**5. Neural structures involved in ethanol-seeking behavior**

In the following sections, several neural structures and circuits involved in ethanol-seeking behavior are discussed. Building off of earlier sections, the studies that are described below involved several different animal models and tools to identify the underlying neurobiology of ethanol seeking. Specifically, this chapter will focus on the neural structures involved in ethanol self-administration and CPP, as these models specifically assess ethanol-seeking behavior. A proposed circuit of the key neural structures implicated in cue-induced ethanol seeking as indexed by self-administration and CPP procedures is included in **Figure 2**.

#### **5.1. Findings from ethanol self-administration studies**

Studies using ethanol self-administration procedures to evaluate the neural mechanisms underlying ethanol seeking have by and large employed intracranial microinfusions. Most of the neural regions that have been evaluated in self-administration studies are part of the mesocorticolimbic system [70] and are typically situated downstream of the VTA. As with many drugs of abuse, acutely administered ethanol excites DA neurons within the VTA through direct and indirect mechanisms [71–74]. Furthermore, the VTA is robustly activated by ethanol-associated cue exposure [75]. Downstream, the nucleus accumbens (NAc) and amygdala receive dopaminergic input from the VTA [76] and considerable evidence suggests that this dopaminergic input to each region underlies associative learning and motivated behavior [77–81]. As such, NAc and amygdala are two of the most well-characterized struc‐ tures in terms of their involvement in ethanol seeking and several of these studies are described below.

The NAc has been routinely implicated in the drug-seeking behavior [82, 83]. Many studies have found a differential involvement of core and shell subdivisions in drug seeking depend‐ ing on the drug of abuse, phase of self-administration, and nature of the procedure and/or stimuli used [84–86]. As with other drugs of abuse, the NAc core and shell have been shown to be differentially involved in cue-induced ethanol seeking. For example, ethanol selfadministration studies have shown that transient inactivation of the NAc core but not shell

is possible for axon collaterals from source region cells to express DREADDs, which given the systemic nature of DREADD activation may lead to activity in sites outside the circuit. Hence,

Overall, several techniques involving classical and contemporary tools have been used to manipulate defined neural circuits. On one hand, classical tools provide indirect manipulation and generally require the use of multiple controls to carefully and appropriately interpret results derived using these strategies. Conversely, contemporary tools involving viralmediated gene transfer confer greater circuit selectivity and have been successfully used to

In the following sections, several neural structures and circuits involved in ethanol-seeking behavior are discussed. Building off of earlier sections, the studies that are described below involved several different animal models and tools to identify the underlying neurobiology of ethanol seeking. Specifically, this chapter will focus on the neural structures involved in ethanol self-administration and CPP, as these models specifically assess ethanol-seeking behavior. A proposed circuit of the key neural structures implicated in cue-induced ethanol seeking as indexed by self-administration and CPP procedures is included in **Figure 2**.

Studies using ethanol self-administration procedures to evaluate the neural mechanisms underlying ethanol seeking have by and large employed intracranial microinfusions. Most of the neural regions that have been evaluated in self-administration studies are part of the mesocorticolimbic system [70] and are typically situated downstream of the VTA. As with many drugs of abuse, acutely administered ethanol excites DA neurons within the VTA through direct and indirect mechanisms [71–74]. Furthermore, the VTA is robustly activated by ethanol-associated cue exposure [75]. Downstream, the nucleus accumbens (NAc) and amygdala receive dopaminergic input from the VTA [76] and considerable evidence suggests that this dopaminergic input to each region underlies associative learning and motivated behavior [77–81]. As such, NAc and amygdala are two of the most well-characterized struc‐ tures in terms of their involvement in ethanol seeking and several of these studies are described

The NAc has been routinely implicated in the drug-seeking behavior [82, 83]. Many studies have found a differential involvement of core and shell subdivisions in drug seeking depend‐ ing on the drug of abuse, phase of self-administration, and nature of the procedure and/or stimuli used [84–86]. As with other drugs of abuse, the NAc core and shell have been shown to be differentially involved in cue-induced ethanol seeking. For example, ethanol selfadministration studies have shown that transient inactivation of the NAc core but not shell

studies using this method should be careful to address this potential caveat.

**5. Neural structures involved in ethanol-seeking behavior**

manipulate serial projections form source to target region.

