Inactivation of ventral hippocampus projections promotes sensitivity to changes in contingency.

The loss of behavioral flexibility is common across a number of neuropsychiatric illnesses. This may be in part due to the loss of the ability to detect or use changes in action-outcome contingencies to guide behavior. There is growing evidence that the ventral hippocampus plays a critical role in the regulation of flexible behavior and reward-related decision making. Here, we investigated the role of glutamatergic projections from the ventral hippocampus in the expression of contingency-mediated reward seeking. We demonstrate that selectively silencing ventral hippocampus projections can restore the use of action-outcome contingencies to guide behavior, while sparing cue-guided behavior and extinction learning. Our findings further indicated that the ability of the ventral hippocampus to promote habitual response strategies may be in part mediated by selective projections from the ventral hippocampus to the nucleus accumbens shell. Together these results implicate glutamatergic projections from the ventral hippocampus in the regulation of behavioral flexibility and suggest that alterations in ventral hippocampus function may contribute to overreliance on habitual response strategy observed in neuropsychiatric illnesses including addiction and obsessive-compulsive disorder.

Mechanisms of Persistent Neurobiological Changes Following Adolescent Alcohol Exposure: NADIA Consortium Findings.

The Neurobiology of Adolescent Drinking in Adulthood (NADIA) Consortium has focused on the impact of adolescent binge drinking on brain development, particularly on effects that persist into adulthood. Adolescent binge drinking is common, and while many factors contribute to human brain development and alcohol use during adolescence, animal models are critical for understanding the specific consequences of alcohol exposure during this developmental period and the underlying mechanisms. Using adolescent intermittent ethanol (AIE) exposure models, NADIA investigators identified long-lasting AIE-induced changes in adult behavior that are consistent with observations in humans, such as increased alcohol drinking, increased anxiety (particularly social anxiety), increased impulsivity, reduced behavioral flexibility, impaired memory, disrupted sleep, and altered responses to alcohol. These behavioral changes are associated with multiple molecular, cellular, and physiological alterations in the brain that persist long after AIE exposure. At the molecular level, AIE results in long-lasting changes in neuroimmune/trophic factor balance and epigenetic-microRNA (miRNA) signaling across glia and neurons. At the cellular level, AIE history is associated in adulthood with reduced expression of cholinergic, serotonergic, and dopaminergic neuron markers, attenuated cortical thickness, decreased neurogenesis, and altered dendritic spine and glial morphology. This constellation of molecular and cellular adaptations to AIE likely contributes to observed alterations in neurophysiology, measured by synaptic physiology, EEG patterns, and functional connectivity. Many of these AIE-induced brain changes replicate findings seen in postmortem brains of humans with alcohol use disorder (AUD). NADIA researchers are now elucidating mechanisms of these adaptations. Emerging data demonstrate that exercise, antiinflammatory drugs, anticholinesterases, histone deacetylase inhibitors, and other pharmacological compounds are able to prevent (administered during AIE) and/or reverse (given after AIE) AIE-induced pathology in adulthood. These studies support hypotheses that adolescent binge drinking increases risk of adult hazardous drinking and influences brain development, and may provide insight into novel therapeutic targets for AIE-induced neuropathology and AUDs.

Loss of δ-GABAA receptor-mediated tonic currents in the adult prelimbic cortex following adolescent alcohol exposure.

Delayed maturation of the adolescent prefrontal cortex may render it particularly vulnerable to insults, including those associated with drugs of abuse. Using a rat model of binge alcohol exposure, the present study examined the effect of adolescent intermittent ethanol (AIE) exposure during postnatal days 28-42 on γ-aminobutyric acid (GABA)ergic neurotransmission in the prelimbic cortex. In control rats, patch-clamp electrophysiology in acute slices obtained at different postnatal ages revealed a developmental increase in the GABAA receptor-mediated tonic current in layer V pyramidal neurons but no change in layers II/III when measured in the adult. In slices from AIE-exposed rats, the amplitude of the tonic current was significantly reduced compared with controls when tested at postnatal days 45, 60 and 90-120. This AIE-induced reduction in tonic current was found to reflect attenuation of currents mediated by δ-subunit containing receptors. Consistent with this, facilitation of the tonic current by bath application of either ethanol or allopregnanolone was attenuated in slices from AIE-exposed adult rats compared with control rats. However, expression of this facilitation as a percent of the amplitude of the total current mediated by δ-GABAA receptors revealed that AIE did not alter their sensitivity to either agonist. Lastly, immunohistochemistry and Western blot analysis revealed no change in the expression of δ-GABAA subunits or their surface expression. Taken together, these studies reveal that AIE exposure results in persistent deficits in δ-GABAA tonic currents in the adult prelimbic cortex that may contribute to deficits in decision-making and behavioral control in adulthood.

