin is a tubulin-binding protein that forms a latticework structure of hexagonal trimers that regulate GABAA receptor clustering at synaptic sites [142, 143]. Gephyrin stabilizes inhibitory synapses and is required for proper function. Genetic reduction of the γ2 subunit of GABAA receptors, a primary binding partner of gephyrin in GABAergic synapses, also severely reduces gephyrin and GABAA receptor clustering required for functional inhibitory synapses [132]. Repression of gephyrin expression causes a similar loss of clustering, revealing an interdependent relationship between the two synaptic binding partners necessary for proper inhibitory synapse formation and function [132, 144, 145]. Increases in endogenous gephyrin in response to compensatory surviving interneuron sprouting may also make the epileptic hippocampus a more receptive environment for new inhibitory synapses to form [146]. Gephyrin is significantly decreased in the first few weeks post-SE followed by a significant increase back towards normal levels at around 1 month post-SE [147]. Following transplantation, a majority of engrafted GABAergic interneuron synaptic boutons were associated with postsynaptic gephyrin clusters, indicating that this vital synaptic scaffolding component may be recruited to sites of new GABAergic synapse formation in the adult hippocampus [31].

Collybistin, another GABAergic synaptic scaffolding component, binds to both gephyrin and Neuroligin 2 (NLGN2) and may facilitate gephyrin-mediated clustering of GABAA receptors. Collybistin is a GDP/GTP-exchange factor that interacts directly with gephyrin in the inhibitory synaptic scaffold [131, 133, 148–150]. Collybistin-deficient mice display reduced clustering of gephyrin and GABAA receptors, reduced synaptic inhibition and altered synaptic plasticity [131, 141].

NLGN2 is part of a family of cell adhesion molecules implicated in synapse formation and stability. NLGN2 localizes only to GABAergic inhibitory synapses, where it is associated with neurexin, its presynaptic binding partner [151–153]. NLGN2 is part of the molecular scaffolding complex that includes collybistin and gephyrin [133]. NLGN2-deficient mice show decreased inhibitory function, as well as a variety of cognitive and behavioural comorbidities, such as increased anxiety, aggression and disruptions in spatial memory formation, similar to those seen in mTLE and other neurological disorders [129, 136, 137, 154–157]. Various studies have shown that binding between NLGN2 and neurexin induces inhibitory synapse formation [130, 158] and stabilization [135, 159], even in non-neuronal cell types [160].

GABAA receptor subunit composition may also play a role in the integration and stabilizing influence of transplanted inhibitory interneurons. Composition of GABAA subunits is impacted by the pathological changes induced in mTLE [161–163]. DG GCs, which are significant propagators of hyperexcitability in mTLE, are particularly enriched in the δ subunit of GABAA receptors in the normal brain; these receptors have a very high affinity for GABA and are strongly involved in tonic inhibition [164–166] at extrasynaptic sites [167]. In general, hippocampal neurons express multiple subunits, including abundant α, β, δ and γ subunits, with δ primarily restricted to GCs of the DG, with additional expression of other subunits in the CA1 and CA3 areas [168]. As such, it is apparent that GABAA receptors in the hippocampus are composed of a diverse pool of subunits that regulate inhibitory input. In mTLE, the composition of GABAA subunits becomes altered. Similar to the upregulation of gephyrin during the chronic phase of mTLE in response to compensatory interneuron sprouting, the γ2 and α subunits also show increased expression in the hippocampus. Conversely, expression of the δ subunit decreases days after the initial epileptic event and remains depressed into the chronic stages of mTLE [169]. It is not known whether synapses formed by surviving inhibitory interneurons are capable of recruiting the necessary subunit composition for proper inhibition, considering the overall depletion of δ subunits compared to γ2 and α. Moreover, whether transplanted, healthy GABAergic inhibitory interneurons can recruit all of the normal subunits to inhibitory synapses is not known. Further investigation of subunit composition within the epileptic hippocampus post transplantation will be necessary to investigate whether transplantation normalizes GABAA receptor composition.

## **8. Conclusion**

The synaptic scaffolding protein gephyrin is a tubulin-binding protein that forms a latticework structure of hexagonal trimers that regulate GABAA receptor clustering at synaptic sites [142, 143]. Gephyrin stabilizes inhibitory synapses and is required for proper function. Genetic reduction of the γ2 subunit of GABAA receptors, a primary binding partner of gephyrin in GABAergic synapses, also severely reduces gephyrin and GABAA receptor clustering required for functional inhibitory synapses [132]. Repression of gephyrin expression causes a similar loss of clustering, revealing an interdependent relationship between the two synaptic binding partners necessary for proper inhibitory synapse formation and function [132, 144, 145]. Increases in endogenous gephyrin in response to compensatory surviving interneuron sprouting may also make the epileptic hippocampus a more receptive environment for new inhibitory synapses to form [146]. Gephyrin is significantly decreased in the first few weeks post-SE followed by a significant increase back towards normal levels at around 1 month post-SE [147]. Following transplantation, a majority of engrafted GABAergic interneuron synaptic boutons were associated with postsynaptic gephyrin clusters, indicating that this vital synaptic scaffolding component may be recruited to sites of new GABAergic synapse forma-

