**2. Evolution of the amygdaloid complex in vertebrates**

comfort. Secondly, the animal should be motivated to escape from predators, cold, sexual competitors and other forms of adversity. To survive as an individual and a species, even our oldest ocean-dwelling ancestors living over 560 million years ago must have been able to react to the environment to feed, evade predators, defend territory and reproduce. Hence, their primitive nervous systems must have regulated the necessary behaviours and incorporated the most essential structures of all today's freely moving Animalia. However, since then the human brain passed through a long evolutionary pathway during which particularly the forebrain showed major changes. The earliest vertebrate brain almost completely lacked the forerunner of the human neocortex and the dorsal parts of the basal ganglia [1, 2]. These newer parts of the brain are believed to determine human behaviour to a high degree and consequently receive most attention in research of processes explaining the genesis of mental disorders (see, e.g. Ref. [3]). This contrasts the involvement in psychic disorders of those behavioural processes described above as also being displayed by the most primitive vertebrates. We want to suggest that these actions are still regulated in humans by brain structures derived from the primitive forebrain of the earliest vertebrates. Therefore, we described the anatomy of the forebrain of the earliest human vertebrate ancestors, which is believed to be comparable with the brain of lampreys [2]. From a comparison of the striatum of lampreys to that of anuran amphibians and younger vertebrates, it can be concluded that the striatum of lampreys is the forerunner of the human nuclear amygdala [4]. In anuran amphibians (frogs and toads), the lamprey's striatum is retrieved as central and medial amygdaloid nuclei, while a later ventral striatum for the first time appears in its direct vicinity [2, 4]. The lamprey's forebrain also contains a structure of which the connections are very well conserved in more recent human ancestors: the habenula. The habenula constitutes—together with the stria medullaris and pineal gland—the epithalamus and consists of medial and lateral parts [5]. The habenula regulates the intensity of reward-seeking and misery-fleeing behaviour probably in all our vertebrate ancestors. In lampreys the activity of the habenula is in turn regulated by a specific structure: the habenula-projecting globus pallidus. It is tempting to speculate that this structure has a similar role in humans, but a clear anatomical human equivalent with the same function has not yet been identified. Based upon the evolution of the basal ganglia in vertebrates and the mechanism of the emotional response, we postulate the existence of two systems regulating the intensity of the aforementioned behaviours [6, 7]. These two circuits include the activities of extrapyramidal and limbic basal ganglia and are collaborating in a yin-and-yang-like fashion. The two basal ganglia systems are linked together by the core and shell parts of the nucleus accumbens (NAcb), which regulates motivation to show reward-seeking and misery-fleeing

The amygdala is believed to address the ability of learning to value sensory information (attentive salience). This capacity is essential to determine which sensory information is of vital importance to react on with reward-seeking or misery-fleeing behaviour. Aberrant salience is believed to be a crucial component of the mechanism of (schizophrenic) psychosis and the antipsychotic effects of dopamine antagonists [8]. We believe that the amygdala regulates the behavioural output by affecting the connection of the ancient forebrain (amygdala, hippocampus) with the monoaminergic centres of the midbrain through the

behaviour, respectively.

110 Schizophrenia Treatment - The New Facets

An important reason for us to become interested in the embryology, connectivity and neuroanatomy of primitive vertebrates is the scientific notion that their primitive brains may reflect earlier evolutionary stages of the current human brain. Hence, the brains of lampreys, sharks, lungfishes, frogs, turtles, opossums, rats and monkeys correspond to the brains of human ancestors from about 560 million years ago until now [9]. The very first vertebrate is supposed to be an animal comparable with modern lamprey [2]. This animal has a head containing a brain and it has vertebrates, but not yet a lower jaw. The lamprey forebrain consists of olfactory bulbs, medial and lateral pallium, subpallium and diencephalon (extensive thalamus). The lateral pallium already forms a primitive hemisphere (**Figure 1**). However, some controversy exists how to divide it into different fields. The question emerges whether the lamprey has a dorsal pallium, a structure which gives rise to the majority of the neocortex in mammals. We have concluded that our earliest vertebrate ancestor (comparable with lamprey) must already have had a dorsal pallium, but it functions as an extension of the medial pallium. Lamprey medial pallium is considered to give rise to the hippocampal complex in tetrapods [10]. The ventral pallium which appears to be present in all vertebrates, but also in hagfishes, is related to olfactory structures. It is included as part of the amygdala in tetrapods [10].