FMRP Mediates Chronic Ethanol-Induced Changes in NMDA, Kv4.2, and KChIP3 Expression in the Hippocampus.

BACKGROUND: Exposure to chronic ethanol (EtOH) results in changes in the expression of proteins that regulate neuronal excitability. This study examined whether chronic EtOH alters the hippocampal expression and function of fragile X mental retardation protein (FMRP) and the role of FMRP in the modulation of chronic EtOH-induced changes in the expression of NMDA receptors and Kv4.2 channels. METHODS: For in vivo studies, C57BL/6J mice underwent a chronic intermittent EtOH (CIE) vapor exposure procedure. After CIE, hippocampal tissue was collected and subjected to immunoblot blot analysis of NMDA receptor subunits (GluN1, GluN2B), Kv4.2, and its accessory protein KChIP3. For in vitro studies, hippocampal slice cultures were exposed to 75 mM EtOH for 8 days. Following EtOH exposure, mRNAs bound to FMRP was measured. In a separate set of studies, cultures were exposed to an inhibitor of S6K1 (PF-4708671 [PF], 6 µM) in order to assess whether EtOH-induced homeostatic changes in protein expression depend upon changes in FMRP activity. RESULTS: Immunoblot blot analysis revealed increases in GluN1 and GluN2B but reductions in Kv4.2 and KChIP3. Analysis of mRNAs bound to FMRP revealed a similar bidirectional change observed as reduction of GluN2B and increase in Kv4.2 and KChIP3 mRNA transcripts. Analysis of FMRP further revealed that while chronic EtOH did not alter the expression of FMRP, it significantly increased phosphorylation of FMRP at the S499 residue that is known to critically regulate its activity. Inhibition of S6K1 prevented the chronic EtOH-induced increase in phospho-FMRP and changes in NMDA subunits, Kv4.2, and KChIP3. In contrast, PF had no effect in the absence of alcohol, indicating it was specific for the chronic EtOH-induced changes. CONCLUSIONS: These findings demonstrate that chronic EtOH exposure enhances translational control of plasticity-related proteins by FMRP, and that S6K1 and FMRP activities are required for expression of chronic EtOH-induced homeostatic plasticity at glutamatergic synapses in the hippocampus.

Role for the Rostromedial Tegmental Nucleus in Signaling the Aversive Properties of Alcohol.

BACKGROUND: While the rewarding effects of alcohol contribute significantly to its addictive potential, it is becoming increasingly appreciated that alcohol's aversive properties also play an important role in the propensity to drink. Despite this, the neurobiological mechanism for alcohol's aversive actions is not well understood. The rostromedial tegmental nucleus (RMTg) was recently characterized for its involvement in aversive signaling and has been shown to encode the aversive properties of cocaine, yet its involvement in alcohol's aversive actions have not been elucidated. METHODS: Adult male and female Long-Evans rats underwent conditioned taste aversion (CTA) procedures where exposure to a novel saccharin solution was paired with intraperitoneal administration of saline, lithium chloride (LiCl), or ethanol (EtOH). Control rats underwent the same paradigm except that drug and saccharin exposure were explicitly unpaired. Saccharin consumption was measured on test day in the absence of drug administration, and rats were sacrificed 90 to 105 minutes following access to saccharin. Brains were subsequently harvested and processed for cFos immunohistochemistry. The number of cFos-labeled neurons was counted in the RMTg and the lateral habenula (LHb)-a region that sends prominent glutamatergic input to the RMTg. RESULTS: In rats that received paired drug and saccharin exposure, EtOH and LiCl induced significant CTA compared to saline to a similar degree in males and females. Both EtOH- and LiCl-induced CTA significantly enhanced cFos expression in the RMTg and LHb but not the hippocampus. Similar to behavioral measures, no significant effect of sex on CTA-induced cFos expression was observed. cFos expression in both the RMTg and LHb was significantly correlated with CTA magnitude with greater cFos being associated with more pronounced CTA. In addition, cFos expression in the RMTg was positively correlated with LHb cFos. CONCLUSIONS: These data suggest that the RMTg and LHb are involved in the expression of CTA and are consistent with previous work implicating the RMTg in aversive signaling. Furthermore, increased cFos expression in the RMTg following EtOH-induced CTA suggests that this region plays a role in signaling alcohol's aversive properties.