Collybistin, another GABAergic synaptic scaffolding component, binds to both gephyrin and Neuroligin 2 (NLGN2) and may facilitate gephyrin-mediated clustering of GABAA receptors. Collybistin is a GDP/GTP-exchange factor that interacts directly with gephyrin in the inhibitory synaptic scaffold [131, 133, 148–150]. Collybistin-deficient mice display reduced clustering of gephyrin and GABAA receptors, reduced synaptic inhibition and altered synaptic

NLGN2 is part of a family of cell adhesion molecules implicated in synapse formation and stability. NLGN2 localizes only to GABAergic inhibitory synapses, where it is associated with neurexin, its presynaptic binding partner [151–153]. NLGN2 is part of the molecular scaffolding complex that includes collybistin and gephyrin [133]. NLGN2-deficient mice show decreased inhibitory function, as well as a variety of cognitive and behavioural comorbidities, such as increased anxiety, aggression and disruptions in spatial memory formation, similar to those seen in mTLE and other neurological disorders [129, 136, 137, 154–157]. Various studies have shown that binding between NLGN2 and neurexin induces inhibitory synapse formation [130, 158] and stabilization [135, 159], even in non-neuronal

GABAA receptor subunit composition may also play a role in the integration and stabilizing influence of transplanted inhibitory interneurons. Composition of GABAA subunits is impacted by the pathological changes induced in mTLE [161–163]. DG GCs, which are significant propagators of hyperexcitability in mTLE, are particularly enriched in the δ subunit of GABAA receptors in the normal brain; these receptors have a very high affinity for GABA and are strongly involved in tonic inhibition [164–166] at extrasynaptic sites [167]. In general, hippocampal neurons express multiple subunits, including abundant α, β, δ and γ subunits, with δ primarily restricted to GCs of the DG, with additional expression of other subunits in the CA1 and CA3 areas [168]. As such, it is apparent that GABAA receptors in the hippocampus are composed of a diverse pool of subunits that regulate inhibitory input. In mTLE,

tion in the adult hippocampus [31].

plasticity [131, 141].

176 Synaptic Plasticity

cell types [160].

While safe and effective stem cell therapies for treating neurological disorders, including severe mTLE, may be years away from the clinic, recent work has increased scientific understanding of how to derive specific types of human neurons for transplantation and how to evaluate functional changes that result. Because human neuron maturation takes many months or years, transplantation studies in rodents are limited in the kinds of information they can provide about the potential therapeutic effects of these cells in clinical populations. Recent studies have utilized a wide range of experimental tools, including electrophysiology, immunohistochemistry, optogenetics, chemogenetics and behavioural assays to assess learning, memory, anxiety, social behaviour and depression. These approaches are aiding studies to evaluate synaptic integration and functionality of human neural stem cell transplants for treating epilepsy.

### **Acknowledgements**

We would like to thank Nicholas Woods, Bryan Luikart and Elizabeth Paquette for their assistance with retroviral labelling of GCs and confocal images. Work in our lab was supported by NINDS grant R15NS072879-01A1, Connecticut Stem Cell Established Investigator Grant and a Challenge Award from Citizens United for Research in Epilepsy (J.R.N.).

### **Author details**

Meghan A. Van Zandt and Janice R. Naegele\* \*Address all correspondence to: jnaegele@wesleyan.edu Wesleyan University, Middletown, CT, USA

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## **Neuroplasticity in Bipolar Disorder: Insights from Neuroimaging**

Marlos Vasconcelos Rocha, Fabiana Nery,

Amanda Galvão-de-Almeida,

Lucas de Castro Quarantini and

Ângela Miranda-Scippa

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67288

#### **Abstract**

Background: Advances in neuroimaging techniques have produced evidence about disrupted frontolimbic circuits related to emotional regulation. These neuroimaging studies may suggest impairments in cellular plasticity in bipolar disorder (BD) patients. However, the long-term use of mood stabilizers may restore these dysfunctions by neurotrophic effects

Objectives: Review the major structures of the brain that underpin this disorder, synthesize the main findings in neuroimaging in BD, and debate neuroplastic effects of psychopharmacological treatment on findings from the neuroimaging studies.

Methods: We undertook a review from neuroimaging in BD. Search words entered were "bipolar disorder", "mania", "depression", "mixed states", "suicide", "psychosis" "lithium", "mood stabilizers", "neuroplasticity", magnetic resonance imaging", "functional magnetic resonance", "FDG-PET","SPECT".

Results: The literature highlighted specific brain areas that support emotional regulation and processing. Moreover, there is heterogeneity across studies and some findings are controversial, but some results suggest neuroplastic effects from the long-term psychopharmacological treatment (particularly mood stabilizers) in bipolar disorder.

Conclusion: The findings in neuroimaging studies suggest there is fronto-limbic circuitry dysregulation in BD; changes in specific brain areas have been replicated in several studies, which may reflect impairments in neuroplastic phenomena. Evidence from neuroimaging studies have been also show that long-term treatment may be

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

associated with metabolic/functional compensation or structural restoration in bipolar responders.