60 Recent Advances in Drug Addiction Research and Clinical Applications

**5.1. Findings from ethanol self-administration studies**

**4.3. Summary**

below.

**Figure 2.** Diagram of the neural circuitry involved in ethanol CPP. Previous studies have demonstrated the involve‐ ment of cortex (ACC), amygdala (BLA/CeA), striatum (NAc), and midbrain (VTA) in ethanol CPP. The present work now demonstrates the involvement of the dorsal and ventral BNST (dBNST and vBNST, respectively) and a direct BNST-VTA circuit. Each of these regions receives dopaminergic input from the VTA (indicated by blue arrows) and is part of a broader mesocorticolimbic dopamine system that underlies reward and motivation. Some of the neurochemi‐ cal signals underlying the involvement of each region in ethanol CPP have also been identified. +, excitatory; -, inhibi‐ tory; blue circles, dopamine cells; green rectangles, glutamate cells; red squares, GABA cells; black boxes, unknown neurochemical signal; green arrows, glutamate projections; μ, μ-opioid receptors; B, GABAB receptor; D1, dopamine D1-like receptor; D2, dopamine D2-like receptor; iGluR, ionotropic glutamate receptors, NMDA, iGluR subtype.

reduces cue-induced reinstatement [87] and context-induced renewal [88] of ethanol seeking. Other studies, however, have demonstrated that NAc shell modulates the expression of cueinduced ethanol seeking [89] and that blockade of DA D1 receptors in the NAc shell but not core reduces the spontaneous recovery of ethanol-seeking behavior following prolonged abstinence [90]. In addition, DA D1 receptor antagonism in NAc core and shell has been reported to block context-induced renewal of extinguished ethanol seeking [91]. These studies vary in their findings of core versus shell involvement in ethanol seeking, likely indicating that the role of NAc subdivisions varies by different phases of self-administration (e.g., acquisition, expression, and reinstatement) and by the use of cues or context. However, this literature overall supports a role for the NAc in general ethanol-seeking behavior and suggests that DA input may underlie its involvement.

The amygdala has also been strongly implicated in drug-seeking behavior, specifically the basolateral amygdala (BLA) and central nucleus of the amygdala (CeA) subdivisions as well as the bed nucleus of the stria terminalis (BNST) of the extended amygdala [92–94]. In ethanol self-administration studies, the inactivation of the BLA has been shown to reduce contextinduced renewal of previously extinguished ethanol seeking [95]. The involvement of the BLA in ethanol seeking may involve a glutamatergic mechanism, as intra-BLA ionotropic glutamate receptor antagonism reduced the expression of ethanol seeking elicited by a discrete cue and invigorated by an ethanol-associated context [96]. Notably, the excitatory transmission from the amygdala to the NAc is believed to underlie reward seeking and suggests that an amyg‐ dala-NAc circuit may be involved in the expression of reward-seeking behavior. For example, the optical stimulation of BLA glutamate to NAc has been shown to be reinforcing, as mice worked to earn additional stimulation of BLA-NAc synaptic inputs [97]. Conversely, the inhibition of BLA-NAc inputs reduced cue-induced responding for sucrose [97]. In addition, pharmacological disconnection has revealed that an amygdala-NAc interaction underlies a stimulus-controlled expression or maintenance of cocaine seeking [59].

The CeA and BNST have generally been implicated in stress-induced ethanol-seeking rein‐ statement but may also play a role in cue-induced ethanol reinstatement. For instance, intra-CeA but not intra-BLA infusion of mifepristone, a glucocorticoid receptor antagonist, has been reported to suppress reinstated ethanol seeking induced by the pharmacological stressor yohimbine [98]. Activation of group II metabotropic glutamate receptors blocks stress- and cue-induced reinstatement of ethanol seeking presumably through CeA and BNST actions [99].

Lastly, additional regions that have been implicated in ethanol seeking using self-administra‐ tion procedures include the dorsomedial striatum [100, 101], medial prefrontal cortex (mPFC) [75], prelimbic cortex, and VTA [102]. Importantly, the VTA, which is the chief source of DA input to NAc, amygdala, and cortical regions, has been shown to be explicitly involved in context- and cue-induced ethanol seeking [103–105]. Overall, self-administration studies have been important in identifying several key neural substrates involved in ethanol-seeking behavior. Several of these structures have also been implicated in cue-induced ethanol seeking through ethanol-induced CPP procedures and thus are discussed in the next section.