Enhancement of extinction learning attenuates ethanol-seeking behavior and alters plasticity in the prefrontal cortex.

Addiction is a chronic relapsing disorder in which relapse is often initiated by exposure to drug-related cues. The present study examined the effects of mGluR5 activation on extinction of ethanol-cue-maintained responding, relapse-like behavior, and neuronal plasticity. Rats were trained to self-administer ethanol and then exposed to extinction training during which they were administered either vehicle or the mGluR5 positive allosteric modulator 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl) or CDPPB. CDPPB treatment reduced active lever responding during extinction, decreased the total number of extinction sessions required to meet criteria, and attenuated cue-induced reinstatement of ethanol seeking. CDPPB facilitation of extinction was blocked by the local infusion of the mGluR5 antagonist 3-((2-methyl-4-thiazolyl)ethynyl) pyridine into the infralimbic (IfL) cortex, but had no effect when infused into the prelimbic (PrL) cortex. Analysis of dendritic spines revealed alterations in structural plasticity, whereas electrophysiological recordings demonstrated differential alterations in glutamatergic neurotransmission in the PrL and IfL cortex. Extinction was associated with increased amplitude of evoked synaptic PrL and IfL NMDA currents but reduced amplitude of PrL AMPA currents. Treatment with CDPPB prevented the extinction-induced enhancement of NMDA currents in PrL without affecting NMDA currents in the IfL. Whereas CDPPB treatment did not alter the amplitude of PrL or IfL AMPA currents, it did promote the expression of IfL calcium-permeable GluR2-lacking receptors in both abstinence- and extinction-trained rats, but had no effect in ethanol-naive rats. These results confirm changes in the PrL and IfL cortex in glutamatergic neurotransmission during extinction learning and demonstrate that manipulation of mGluR5 facilitates extinction of ethanol cues in association with neuronal plasticity.

Chronic alcohol disrupts dopamine receptor activity and the cognitive function of the medial prefrontal cortex.

Dopamine (DA) receptors in the medial prefrontal cortex (mPFC) exert powerful effects on cognition by modulating the balance between excitatory and inhibitory neurotransmission. The present study examined the impact of chronic intermittent ethanol (CIE) exposure on cognitive function and DA receptor-mediated neurotransmission in the rat mPFC. Consistent with alterations in executive function in alcoholics, CIE-exposed rats exhibited deficits in behavioral flexibility in an operant set-shifting task. Since alterations in dopaminergic neurotransmission in the mPFC have been implicated in a number of behavioral disorders including addiction, studies were then performed in the adult acute slice preparation to examine changes in DA receptor function in the mPFC following CIE exposure. In slices obtained from control rats, DA receptor stimulation was observed to exert complex actions on neuronal firing and synaptic neurotransmission that were not only dependent upon the particular receptor subtype but also whether it was a pyramidal cell or a fast-spiking interneuron. In contrast to slices from control rats, there was a near complete loss of the modulatory actions of D2/D4 receptors on cell firing and neurotransmission in slices obtained immediately, 1 and 4 weeks after the last day of CIE exposure. This loss did not appear to be associated with changes in receptor expression. In contrast, CIE exposure did not alter D1 receptor function or mGluR1 modulation of firing. These studies are consistent with the suggestion that chronic alcohol exposure disrupts cognitive function at least in part through disruption of D2 and D4 receptor signaling in mPFC.

A unifying model of the role of the infralimbic cortex in extinction and habits.

The infralimbic prefrontal cortex (IL) has been shown to be critical for the regulation of flexible behavior, but its precise function remains unclear. This region has been shown to be critical for the acquisition, consolidation, and expression of extinction learning, leading many to hypothesize that IL suppresses behavior as part of a "stop" network. However, this framework is at odds with IL function in habitual behavior in which the IL has been shown to be required for the expression and acquisition of ongoing habitual behavior. Here, we will review the current state of knowledge of IL anatomy and function in behavioral flexibility and provide a testable framework for a single IL mechanism underlying its function in both extinction and habit learning.