**Keywords:** bipolar disorder, neuroimaging, treatment, neuroplasticity

## **1. Introduction**

Bipolar disorder (BD) affects around 3% of the population [1] and is a serious multifactorial disease, caused by combination of genetic vulnerability and environmental stressors with abnormalities in neurotransmitter and neuroendocrine systems, and intracellular signaling pathways as well. Clinically, BD is characterized by recurrent changes of thought, behavior, cognition, mood, and desynchronization of circadian rhythm, which imply in affective phases—mania, hypomania, depression, and mixed states. As a result, BD is a condition often difficult to diagnose, since at least 50% of patients with BD have an initial episode of depression and 35% may have a delay in their diagnosis in up to 10 years [2]. In this context, samples more homogeneous in neuroimaging studies in BD may allow better understanding of BD pathophysiology, through the establishment of putative associations between areas and neuronal circuits and clinical phenotypes, and also to clarify the utility of the various neuroimaging methods for determining potential neurobiological markers of BD.

In this sense, some authors propose concepts in neuroimaging biomarkers for mood disorders, such as *prognostic biomarkers* that characterize the risk for onset or progression of the disease, *predictive biomarkers* associated with the likelihood of therapeutic response, and *pharmacodynamic biomarkers*, which show biological response related to drug treatment [3].

The majority of neuroimaging studies in BD have been demonstrated abnormalities in different cortical and subcortical areas involved in emotional processing and regulation, while *postmortem* histopathological studies of these regions have shown abnormal reductions of synaptic markers and glial cells in prefrontal cortex and hippocampus and point to a dysfunction of the complex intracellular mechanisms, which involve second messengers systems, regulation of the genic expression and synthesis of trophic factors [4]. Overall, these neuropathological and neuroimaging studies may suggest impairments in cellular plasticity and resilience in patients who suffer from mood disorders.

Conversely, a substantial body of evidence suggests that long-term psychopharmacological treatment with antidepressants and mood stabilizers—in particular lithium and valproate may compensate for this dysfunction by reducing the pathological limbic activity subjacent to affective symptoms and by regulating gene expression of neurotrophic factors that exert neuroplastic effects within the pathways modulating emotional expression. Such effects may be associated with structural restoration or enlargement of specific brain areas in chronically treated BD patients evaluated in multiple neuroimaging studies, when compared to healthy controls [5–7].

Keeping these issues in mind, the purpose of this chapter is to review the major cortical and subcortical structures of the brain that underpin this disorder, describe the main findings in structural and functional neuroimaging in BD, and synthesize impaired major cellular plasticity mechanisms and potential neuroplastic effects of mood stabilizers on structural and functional findings from the neuroimaging studies.

## **2. Circuits and neuronal models of emotion regulation**

associated with metabolic/functional compensation or structural restoration in bipolar

Bipolar disorder (BD) affects around 3% of the population [1] and is a serious multifactorial disease, caused by combination of genetic vulnerability and environmental stressors with abnormalities in neurotransmitter and neuroendocrine systems, and intracellular signaling pathways as well. Clinically, BD is characterized by recurrent changes of thought, behavior, cognition, mood, and desynchronization of circadian rhythm, which imply in affective phases—mania, hypomania, depression, and mixed states. As a result, BD is a condition often difficult to diagnose, since at least 50% of patients with BD have an initial episode of depression and 35% may have a delay in their diagnosis in up to 10 years [2]. In this context, samples more homogeneous in neuroimaging studies in BD may allow better understanding of BD pathophysiology, through the establishment of putative associations between areas and neuronal circuits and clinical phenotypes, and also to clarify the utility of the various neuroimag-

In this sense, some authors propose concepts in neuroimaging biomarkers for mood disorders, such as *prognostic biomarkers* that characterize the risk for onset or progression of the disease, *predictive biomarkers* associated with the likelihood of therapeutic response, and *pharmacodynamic biomarkers*, which show biological response related to drug treatment [3].

The majority of neuroimaging studies in BD have been demonstrated abnormalities in different cortical and subcortical areas involved in emotional processing and regulation, while *postmortem* histopathological studies of these regions have shown abnormal reductions of synaptic markers and glial cells in prefrontal cortex and hippocampus and point to a dysfunction of the complex intracellular mechanisms, which involve second messengers systems, regulation of the genic expression and synthesis of trophic factors [4]. Overall, these neuropathological and neuroimaging studies may suggest impairments in cellular plasticity and

Conversely, a substantial body of evidence suggests that long-term psychopharmacological treatment with antidepressants and mood stabilizers—in particular lithium and valproate may compensate for this dysfunction by reducing the pathological limbic activity subjacent to affective symptoms and by regulating gene expression of neurotrophic factors that exert neuroplastic effects within the pathways modulating emotional expression. Such effects may be associated with structural restoration or enlargement of specific brain areas in chronically treated BD patients evaluated in multiple neuroimaging studies, when compared to healthy controls [5–7]. Keeping these issues in mind, the purpose of this chapter is to review the major cortical and subcortical structures of the brain that underpin this disorder, describe the main findings in

**Keywords:** bipolar disorder, neuroimaging, treatment, neuroplasticity

ing methods for determining potential neurobiological markers of BD.

resilience in patients who suffer from mood disorders.

responders.