**Figure 1.** Central nervous system of lamprey.

#### **2.1. Evolution of the cerebral cortex**

Studying the development of the human cerebral cortex during subsequent steps of evolution brings a few typical problems. Firstly, in reptiles and birds, the neocortex developed in another direction than in mammals [10]. Secondly, a large part of the cerebral cortex in mammals is laminated (organised in three or six different layers), while the pallium in nonmammals is primary organised in a non-laminar fashion. However, by integrating comparative neuroanatomy with comparative embryology and developmental genetics, a clear picture can be created on the evolutionary development of human cerebral cortex [10–12].

In human embryos the future insula is the first 'neo'cortical structure to develop [13, 14]. The primordial insula is initially located on the free lateral surface of the cerebral hemisphere and adjoining the cortical amygdaloid regions on one side and olfactory cortical regions on the other [13, 15]. This is highly comparable to the position of the dorsal pallium in lamprey hemispheres [16]. According to the von Baerian theory, embryos of later-descendent species resemble the embryos of earlier-descendant species to the point of their divergence. So, it may be concluded that the human insula is the most ancient part of the neocortex. In addition, the lamprey has a small but well-developed dorsal thalamus (the part forming the proper so-called thalamus in humans), which connects the tectum (e.g. somatosensory and viscerosensory information) and optic tract (visual information) with caudal parts of the pallium (mainly hippocampal primordium and subhippocampal lobe, i.e. medial pallium) [17].

In amphibians, the dorsal pallium has significantly expanded in comparison to lamprey dorsal pallium, and it covers almost the entire roof of the hemisphere in these animals [18]. Fibres of neurons within these dorsal pallial fields run ipsilaterally to other pallial regions (medial, lateral, ventral), to the septum and to the amphibian striatopallidum [18]. However, fibres running from the thalamus to the primitive cerebral cortex are certainly not restricted to the newly present dorsal fields [19–21]. Anterior parts of the anuran thalamus are projecting widely within the forebrain, while, e.g. visual information is also projected to the hypothalamus and brainstem [19]. From electrical recording and anatomical labelling experiments, it can be concluded that the anuran dorsal pallium does not yet has achieved its human input processing and output generating role [19–21] but is still part of a more extensive 'limbic' behavioural control system including almost all pallial and subpallial regions.

In more recent jawed vertebrates, the input to the dorsal thalamus largely increases and this leads to a significant expansion of the dorsal pallium [22, 23]. However, this expansion occurs along different lines in non-synapsid (reptiles, birds) and synapsid (mammals) animals [12, 23]. Both groups derive from a common sauropsid ancestor with a turtle-like brain. The dorsal thalamus consists of two divisions called lemnothalamus and collothalamus, dependent upon their brainstem input structures. Within the mammalian line, both thalamic divisions with their corresponding cortical fields developed [23]. These cortical fields comprise the subicular, cingulate, prefrontal, sensorimotor and related cortices of mammals. The described expansion resulted probably in a total displacement of ventral and medial pallial fields. The medial pallium became hippocampus and the ventral pallium became the most caudal edge of the frontal lobe (including olfactory tubercle) and cortical regions of the amygdaloid complex in the temporal lobe.

Secondary to the described synapsid development of the dorsal pallium, lamination of the cerebral cortex occurred [10, 24]. This lamination is absent in non-mammals and not restricted to new, originally dorsal, pallial fields [10]. Due to this lamination, the human neocortex consists of horizontal layers, intersected by vertical (or radial) columns that are stereotypically interconnected in the vertical dimension and comprise processing units [24, 25]. The expansion and elaboration of the cerebral neocortex during evolutionary development from primitive mammals to humans resulted in formation of new areas with new connections creating numerous extensive networks regulating different new or more sophisticated types of behaviour [24]. However, the majority of this progress is of relatively recent date.

#### **2.2. Evolution of subcortical structures**

**2.1. Evolution of the cerebral cortex**

112 Schizophrenia Treatment - The New Facets

plex in the temporal lobe.

Studying the development of the human cerebral cortex during subsequent steps of evolution brings a few typical problems. Firstly, in reptiles and birds, the neocortex developed in another direction than in mammals [10]. Secondly, a large part of the cerebral cortex in mammals is laminated (organised in three or six different layers), while the pallium in nonmammals is primary organised in a non-laminar fashion. However, by integrating comparative neuroanatomy with comparative embryology and developmental genetics, a clear picture

In human embryos the future insula is the first 'neo'cortical structure to develop [13, 14]. The primordial insula is initially located on the free lateral surface of the cerebral hemisphere and adjoining the cortical amygdaloid regions on one side and olfactory cortical regions on the other [13, 15]. This is highly comparable to the position of the dorsal pallium in lamprey hemispheres [16]. According to the von Baerian theory, embryos of later-descendent species resemble the embryos of earlier-descendant species to the point of their divergence. So, it may be concluded that the human insula is the most ancient part of the neocortex. In addition, the lamprey has a small but well-developed dorsal thalamus (the part forming the proper so-called thalamus in humans), which connects the tectum (e.g. somatosensory and viscerosensory information) and optic tract (visual information) with caudal parts of the pallium (mainly hippocampal primordium and subhippocampal lobe, i.e. medial pallium) [17].