#### **5.2. Findings from ethanol CPP studies**

Studies on the neural mechanism of ethanol CPP can be grouped into several categories that include acquisition, expression, extinction, and reinstatement. Acquisition studies are those that assess the development of ethanol place preference. These typically include procedures where manipulations occurred during the conditioning or training phase, where animals learn to associate contextual cues with ethanol reward. Conversely, expression studies involve manipulations that occur after the conditioning phase and before preference testing. Expres‐ sion studies, in particular, are useful in assessing ethanol-seeking behavior and conditioned reward. Below, findings from each of these types of ethanol CPP studies are discussed. Of note, relatively few laboratories study the primary and conditioned rewarding properties of ethanol using a CPP procedure. This is partly due to the difficult and unreliable nature of ethanol place conditioning in rats [21, 106] and the relatively weak ethanol CPP obtained with commonly used inbred mouse strains, such as C57BL/6J [107, 108]. In view of this, many of the studies described below have used male DBA/2J mice. This inbred strain will rapidly and reliably develop and ethanol CPP, even with a minimal amount of conditioning sessions [34, 36, 108]. In this section, we provide an overview of studies that have directly examined the neural areas underlying the acquisition and expression of ethanol place preference.

#### *5.2.1. Acquisition*

self-administration studies, the inactivation of the BLA has been shown to reduce contextinduced renewal of previously extinguished ethanol seeking [95]. The involvement of the BLA in ethanol seeking may involve a glutamatergic mechanism, as intra-BLA ionotropic glutamate receptor antagonism reduced the expression of ethanol seeking elicited by a discrete cue and invigorated by an ethanol-associated context [96]. Notably, the excitatory transmission from the amygdala to the NAc is believed to underlie reward seeking and suggests that an amyg‐ dala-NAc circuit may be involved in the expression of reward-seeking behavior. For example, the optical stimulation of BLA glutamate to NAc has been shown to be reinforcing, as mice worked to earn additional stimulation of BLA-NAc synaptic inputs [97]. Conversely, the inhibition of BLA-NAc inputs reduced cue-induced responding for sucrose [97]. In addition, pharmacological disconnection has revealed that an amygdala-NAc interaction underlies a

The CeA and BNST have generally been implicated in stress-induced ethanol-seeking rein‐ statement but may also play a role in cue-induced ethanol reinstatement. For instance, intra-CeA but not intra-BLA infusion of mifepristone, a glucocorticoid receptor antagonist, has been reported to suppress reinstated ethanol seeking induced by the pharmacological stressor yohimbine [98]. Activation of group II metabotropic glutamate receptors blocks stress- and cue-induced reinstatement of ethanol seeking presumably through CeA and BNST actions [99].

Lastly, additional regions that have been implicated in ethanol seeking using self-administra‐ tion procedures include the dorsomedial striatum [100, 101], medial prefrontal cortex (mPFC) [75], prelimbic cortex, and VTA [102]. Importantly, the VTA, which is the chief source of DA input to NAc, amygdala, and cortical regions, has been shown to be explicitly involved in context- and cue-induced ethanol seeking [103–105]. Overall, self-administration studies have been important in identifying several key neural substrates involved in ethanol-seeking behavior. Several of these structures have also been implicated in cue-induced ethanol seeking

through ethanol-induced CPP procedures and thus are discussed in the next section.

Studies on the neural mechanism of ethanol CPP can be grouped into several categories that include acquisition, expression, extinction, and reinstatement. Acquisition studies are those that assess the development of ethanol place preference. These typically include procedures where manipulations occurred during the conditioning or training phase, where animals learn to associate contextual cues with ethanol reward. Conversely, expression studies involve manipulations that occur after the conditioning phase and before preference testing. Expres‐ sion studies, in particular, are useful in assessing ethanol-seeking behavior and conditioned reward. Below, findings from each of these types of ethanol CPP studies are discussed. Of note, relatively few laboratories study the primary and conditioned rewarding properties of ethanol using a CPP procedure. This is partly due to the difficult and unreliable nature of ethanol place conditioning in rats [21, 106] and the relatively weak ethanol CPP obtained with commonly used inbred mouse strains, such as C57BL/6J [107, 108]. In view of this, many of the studies described below have used male DBA/2J mice. This inbred strain will rapidly and reliably develop and ethanol CPP, even with a minimal amount of conditioning sessions [34, 36, 108].