194 Synaptic Plasticity

**1. Introduction**


The ACC is divided functionally into ventral or "affective" region (more anterior portions of BA 25 [paragenual] and BA 24 [subgenual]) and dorsal or "cognitive" region (posterior prelimbic area [BA 32] and more posterior portions of BA 24).

The affective division of the ACC has connections with the amygdala, the periaqueductal gray matter, the anterior thalamic nuclei, the ventral striatum, and the insula; it contributes to the regulation of endocrine and autonomic functions, generation of appropriate social behavior, and part of the global emotional response by activation of somatic and visceral states relevant to emotional experience; cognitive division includes the posterior portions of BA 24 and 32 and connects to the periaqueductal gray matter and primary and associative cortical motor areas; it is associated with inhibition responses [10] and monitoring conflicts [11, 12].


Compounding these circuits, the cerebellum receives cortical projections from nuclei located in the base of the pons; the fibers of the pontine nuclei decussate and follow the middle cerebellar peduncle to specific cerebellar targets: while the motor cortex projects to the cerebellum (paravermian region) through lateral pontine nuclei, associative cortical areas of the prefrontal, parietal, and temporal regions as well as ACC reach the cerebellum through pontine nuclei medial [13].

The amygdala is divided into three major sections: basolateral, corticomedial, and central. The basolateral nucleus participates in the sensory information integration from external and internal environments, which are linked to learned information and are processed by associative cortical areas, with subsequent planning, selection, and implementation of the action; corticomedial nucleus contributes to the presence of emotional attributes related to sensory and nociceptive stimuli; and the central nucleus is the convergence site of all signs of the amygdala. The amygdala regulates the fight, flight, or freeze behaviors together with the periaqueductal gray matter and contributes to motor and autonomic responses to emotional stimuli [14].

Among several neural circuit models related to processes of emotional perception and regulation proposed in the literature, the Mary Phillips and coworkers' model highlights over others [15, 16]. This model proposes the existence of two neuronal systems: a ventral system comprising subcortical (the amygdala, the insula, the ventral striatum) and cortical structures (the hippocampus, the anterior cingulate, and prefrontal cortex) and would be linked to identification of the emotional meaning of a stimulus associated with generation of affective states and autonomic regulation; the dorsal system would be represented by dorsal regions of the anterior cingulate and PFC as well as the hippocampus and would support cognitive processes such as selective attention, planning, performance monitoring, and voluntary regulation of emotional states.

The assessment of neuroimaging findings in BD allows to corroborate the relevance of this model, from the identification of dysfunction in different cortical and subcortical areas—as already stated, structures involved in processing and emotion regulation—abnormal increase in activity of the amygdala during performance of emotional and non-emotional tasks; abnormal decrease in activity of the VLPFC and orbital frontal cortex (OFC); and abnormal decrease in functional connectivity between the amygdala and the prefrontal cortex during emotional regulation tasks. Moreover, in studies involving reward paradigms (anticipation of reward), there is abnormal increase in activity of the ventral striatum, the VLPFC and OFC [17, 18].

## **3. Main findings of neuroimaging in BD**

#### **3.1. White matter**

The white matter (WM) hyperintensity is a change often described in BD patients, both in adult [19] and pediatric [20] samples. Among the WM association bundles, the corpus callosum (CC) is one of the structures of great interest in BD research. In this region, studies using diffusion tensor technique (DTI) often show loss of structural integrity of the CC in its various segments (genu, body, or splenius) [21, 22]. Moreover, a recent study conducted by our group evaluated bipolar patients type I euthymic and showed reduction of CC in the areas of the genu and isthmus when compared to healthy controls, confirming data from other studies [23, 24], but with no significant difference between suicide and non-suicidal [25]; a meta-analysis documented the volume reduction of this structure in bipolar patients [26]. Finally, another study found that bipolar patients without suicide attempt had lower values of fractional anisotropy (FA) in the genu and body of the CC when compared to unipolar depressed and healthy controls, and bipolar suicide patients had reduction of FA in all regions of the CC when compared to healthy controls [27]. In addition, more recent studies have shown that in euthymic and non-euthymic bipolar patients with a history of psychotic symptoms was observed higher area of the rostrum of the CC [28] and lower FA in the body of the CC in bipolar euthymic or depressed patients [29]; importantly, changes of the CC have also been described in children and adolescents with BD [30] and in groups of risk for BD, such as first-degree relatives [31].

Furthermore, loss of functional integrity was verified in other associative bundles of white matter, for instance, uncinate fasciculus (which connects the orbital frontal cortex and areas of the ventromedial prefrontal cortex to the amygdala and hippocampus) was studied in some works, in which the results are inconsistent, with bilateral reduction of AF [32, 33] or increased AF to the left in this region [34]. Finally, lower FA in the left orbital frontal WM among patients with attempted suicide, a finding that correlated with higher impulsivity score [35].