In amphibians, the dorsal pallium has significantly expanded in comparison to lamprey dorsal pallium, and it covers almost the entire roof of the hemisphere in these animals [18]. Fibres of neurons within these dorsal pallial fields run ipsilaterally to other pallial regions (medial, lateral, ventral), to the septum and to the amphibian striatopallidum [18]. However, fibres running from the thalamus to the primitive cerebral cortex are certainly not restricted to the newly present dorsal fields [19–21]. Anterior parts of the anuran thalamus are projecting widely within the forebrain, while, e.g. visual information is also projected to the hypothalamus and brainstem [19]. From electrical recording and anatomical labelling experiments, it can be concluded that the anuran dorsal pallium does not yet has achieved its human input processing and output generating role [19–21] but is still part of a more extensive 'limbic'

In more recent jawed vertebrates, the input to the dorsal thalamus largely increases and this leads to a significant expansion of the dorsal pallium [22, 23]. However, this expansion occurs along different lines in non-synapsid (reptiles, birds) and synapsid (mammals) animals [12, 23]. Both groups derive from a common sauropsid ancestor with a turtle-like brain. The dorsal thalamus consists of two divisions called lemnothalamus and collothalamus, dependent upon their brainstem input structures. Within the mammalian line, both thalamic divisions with their corresponding cortical fields developed [23]. These cortical fields comprise the subicular, cingulate, prefrontal, sensorimotor and related cortices of mammals. The described expansion resulted probably in a total displacement of ventral and medial pallial fields. The medial pallium became hippocampus and the ventral pallium became the most caudal edge of the frontal lobe (including olfactory tubercle) and cortical regions of the amygdaloid com-

behavioural control system including almost all pallial and subpallial regions.

can be created on the evolutionary development of human cerebral cortex [10–12].

The lamprey telencephalon can be divided into a dorsal, pallial region and a ventral, subpallial region. This subpallial part largely consists from the striatum, septum and preoptic area [17]. Sten Grillner and collaborators have demonstrated that the complete basal ganglia circuitry is already present in these phylogenetically oldest vertebrates [26, 27]. Moreover, these animals possess a subpallial structure (**Figure 2**), the habenula-projecting globus pallidus (GPh), which has an essential role in selecting behaviours that are either rewarding and should be continued or are not rewarding and should be abandoned [28]. We have hypothesised that lamprey striatal subpallium is included in nuclear amygdala of mammals [2]. However, some controversy exists concerning the fate of the GPh in more recent vertebrates. Lamprey also

**Figure 2.** Position of the striatum and habenula-projecting globus pallidus of lamprey.

have an epithalamus which is very similar to its homologue in more modern animals [2]. This includes the output structures of the habenula via fasciculus retroflexus to midbrain nuclei.

It was formerly believed that the forebrain (especially its basal ganglia) underwent important changes during the evolution from anamniotes (lampreys, fishes, amphibians) to amniotes (reptiles, birds and mammals). However, the organisation of the basal ganglia is more conserved than previously thought [29]. The two main components of the basal ganglia develop embryologically from two different areas: the lateral ganglionic eminence (giving rise to the striatum) and the medial ganglionic eminence (giving rise to the pallidum). During embryological development different genes are brought to expression in order to regulate the regionalisation of these two areas. In particular, the transcription factor Nkx2.1 is expressed in the medial ganglionic eminence (and not the lateral one), and the expression of this gene in combination with that of others has been used to characterise pallidal basal ganglia in embryos of amniotes and anamniotes [29]. These authors were able to identify striatal and pallidal regions in cartilaginous fishes, ray-finned fishes, lungfishes and amphibians. However, in the subpallium of lampreys, a pallidal region could not be identified because these animals lack an Nkx2.1-expressing zone. Therefore, it was suggested that the pallidum is still absent in agnathans and first appeared during or after the transition from jawless to jawed vertebrates [30]. However, since Stephenson-Jones and colleagues [26, 27] have demonstrated the existence of a complete extrapyramidal circuitry in the subpallium of lamprey, this proposal of Moreno and co-workers [30] has to be rejected. In lampreys pallidal structures do exist in spite of the absence of expression of Nkx2.1 during the embryonic development of this structure.