**5.2. Findings from ethanol CPP studies**

stimulus-controlled expression or maintenance of cocaine seeking [59].

62 Recent Advances in Drug Addiction Research and Clinical Applications

As with self-administration studies, the NAc and amygdala are the two most evaluated regions in terms of their involvement in ethanol CPP acquisition. Previously, the involvement of the NAc in ethanol place preference acquisition has been investigated. In one study, bilateral electrolytic lesions of the NAc before CPP training disrupted the acquisition of CPP [109]. In a later study, bilateral NAc infusions of the D1-like receptor antagonist SCH-23390 disrupted the development of ethanol CPP [110]. This finding is similar to a finding reported in rats showing that nonselective DA antagonism prevented CPP induced by intracerebroventricular (icv) infusions of ethanol [111]. Combined, these studies indicate that the NAc is necessary for establishing associative relationships between ethanol reward and environmental cues likely through a DA D1-like receptor-dependent mechanism. Other work has looked at the role of the amygdala in ethanol CPP acquisition [109]. Using electrolytic lesions, the amygdala was ablated bilaterally before CPP training. Amygdala lesions disrupted acquisition (and/or expression) of ethanol place preference, suggesting that this region is also involved in ethanol cue learning.

Together, these findings demonstrate that these structures downstream the VTA are necessary for the development of ethanol CPP. Moreover, NAc involvement in ethanol CPP is directly attributed to dopaminergic innervation, as activity at D1-like receptors in this region is necessary for acquisition. One consideration of these results is that acquisition studies involving microinjections can be problematic, as the additional handling required to focally administer a drug can interfere with ethanol CPP [110]. Although they do not require added handling prior, lesions are also problematic when administered preconditioning, as it unclear whether they affected the acquisition or expression phase.

#### *5.2.2. Expression*

Over the last decade, studies have investigated the involvement of several brain areas in ethanol place preference expression. These include the VTA, NAc, amygdala, and anterior cingulate cortex (ACC). Each of these structures is thought to be involved in reward and motivation partly through DA mechanisms, and as such, each is part of a broader mesocorti‐ colimbic DA system.

Although VTA DA cells are initially activated by rewarding stimuli, this activation diminishes over time with repeated reward exposures and subsequent learning [112]. Eventually, DA cells are no longer activated by the reward itself and instead become robustly activated by envi‐ ronmental stimuli that have become associated with the reward and predict its delivery [113, 114]. Thus, the involvement of this conditioned DA response in ethanol CPP expression can be supported by studies focusing on the VTA or downstream sites as are described below.

One pharmacological microinjection study separately infused the nonselective opioid receptor antagonist methylnaloxonium and the GABAB receptor agonist baclofen into the VTA to assess the participation of the VTA in ethanol CPP expression. Methylnaloxonium decreased the magnitude of ethanol CPP, whereas baclofen blocked preference expression entirely. These findings suggested that the VTA activity is necessary for the expression of ethanol place preference. Moreover, GABAergic and opioidergic activity appear to underlie the involvement of the VTA in ethanol CPP expression, presumably through the local modulation of DA cell activity.

Although involvement of the NAc in ethanol place preference acquisition is clear, its involve‐ ment in expression is more complicated. In early work, an intra-NAc infusion of methylna‐ loxonium failed to impact ethanol preference expression, suggesting a lack of NAc opioid receptor involvement in the conditioned ethanol reward [115]. A later study directed bilateral electrolytic lesions at the NAc after ethanol CPP conditioning and before testing to isolate the involvement of this structure in expression [109]. Overall, lesions made at this time point did not affect ethanol place preference, suggesting that the NAc may not be critical in ethanol CPP expression.