Taken together, these findings suggest that WM abnormalities in BD may compromise the interhemispheric neuronal transmission and subsequent emotional processing/regulation which may represent a potential anatomical biomarker of the disease—and precede the onset of bipolar disorder and predispose to brain development changes during the neurodevelopmental process of the central nervous system (CNS) in children and adolescents.

#### **3.2. Frontal lobe**

Compounding these circuits, the cerebellum receives cortical projections from nuclei located in the base of the pons; the fibers of the pontine nuclei decussate and follow the middle cerebellar peduncle to specific cerebellar targets: while the motor cortex projects to the cerebellum (paravermian region) through lateral pontine nuclei, associative cortical areas of the prefrontal, parietal, and temporal regions as well as ACC reach the cerebellum through pon-

The amygdala is divided into three major sections: basolateral, corticomedial, and central. The basolateral nucleus participates in the sensory information integration from external and internal environments, which are linked to learned information and are processed by associative cortical areas, with subsequent planning, selection, and implementation of the action; corticomedial nucleus contributes to the presence of emotional attributes related to sensory and nociceptive stimuli; and the central nucleus is the convergence site of all signs of the amygdala. The amygdala regulates the fight, flight, or freeze behaviors together with the periaqueductal gray matter and contributes to motor and autonomic responses to emotional

Among several neural circuit models related to processes of emotional perception and regulation proposed in the literature, the Mary Phillips and coworkers' model highlights over others [15, 16]. This model proposes the existence of two neuronal systems: a ventral system comprising subcortical (the amygdala, the insula, the ventral striatum) and cortical structures (the hippocampus, the anterior cingulate, and prefrontal cortex) and would be linked to identification of the emotional meaning of a stimulus associated with generation of affective states and autonomic regulation; the dorsal system would be represented by dorsal regions of the anterior cingulate and PFC as well as the hippocampus and would support cognitive processes such as selective attention, planning, performance monitoring, and voluntary regu-

The assessment of neuroimaging findings in BD allows to corroborate the relevance of this model, from the identification of dysfunction in different cortical and subcortical areas—as already stated, structures involved in processing and emotion regulation—abnormal increase in activity of the amygdala during performance of emotional and non-emotional tasks; abnormal decrease in activity of the VLPFC and orbital frontal cortex (OFC); and abnormal decrease in functional connectivity between the amygdala and the prefrontal cortex during emotional regulation tasks. Moreover, in studies involving reward paradigms (anticipation of reward), there is abnormal increase in activity of the ventral striatum, the VLPFC and OFC [17, 18].

The white matter (WM) hyperintensity is a change often described in BD patients, both in adult [19] and pediatric [20] samples. Among the WM association bundles, the corpus callosum (CC) is one of the structures of great interest in BD research. In this region, studies using diffusion tensor

tine nuclei medial [13].

196 Synaptic Plasticity

stimuli [14].

lation of emotional states.

**3.1. White matter**

**3. Main findings of neuroimaging in BD**

The anterior cingulate cortex (ACC), dorsolateral prefrontal cortex (DLPFC), and orbital frontal cortex (OFC) represent the most widely studied frontal lobe areas in BD research.

Studies assessed the ACC through MRI showed volumetric reduction of the subgenual AC (sgACC) (areas 24 and 25 Brodmann), a finding confirmed in a meta-analysis [36]. In studies with proton magnetic resonance spectroscopy (H+ MRS), reduction of the N-acetyl-aspartate (NAA) and increase of the choline in the ACC are the most consistent results [37]. Interestingly, some studies showed that long-term treatment with lithium increased the sgACC volume [6] and was associated to higher levels of NAA in this region [38, 39]. Additionally, Functional changes on PET studies were observed both in patients at resting state as in those undergoing activation tasks in the CC: most of them show hyperactivity ACC, notably the sgACC in patients in depressive [40] and manic [41] states.

Studies that evaluated the pregenual ACC (pgACC) also reported abnormalities, with reduction of the left pgACC volume, both in adults [42, 43] and in adolescents [44]. Another H+ MRS study of this region detected in patients with mania increased relationship glutamine/glutamate, a finding that may be related to impairment of glial-neuronal cell interaction in the interpretation of the authors [45].

Some studies have demonstrated reduction in DLPFC volume in adults [46, 47] and pediatric samples [48], whereas other studies using H+ MRS reported reduction of NAA in this region [49, 50]. At last, hypometabolism of the DLPFC with <sup>18</sup>FDG-PET and functional magnetic resonance imaging (fMRI) in patients with BD was found, both in mania [51] and in depression [52–54].

Other studies evaluating the OFC showed reduction of its volume both in adults [46, 55–57] and in pediatric patients [58, 59], but the heterogeneity of their samples may limit the interpretation of results. A H+ MRS study of this region demonstrated the decrease of the NAA and the choline in hospitalized non-euthymic bipolar patients (mixed or manic episode), but a minority was in use of lithium [60], whereas our group reported normal metabolic levels in medial orbital frontal cortex in BD I euthymic outpatients with and without suicidal behavior [7]. In fact, in our sample, 30.2% of the subjects were prescribed the first mood stabilizer in the year after the first affective episode, 22% after the first year before the fifth year, and 47.8% 5 years after the first affective episode, and it was demonstrated a higher prevalence of suicide attempts in the latter group [61]. These results support the protective clinical effect of the use of mood stabilizers on the suicidal behavior.