The nuclear part of the amygdaloid complex is another derivative of the lateral ganglionic eminence, and the development of the amphibian amygdaloid complex has been studied in detail by Moreno and González [4, 31–33]. Within the anuran forebrain, the striatum (anterior) is continuous with the central and medial amygdala (posterior) and clearly separated from pallidum, bed nucleus of the stria terminalis and septum [4]. In humans, the bed nucleus of the stria terminalis is continuous with the connecting extended amygdala on one side and with the shell part of the nucleus accumbens (NAcbS) on the other [2, 25]. As a matter of fact, the original concept described the centromedial amygdala and bed nucleus of the stria terminalis both as part of the extended amygdala [34]. The investigations made clear that the organisation of the ancestral tetrapod (amphibian-like) amygdaloid complex is retained within more recent ancestors [35]. Evolution of the anamnio-amniotic (mammalian) striatum probably occurred in a modular sense when a more lateral part of the striatum was added every time when a cortical part with a new function was added to the expanding the neocortex [1, 36]. The amygdaloid complex derives from pallial and subpallial territories. Pallial (corticoid) structures include the cortical amygdala (olfactory and vomeronasal) and the basolateral complex deep to it [37]. These pallial components originate from lateral and ventral pallial regions and are also maintained during evolution of amniotic vertebrates [35, 37].

An important discovery during studying the embryological development of anuran basal ganglia was the finding that the bed nucleus of the stria terminalis (BST) and part of the septum are also of pallidal instead of striatal origin [29, 38]. This is interesting because the BST is a suitable structure to execute the functions of the limbic component of lamprey habenula-projecting globus pallidus. The architecture and connectivity of the rat BST has been studied in detail by Larry Swanson and collaborators. It becomes evident that the BST is an extremely complex set of nuclei, which can be separated into dorsal, lateral and ventral areas [39]. These nuclei receive input from the central amygdaloid nucleus (innervating various parts of the anterior BST division) and medial amygdaloid nucleus (preferentially innervating the posterior BST division), but not from the superficial and deep corticoid nuclei of the amygdala [40]. It is concluded that BST is a rostral differentiation of the pallidum receiving massive GABAergic input from centromedial amygdala and giving again GABAergic output to brainstem motor systems and thalamocortical re-entrant loops [40]. Viewed broadly, BST posterior division cell groups share massive bidirectional connections with the medial amygdaloid nucleus and other amygdaloid components of the accessory olfactory system, and they send massive projections to hypothalamic control centres regulating reproduction and defence [41]. The BST anterolateral group projects to the ventral autonomic control network, to midbrain structures modulating the expression of orofacial and locomotor somatosensory responses and to the ventral striatopallidal system. This suggests that the anterolateral group is primary involved in appetitive feeding (eating and drinking) behaviour [41]. The lateral habenula hardly receives any fibres from these BST areas. However, the anteromedial BST division projects to the lateral habenula [41, 42]. In our opinion, it is very well possible that the anteromedial division of the rat BST contains glutamatergic neurons which are running to the lateral habenula and have similar function as lamprey GPh neurons. Moreover, the anterior BST division receives input from hippocampus (ventral subiculum) and infralimbic cortex (comparable with the human subgenual anterior cingulate cortex, Brodmann area 25 (BA25)). This connectivity probably corresponds to the cortical input to the habenula-projecting globus pallidus.

#### **2.3. Conclusion: evolution of the amygdaloid complex**

have an epithalamus which is very similar to its homologue in more modern animals [2]. This includes the output structures of the habenula via fasciculus retroflexus to midbrain nuclei.

114 Schizophrenia Treatment - The New Facets

It was formerly believed that the forebrain (especially its basal ganglia) underwent important changes during the evolution from anamniotes (lampreys, fishes, amphibians) to amniotes (reptiles, birds and mammals). However, the organisation of the basal ganglia is more conserved than previously thought [29]. The two main components of the basal ganglia develop embryologically from two different areas: the lateral ganglionic eminence (giving rise to the striatum) and the medial ganglionic eminence (giving rise to the pallidum). During embryological development different genes are brought to expression in order to regulate the regionalisation of these two areas. In particular, the transcription factor Nkx2.1 is expressed in the medial ganglionic eminence (and not the lateral one), and the expression of this gene in combination with that of others has been used to characterise pallidal basal ganglia in embryos of amniotes and anamniotes [29]. These authors were able to identify striatal and pallidal regions in cartilaginous fishes, ray-finned fishes, lungfishes and amphibians. However, in the subpallium of lampreys, a pallidal region could not be identified because these animals lack an Nkx2.1-expressing zone. Therefore, it was suggested that the pallidum is still absent in agnathans and first appeared during or after the transition from jawless to jawed vertebrates [30]. However, since Stephenson-Jones and colleagues [26, 27] have demonstrated the existence of a complete extrapyramidal circuitry in the subpallium of lamprey, this proposal of Moreno and co-workers [30] has to be rejected. In lampreys pallidal structures do exist in spite of the absence of expression of Nkx2.1 during the embryonic development of this structure.