However, additional pharmacological procedures have shown a more specific role for the NAc in CPP expression. In one study, intra-NAc antagonism of D1- and D2-like receptors prevented the expression of CPP induced by icv ethanol [111]. In another study, NAc DA (D1- and D2 like) and glutamate (NMDA) receptors were blocked during the CPP expression test using either flupenthixol or AP-5, respectively [116]. Although DA receptor antagonism did not affect ethanol place preference, NMDA receptor antagonism reduced it, suggesting that the involvement of the NAc in expression is specific to activity at NMDA receptors. This effect was reproduced in another study using only unilaterally administered AP5, further indicating the importance of NAc NMDA receptor involvement in ethanol place preference expression [60]. Notably, this study was designed to examine glutamate input to NAc from amygdala. Although findings appeared to demonstrate that amygdala disconnection from NAc blocked ethanol CPP expression, reduced CPP in mice unilaterally infused with AP5 in NAc prevented this interpretation. Overall, these findings have established a role for NAc NMDA, but not DA receptors, in ethanol place preference expression and suggest that glutamatergic input from amygdala may also be involved. Although DA input from VTA to NAc is a hypothesized mechanism underlying drug seeking, these results suggest that it does not underlie ethanol seeking, at least as indexed by CPP. Finally, these studies serve to demonstrate that manipu‐ lations more selective than global inactivation or deletion of a structure may be necessary to appropriately gauge that the importance of structure in behavior.

Accordingly, the role of the amygdala in ethanol CPP expression has also been addressed. Like lesions made before conditioning, the bilateral electrolytic lesion of the amygdala made before the test phase blocked ethanol place preference expression [109]. In addition, when bilaterally infused into the amygdala, the D1- and D2-like DA receptor agonist flupenthixol blocked ethanol CPP expression [116]. Together, these results illustrate the role of the amygdala in ethanol-seeking behavior and indicate the importance of dopaminergic input to this structure for ethanol CPP expression.

The BNST also has a role in modulating the expression of ethanol reward. Exposure to ethanol causes changes in glutamate synaptic plasticity [117], increases extracellular DA levels in the BNST [118], and activates BNST cells [119]. In particular, the inhibition of the BNST during CPP expression using electrolytic lesions, coinfusion of GABAA and GABAB receptor agonists muscimol and baclofen, and activation of inhibitory DREADDs (hM4Di-DREADD) lead to a loss or reduction of the expression of ethanol CPP. All of these studies suggest the importance of the BNST to ethanol seeking.

Finally, ACC involvement in expression was assessed by bilaterally infusing the nonspecific opioid receptor antagonist methylnaloxonium into the ACC before the ethanol CPP test [120]. The intra-ACC infusion of methylnaloxonium disrupted ethanol place preference expression, reducing its magnitude at the lowest dose and abolishing it at the highest dose. Hence, the ACC appears to modulate ethanol CPP expression through an opioidergic mechanism.

In summary, findings from expression studies have demonstrated that the VTA, NAc, amygdala, BNST, and ACC are all structures involved in ethanol place preference expression. Infusion of a mixed opioid receptor antagonist into VTA and ACC but not NAc interfered with ethanol CPP expression. Similarly, lesions and mixed DA receptor antagonism disrupted expression when targeted to the amygdala and not NAc. Involvement of the NAc in ethanol CPP expression appeared to be confined to activity NMDA receptors only, suggesting that a more explicit neurochemical mechanism underlies its involvement in ethanol place preference expression.

#### *5.2.3. Extinction and reinstatement*

the participation of the VTA in ethanol CPP expression. Methylnaloxonium decreased the magnitude of ethanol CPP, whereas baclofen blocked preference expression entirely. These findings suggested that the VTA activity is necessary for the expression of ethanol place preference. Moreover, GABAergic and opioidergic activity appear to underlie the involvement of the VTA in ethanol CPP expression, presumably through the local modulation of DA cell

64 Recent Advances in Drug Addiction Research and Clinical Applications

Although involvement of the NAc in ethanol place preference acquisition is clear, its involve‐ ment in expression is more complicated. In early work, an intra-NAc infusion of methylna‐ loxonium failed to impact ethanol preference expression, suggesting a lack of NAc opioid receptor involvement in the conditioned ethanol reward [115]. A later study directed bilateral electrolytic lesions at the NAc after ethanol CPP conditioning and before testing to isolate the involvement of this structure in expression [109]. Overall, lesions made at this time point did not affect ethanol place preference, suggesting that the NAc may not be critical in ethanol CPP