Finally, in patients with mania, hypoactivation of the VLPFC and the OFC in fMRI studies was documented [62, 63].

#### **3.3. Amygdala**

Volumetric abnormalities of amygdala are among the most common findings, especially in adolescent samples, in which smaller volumes were reported, but with controversial results among adults. While the reduction of the amygdala among adolescents may represent an anatomical characteristic finding of this clinical subgroup, inconsistent results in adults may result from clinical course, the proportion of adult patients with early BD compounding the sample or neuroplastic effects associated with treatment [15].

The studies of fMRI suggest abnormality of the amygdala in response to a variety of experimental paradigms (resting state, processing of emotional stimuli, and cognitive tasks with or without emotional valence) in the context of the various affective states [11]. On the other hand, some authors reported the absence of hyperactivity of the amygdala during euthymia, which may reflect normalization of the amygdala function induced by long-term treatment, possible evidence of neuroplasticity [64].

#### **3.4. Cerebellum**

Most structural neuroimaging studies of the cerebellum showed a reduction in the volume of sub-regions of the cerebellar vermis [65–68], and reduction of the cerebellar volume may be associated with genetic predisposition to BD [69]. Additionally, reduction of the density of the gray matter of the cerebellar vermis has been reported in untreated BD patients, but not in patients under treatment, which may suggest possible neuroprotective effects associated with psychopharmacological drug use [70].

#### **3.5. Hippocampus**

Another H+

198 Synaptic Plasticity

MRS study of this region detected in patients with mania increased relation-

MRS reported reduction of NAA in this region [49, 50]. At

MRS study of this region demonstrated the decrease of the NAA

ship glutamine/glutamate, a finding that may be related to impairment of glial-neuronal

Some studies have demonstrated reduction in DLPFC volume in adults [46, 47] and pediatric sam-

last, hypometabolism of the DLPFC with <sup>18</sup>FDG-PET and functional magnetic resonance imaging

Other studies evaluating the OFC showed reduction of its volume both in adults [46, 55–57] and in pediatric patients [58, 59], but the heterogeneity of their samples may limit the inter-

and the choline in hospitalized non-euthymic bipolar patients (mixed or manic episode), but a minority was in use of lithium [60], whereas our group reported normal metabolic levels in medial orbital frontal cortex in BD I euthymic outpatients with and without suicidal behavior [7]. In fact, in our sample, 30.2% of the subjects were prescribed the first mood stabilizer in the year after the first affective episode, 22% after the first year before the fifth year, and 47.8% 5 years after the first affective episode, and it was demonstrated a higher prevalence of suicide attempts in the latter group [61]. These results support the protective clinical effect of the use

Finally, in patients with mania, hypoactivation of the VLPFC and the OFC in fMRI studies

Volumetric abnormalities of amygdala are among the most common findings, especially in adolescent samples, in which smaller volumes were reported, but with controversial results among adults. While the reduction of the amygdala among adolescents may represent an anatomical characteristic finding of this clinical subgroup, inconsistent results in adults may result from clinical course, the proportion of adult patients with early BD compounding the

The studies of fMRI suggest abnormality of the amygdala in response to a variety of experimental paradigms (resting state, processing of emotional stimuli, and cognitive tasks with or without emotional valence) in the context of the various affective states [11]. On the other hand, some authors reported the absence of hyperactivity of the amygdala during euthymia, which may reflect normalization of the amygdala function induced by long-term treatment,

Most structural neuroimaging studies of the cerebellum showed a reduction in the volume of sub-regions of the cerebellar vermis [65–68], and reduction of the cerebellar volume may be associated with genetic predisposition to BD [69]. Additionally, reduction of the density of the gray matter of the cerebellar vermis has been reported in untreated BD patients, but not in

(fMRI) in patients with BD was found, both in mania [51] and in depression [52–54].

cell interaction in the interpretation of the authors [45].

ples [48], whereas other studies using H+

of mood stabilizers on the suicidal behavior.

possible evidence of neuroplasticity [64].

sample or neuroplastic effects associated with treatment [15].

pretation of results. A H+

was documented [62, 63].

**3.3. Amygdala**

**3.4. Cerebellum**

Data from a meta-analysis that summarized the results of 25 studies of hippocampal structure have found reduced hippocampal volume, especially in bipolar adolescent samples and reported apparent relationship between increased hippocampal volume and lithium therapy, which may explain the non-significant difference in hippocampal volume in most studies with samples of adult patients when compared to healthy controls [71].

## **4. Evidence of impairment of cellular resilience and plasticity in BD**

There is growing evidence in literature of changes of neuroprotective processes and cellular plasticity and resilience pathways in BD from morphometric and neuropathological studies. Particularly, several mechanisms have founded to be involved as putative etiologic theories that underlie the neurobiological basis of BD, including proinflammatory cytokines, intracellular signaling cascades, and disrupted neurotrophic factor pathways.