The nuclear part of the amygdaloid complex is another derivative of the lateral ganglionic eminence, and the development of the amphibian amygdaloid complex has been studied in detail by Moreno and González [4, 31–33]. Within the anuran forebrain, the striatum (anterior) is continuous with the central and medial amygdala (posterior) and clearly separated from pallidum, bed nucleus of the stria terminalis and septum [4]. In humans, the bed nucleus of the stria terminalis is continuous with the connecting extended amygdala on one side and with the shell part of the nucleus accumbens (NAcbS) on the other [2, 25]. As a matter of fact, the original concept described the centromedial amygdala and bed nucleus of the stria terminalis both as part of the extended amygdala [34]. The investigations made clear that the organisation of the ancestral tetrapod (amphibian-like) amygdaloid complex is retained within more recent ancestors [35]. Evolution of the anamnio-amniotic (mammalian) striatum probably occurred in a modular sense when a more lateral part of the striatum was added every time when a cortical part with a new function was added to the expanding the neocortex [1, 36]. The amygdaloid complex derives from pallial and subpallial territories. Pallial (corticoid) structures include the cortical amygdala (olfactory and vomeronasal) and the basolateral complex deep to it [37]. These pallial components originate from lateral and ventral pallial regions and are also maintained during evolution of amniotic vertebrates [35, 37].

An important discovery during studying the embryological development of anuran basal ganglia was the finding that the bed nucleus of the stria terminalis (BST) and part of the septum are also of pallidal instead of striatal origin [29, 38]. This is interesting because the BST is a suitable structure to execute the functions of the limbic component of lamprey The endbrain (telencephalon) of the very first vertebrates can be considered to be the evolutionary starting point of the human amygdaloid complex. Its pallium largely consisted of ventral, lateral and medial fields. Its dorsal pallium was not contributing to a very significant extent. Its subpallium contained a striatopallidal complex for motor control and a habenula-projecting globus pallidus for decision-making. During evolution to an amphibian-like ancestor, the dorsal pallium developed to a significant extent, but it can still be considered an extension of the medial pallium. This can be concluded from its connectivity with other pallial and subpallial structures as well as from its input received from the dorsal thalamus. The medial pallium later developed into the hippocampus. At a subpallidal level, the primitive striatopallidal complex becomes nuclear amygdala and bed nucleus of the stria terminalis, respectively. This limbic striatopallidal structure will later become the human extended amygdala of Heimer [34]. Next to the amygdaloid complex, a new ventral and dorsal striatopallidal complex arises in amphibians which will form the extrapyramidal system in our mammalian ancestors. In our opinion it actually took until the evolution of our mammalian ancestors before the dorsal pallium was actually transformed into the current neocortex. The massive growth of this neocortex resulted in a C-shaped and outside-inward curving of the cerebral hemispheres. The medial pallium became hippocampus and the ventral pallium superficial and deep corticoid amygdala. This means that almost the entire cerebral hemisphere is of quite recent origin. This is probably also true for the limbic cortical-subcorti-

**Figure 3.** Position of the limbic basal ganglia (extended amygdala and nucleus accumbens shell) relative to the extrapyramidal striatum (caudate nucleus, putamen, nucleus accumbens core) and hippocampus.

cal-cortical connectivity we have previously suggested [2, 6]. Corticoid amygdaloid output reaches the hypothalamus and brainstem (to minor extent directly and) largely along nuclear amygdala (striatal amygdala) and bed nucleus of the stria terminalis (pallidal amygdala). This directly results from the regulation of vegetative and motor behaviour by the striatum instead of the pallium in lamprey [2]. However, the human frontal neocortex is reached through connectivity with the dorsal thalamus. This last connectivity must have developed later during the evolution of the mammalian forebrain. The amygdaloid equivalent of the habenula-projecting globus pallidus is probably localised within the bed nucleus of the stria terminalis.

The final picture of the position of the human limbic and extrapyramidal basal ganglia is given in **Figure 3**.