However, additional pharmacological procedures have shown a more specific role for the NAc in CPP expression. In one study, intra-NAc antagonism of D1- and D2-like receptors prevented the expression of CPP induced by icv ethanol [111]. In another study, NAc DA (D1- and D2 like) and glutamate (NMDA) receptors were blocked during the CPP expression test using either flupenthixol or AP-5, respectively [116]. Although DA receptor antagonism did not affect ethanol place preference, NMDA receptor antagonism reduced it, suggesting that the involvement of the NAc in expression is specific to activity at NMDA receptors. This effect was reproduced in another study using only unilaterally administered AP5, further indicating the importance of NAc NMDA receptor involvement in ethanol place preference expression [60]. Notably, this study was designed to examine glutamate input to NAc from amygdala. Although findings appeared to demonstrate that amygdala disconnection from NAc blocked ethanol CPP expression, reduced CPP in mice unilaterally infused with AP5 in NAc prevented this interpretation. Overall, these findings have established a role for NAc NMDA, but not DA receptors, in ethanol place preference expression and suggest that glutamatergic input from amygdala may also be involved. Although DA input from VTA to NAc is a hypothesized mechanism underlying drug seeking, these results suggest that it does not underlie ethanol seeking, at least as indexed by CPP. Finally, these studies serve to demonstrate that manipu‐ lations more selective than global inactivation or deletion of a structure may be necessary to

Accordingly, the role of the amygdala in ethanol CPP expression has also been addressed. Like lesions made before conditioning, the bilateral electrolytic lesion of the amygdala made before the test phase blocked ethanol place preference expression [109]. In addition, when bilaterally infused into the amygdala, the D1- and D2-like DA receptor agonist flupenthixol blocked ethanol CPP expression [116]. Together, these results illustrate the role of the amygdala in ethanol-seeking behavior and indicate the importance of dopaminergic input to this structure

The BNST also has a role in modulating the expression of ethanol reward. Exposure to ethanol causes changes in glutamate synaptic plasticity [117], increases extracellular DA levels in the

appropriately gauge that the importance of structure in behavior.

for ethanol CPP expression.

activity.

expression.

Unlike the acquisition and expression of ethanol CPP, the neural correlates of extinction of ethanol CPP is relatively understudied. The only region consistently studied for its role in ethanol CPP is the mPFC.

The mPFC is known to be involved in the acquisition and extinction of Pavlovian conditioned fear and drug memories [121]; thus, it is not surprising that disruption of its activity impairs the extinction of ethanol CPP. Several studies found that lesions or inhibition of the mPFC, but not the ACC, following acquisition blocks the extinction of CPP [122, 123]. These studies suggest the importance of this mPFC in the formation of inhibitory ethanol seeking memories. Additionally, one study links the NAc core with extinction of ethanol seeking. Lesions of the NAc core were found to have no effect on the expression of ethanol CPP but caused a rapid loss of responding during extinction [109], which suggest that the NAc core is associated with inhibitory ethanol memory, but further research will be needed to confirm the role of the NAc in extinction.

Like extinction, the brain regions involved in the reinstatement of ethanol CPP are not well studied, but there are several studies that implicate particular brain regions. One such study found an increase in c-Fos activity in the BLA of rats following cued reinstatement of ethanol seeking in a discriminative stimulus operant paradigm [75]. Although this is not a direct proof of the necessity of the BLA in ethanol seeking in CPP, another study found that the direct manipulation of the BLA alters cued reinstatement of cocaine seeking [59]. Together, these studies suggest the importance of the BLA in ethanol reinstatement and likely in the reinstate‐ ment of ethanol CPP. Other regions, such as the VTA, amygdala, and BNST, are likely involved in the extinction and reinstatement of ethanol CPP, as they are important for the extinction and reinstatement of CPP of other addictive substances. Investigating the involvement of these regions in extinction and reinstatement of ethanol CPP will be an important step for under‐ standing the circuitry of relapse.