More specifically, inflammatory mediators, such as interleukins, tumor necrosis factor alpha (TNF-a), and C-reactive protein, may influence several aspects of the pathophysiology of BD through changes in regulation of neuronal excitability, neuronal survival, synaptic transmission, and plasticity [72, 73]. Several studies have demonstrated a low-grade proinflammatory state in BD during euthymia [74–77], whereas both mania and depression seem to be associated with even more increased circulating cytokines [78, 79]. In addition, it has been suggested that proinflammatory cytokines may be one of the mechanisms of progression of BD, according to some studies [77, 80].

In terms of dysfunction of intracellular signaling cascades, there is a solid evidence of impaired regulation of calcium signaling and increased intracellular calcium levels, with subsequent loss of modulation of neuronal and glial activity, increased oxidative stress, and shortened survival cell [81–83]. Besides, Bcl-2, a protein with both antiapoptotic and neuroprotective properties highly expressed in the limbic system [84, 85], is associated with calcium regulation, reducing its release; Bcl-2 polymorphism AA was associated with both higher cytosolic calcium levels in lymphoblasts [86] and age-related decreases in brain gray matter volume [87].

Additional important signaling cascades involved in BD pathophysiology are those associated with members of the neurotrophin family, especially brain-derived neurotrophic factor (BDNF), which exerts its biological effects through activation of intracellular systems, including the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway [88].

BDNF is essential for neuroplastic phenomena, such as neurogenesis, neuronal survival, normal maturation of neural development pathways, and synaptic plasticity and dendritic growth in adulthood as well [89], and it has been demonstrated circulating BDNF is reduced during manic and depressive states [90, 91], while ERK/MAPK pathway is an important intracellular mediator of biological effects of neurotrophic factors, acting on several proteins involved in cellular plasticity, such as glycogen synthase K-3 (GSK-3)—a major regulator of apoptosis—and cAMP response element-binding protein (CREB), which is a facilitator of the expression of neurotrophic/neuroprotective proteins such as Bcl-2 and BDNF [92, 93].

Information about histopathological abnormalities of neuronal and glial cells from *postmortem* studies in mental illness is scarce and its interpretation may be limited due to employment of different techniques and presence of confounding factors such as illicit-drugs and alcohol abuse [94]. However, particularly in BD patients, these abnormalities seem to be concentrated on frontolimbic regions associated with emotional regulation, including the DLPFC and ACC [95].

Although histopathological findings vary among regions and layers of the prefrontal cortex in BD patients, the majority of *postmortem* studies points up reductions in the neuronal density and size, glial cell density and changes in protein expression (implicated in the regulation of synaptic plasticity), which likely result from a combination of dendritic atrophy and/or cell loss in the DLPFC and ACC [95].

In this context, it is possible to hypothesize a link among the neuropathological, neuroimaging, and clinical findings, which dendritic atrophy and cell loss may result in reductions of volume in prefrontal areas, as abnormal synaptic interactions among cortical and subcortical brain structures may result in structural and functional intra- and interhemispheric disconnections and culminate in more vulnerability to stressful stimuli from environment, emotional dysregulation, and BD-related affective, cognitive, and behavioral symptoms [96].

However, lithium and valproic acid, respectively, through inhibition of glycogen synthase kinase-3 (GSK-3) and the histone deacetylases (HDACs), regulate the transcription and expression of neurotrophic, angiogenic, and neuroprotective proteins, such as BDNF, glial cell line-derived neurotrophic factor (GDNF), and angiogenic vascular endothelial growth factor (VEGF). Also, lithium in particular acts on factors that affect apoptotic signaling, such as Bcl-2, p53, Bax, caspase, and heat shock proteins (HSP); both lithium and valproate activate ERK/MAPK pathway. Finally, lithium contributes to induction of the ubiquitin-proteasome system and autophagy, two major intracellular quality control mechanisms for protein clearance that prevent abnormal protein accumulation. Overall, these findings highlight the properties of lithium and probably other mood stabilizers to suppress cell death, attenuate neuroinflammation, and promote angiogenesis and cellular plasticity in BD patients, which contribute to the reduction of neuronal loss [5].

However, not all neuroimaging studies show benefits from long-term use of mood stabilizers. For instance, in a study that evaluated both medicated with antipsychotics or lithium manic (most hospitalized) and outpatient euthymic patients and healthy controls using fMRI demonstrated loss of functional connectivity between amygdala and ACC in manic, but not in euthymic patients; according to its authors, these findings may suggest a state-dependent neuronal dysfunction [97], but these results may be a marker of treatment non-response, since all patients were medicated.

This latter hypothesis has been brought up a longitudinal study in which bipolar I patients were assigned to euthymic, responders, and non-responders to lithium therapy. When baseline and after treatment volumes of the hippocampus, amygdala, PFC, DLPFC, and ACC volumes were compared, there was a significant enlargement in the left PFC and DLPF in bipolar I patients who responded to treatment, and the left hippocampus and right ACC volumes were decreased in non-responders [98].

## **5. Conclusion**

growth in adulthood as well [89], and it has been demonstrated circulating BDNF is reduced during manic and depressive states [90, 91], while ERK/MAPK pathway is an important intracellular mediator of biological effects of neurotrophic factors, acting on several proteins involved in cellular plasticity, such as glycogen synthase K-3 (GSK-3)—a major regulator of apoptosis—and cAMP response element-binding protein (CREB), which is a facilitator of the expression of neurotrophic/neuroprotective proteins such as Bcl-2 and

Information about histopathological abnormalities of neuronal and glial cells from *postmortem* studies in mental illness is scarce and its interpretation may be limited due to employment of different techniques and presence of confounding factors such as illicit-drugs and alcohol abuse [94]. However, particularly in BD patients, these abnormalities seem to be concentrated on frontolimbic regions associated with emotional regulation, including the DLPFC

Although histopathological findings vary among regions and layers of the prefrontal cortex in BD patients, the majority of *postmortem* studies points up reductions in the neuronal density and size, glial cell density and changes in protein expression (implicated in the regulation of synaptic plasticity), which likely result from a combination of dendritic atrophy and/or cell

In this context, it is possible to hypothesize a link among the neuropathological, neuroimaging, and clinical findings, which dendritic atrophy and cell loss may result in reductions of volume in prefrontal areas, as abnormal synaptic interactions among cortical and subcortical brain structures may result in structural and functional intra- and interhemispheric disconnections and culminate in more vulnerability to stressful stimuli from environment, emotional dysregulation, and BD-related affective, cognitive, and behavioral symptoms [96].

However, lithium and valproic acid, respectively, through inhibition of glycogen synthase kinase-3 (GSK-3) and the histone deacetylases (HDACs), regulate the transcription and expression of neurotrophic, angiogenic, and neuroprotective proteins, such as BDNF, glial cell line-derived neurotrophic factor (GDNF), and angiogenic vascular endothelial growth factor (VEGF). Also, lithium in particular acts on factors that affect apoptotic signaling, such as Bcl-2, p53, Bax, caspase, and heat shock proteins (HSP); both lithium and valproate activate ERK/MAPK pathway. Finally, lithium contributes to induction of the ubiquitin-proteasome system and autophagy, two major intracellular quality control mechanisms for protein clearance that prevent abnormal protein accumulation. Overall, these findings highlight the properties of lithium and probably other mood stabilizers to suppress cell death, attenuate neuroinflammation, and promote angiogenesis and cellular plasticity in BD patients, which

However, not all neuroimaging studies show benefits from long-term use of mood stabilizers. For instance, in a study that evaluated both medicated with antipsychotics or lithium manic (most hospitalized) and outpatient euthymic patients and healthy controls using fMRI demonstrated loss of functional connectivity between amygdala and ACC in manic, but not in euthymic patients;

BDNF [92, 93].

200 Synaptic Plasticity

and ACC [95].

loss in the DLPFC and ACC [95].

contribute to the reduction of neuronal loss [5].

In summary, the main findings in structural and functional neuroimaging studies suggest that there is frontolimbic circuitry dysregulation in BD, characterized by impairment of control of subcortical regions by cortical ones; changes in specific brain areas have been replicated in several studies, which may reflect impairments in physiological neuroplastic phenomena in the central nervous system. However, growing body of evidence from neuroimaging studies also shows that long-term treatment with mood stabilizers may be associated with metabolic/functional compensation or structural restoration, at least in bipolar responders, and neuroimaging techniques may be considered as a potential tool for establishing prognostic, predictive, or pharmacodynamic biomarkers in BD in the future.

## **Author details**

Marlos Vasconcelos Rocha1, 2\*, Fabiana Nery2, 3, Amanda Galvão-de-Almeida2, 3, Lucas de Castro Quarantini2, 3 and Ângela Miranda-Scippa2, 3

\*Address all correspondence to: mfvrocha@gmail.com

1 Department of Cognitive Neurology, University Hospital Professor Edgard Santos, Salvador, Bahia, Brazil

2 Program of Mood and Anxiety Disorders (CETHA), Federal University of Bahia, Bahia, Brazil

3 Department of Neuroscience and Mental Health, Federal University of Bahia, Salvador, Bahia, Brazil

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208 Synaptic Plasticity


## *Edited by Thomas Heinbockel*

Nerve cells form thousands of contact points, the synapses, to communicate information with other neurons and target cells. Synapses are sites for changes in brain function through modification of synaptic transmission termed synaptic plasticity. The study of synaptic plasticity has flourished over the years with the advancement of technical breakthroughs and is a timely scientific endeavor today just like it was several decades ago. This book contributes to our understanding of synaptic plasticity at the molecular, biochemical, and cellular systems and behavioral level and informs the reader about its clinical relevance. The book contains ten chapters in three sections: (1) "Mechanisms of Synaptic Plasticity," (2) "Neural Plasticity," and (3) "Plasticity and Neurological Diseases." The book provides detailed and current reviews in these different areas written by experts in their respective fields. The mechanisms of synaptic plasticity and its relation to neurological diseases are featured prominently as a recurring theme throughout most chapters. This book will be most useful for neuroscientists and other scientists alike and will contribute to the training of current and future students who find the plastic nervous system as fascinating as many generations before them.

Synaptic Plasticity

Synaptic Plasticity

*Edited by Thomas Heinbockel*

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