#### **5.3. Summary**

In summary, these studies show the importance of several brain regions heavily implicated in drug reward to ethanol seeking as well. Most of these studies evaluated the structures situated downstream the VTA that have been implicated in drug seeking based on their efferent dopaminergic input. In addition to these studies demonstrating that downstream sites are involved, findings from Bechtholt and Cunningham [115] further illustrate the importance of the VTA in ethanol-seeking behavior. As opioid receptors are situated presynaptically on local GABAergic inputs to DA cells, the authors hypothesized that methylnaloxonium disinhibited VTA GABAergic interneurons. This likely inhibited VTA DA activity, thereby reducing ethanol place preference expression. Conversely, baclofen presumably reduced CPP by acting directly on VTA DA cells, as they express GABAB receptors. Thus, the VTA is a critical structure for drug and, specifically, ethanol cue associations. Although these findings and proposed mechanisms underscore the importance of VTA DA in ethanol CPP expression, they provide little information on the inputs to the VTA that modulate DA cell activity. Thus, the origins and neurochemical sources of VTA DA cell innervation involved in ethanol-seeking behavior remain unknown. Accordingly, the next section discusses the involvement of VTA input in reward and identifies several inputs that may be of importance and deserve further study for their role in ethanol CPP.

#### **5.4. Inputs to the VTA**

Excitatory (glutamatergic) afferents of the VTA arise from virtually all structures to which this region projects, with the exception of the NAc and lateral septum (LS), which provide strong GABAergic inputs [124]. This suggests that there is a broad network of excitatory reciprocal projections to and from the VTA, with much of the glutamatergic input to VTA neurons arising from subcortical regions that include but are not limited to amygdala, mesopontine nuclei, lateral habenula, and hypothalamus [125]. Although this reciprocal flow of neurotransmission is also found between the VTA and cortex, the PFC serves as the only cortical source of glutamate to the VTA [126]. Importantly, these glutamatergic afferents play a critical role in regulating VTA neuron firing. Specifically, glutamatergic input to the VTA appears to be critical for behaviorally relevant burst firing of VTA DA neurons [127]. The resulting phasic release of DA from the VTA is intimately associated with goal-directed behaviors and drug reward [128].

In addition to the contemporary methods described earlier, recent advances in immunohisto‐ logical techniques have facilitated a more precise mapping of afferent and efferent projections of the VTA. As a result, an updated view on the role of broad neural circuit activity (including that of the VTA) in relation to behavior has been formed. In this current view, the net result of cell-specific projections onto cell-specific targets is accounted for and the subsequent plotting of these circuits suggests a complex topographical map. This map indicates the existence of an intricate network of connectivity designed to tightly regulate the activity of neuronal ensem‐ bles, which in turn orchestrate complex and divergent behaviors, even from within the same circuits. In the VTA specifically, not all inputs are alike in their behavioral consequences. Here, DA activity is governed by a complex network of cell-type-specific neuron-to-neuron connec‐ tions, the net effect of which can result in vastly different motivational states [129, 130].

Several lines of evidence suggest the importance of monosynaptic inputs to VTA DA neurons (one-step inputs) in governing motivational behaviors. For example, investigation of the laterodorsal tegmental (LDT) nucleus has revealed that 80% of its glutamatergic afferents synapse onto VTA DA neurons directly [131]. The importance of these glutamatergic inputs have been corroborated through electrophysiological analysis, which has shown that this region is essential to VTA DA cell burst firing [132]. Moreover, in vivo stimulation of LDT glutamate afferents has been reported to selectively terminate on and stimulate a distinct population of VTA DA neurons, which thereby generate reward [130].

The BNST is another region upstream the VTA that has been identified as critically involved in regulating the activity of DA cell activity [133–135]. Specifically, the BNST positively modulates VTA DA activity putatively through two primary and distinct mechanisms: (1) a direct glutamate projection to VTA DA and (2) a direct GABA projection to VTA GABA [129, 135–137]. Of note, more recent evidence derived from studies using more advanced and selective tools suggests that a BNST GABA input to VTA GABA is the predominant source of the modulation of the BNST of the VTA [129]. Of relevance, behavior in rodents has demon‐ strated that the BNST underlies cue-elicited drug seeking. For example, transient inactivation of the BNST has been shown to prevent cue-induced reinstatement of cocaine seeking [138]. Direct projections from BNST to VTA appear to be important for cocaine-related behaviors. For example, disconnection of the BNST-VTA pathway has been shown to reduce expression of cocaine CPP [62], but the role of that projection in ethanol seeking still remains unknown and further study of this projection and other VTA inputs will advance our understanding of the larger neural network driving ethanol seeking.