Adolescent alcohol exposure alters GABAA receptor subunit expression in adult hippocampus.

BACKGROUND: The long-term consequences of adolescent alcohol abuse that persist into adulthood are poorly understood and have not been widely investigated. We have shown that intermittent exposure to alcohol during adolescence decreased the amplitude of GABAA receptor (GABAA R)-mediated tonic currents in hippocampal dentate granule cells in adulthood. The aim of this study was to investigate the enduring effects of chronic intermittent alcohol exposure during adolescence or adulthood on the expression of hippocampal GABAA Rs. METHODS: We used a previously characterized tissue fractionation method to isolate detergent resistant membranes and soluble fractions, followed by Western blots to measure GABAA R protein expression. We also measured mRNA levels of GABAA R subunits using quantitative real-time polymerase chain reaction. RESULTS: Although the protein levels of α1-, α4-, and δ-GABAA R subunits remained stable between postnatal day (PD) 30 (early adolescence) and PD71 (adulthood), the α5-GABAA R subunit was reduced across that period. In rats that were subjected to adolescent intermittent ethanol (AIE) exposure between PD30 and PD46, there was a significant reduction in the protein levels of the δ-GABAA R, in the absence of any changes in mRNA levels, at 48 hours and 26 days after the last ethanol (EtOH) exposure. Protein levels of the α4-GABAA R subunit were significantly reduced, but mRNA levels were increased, 26 days (but not 48 hours) after the last AIE exposure. Protein levels of α5-GABAA R were not changed by AIE, but mRNA levels were reduced at 48 hours but normalized 26 days after AIE. In contrast to the effects of AIE, chronic intermittent ethanol (CIE) exposure during adulthood had no effect on expression of any of the GABAA R subunits examined. CONCLUSIONS: AIE produced both short- and long-term alterations of GABAA R subunits mRNA and protein expression in the hippocampus, whereas CIE produced no long-lasting effects on those measures. The observed reduction of protein levels of the δ-GABAA R, specifically, is consistent with previously reported altered hippocampal GABAA R-mediated electrophysiological responses after AIE. The absence of effects of CIE underscores the emerging view of adolescence as a time of distinctive vulnerability to the enduring effects of repeated EtOH exposure.

Adolescent Alcohol Exposure Promotes Mechanical Allodynia and Alters Synaptic Function at Inputs from the Basolateral Amgydala to the Prelimbic Cortex.

Binge drinking is common among adolescents despite mounting evidence linking it to various adverse health outcomes that includes heightened pain perception. The prelimbic (PrL) cortex is vulnerable to insults from adolescent alcohol exposure and receives input from the basolateral amygdala (BLA) while sending projections to the ventrolateral periaqueductal gray (vlPAG) - two brain regions implicated in nociception. In this study, adolescent intermittent ethanol (AIE) exposure was carried out in male and female rats using a vapor inhalation procedure. Mechanical and thermal sensitivity, assessed throughout adolescence and into adulthood, revealed that AIE exposure induced protracted mechanical allodynia in both male and female rats. However, a carrageenan inflammatory paw pain challenge in adult rats revealed that AIE did not further augment carrageenan-induced hyperalgesia. To investigate synaptic function at BLA inputs onto defined populations of PrL neurons, retrobeads and viral labelling were combined with optogenetics and slice electrophysiology. Recordings from retrobead labelled cells in the PrL revealed AIE reduced BLA driven feedforward inhibition of neurons projecting from the PrL to the vlPAG (PrLPAG neurons), resulting in augmented excitation/inhibition (E/I) balance and increased intrinsic excitability. Consistent with this finding, recordings from virally tagged PrL parvalbumin interneurons (PVINs) demonstrated that AIE exposure reduced both E/I balance at BLA inputs onto PVINs and PVIN intrinsic excitability when assessed in adulthood. These findings provide compelling evidence that AIE and acute pain alter synaptic function and intrinsic excitability within a prefrontal nociceptive circuit.

Central Amygdala Astrocyte Plasticity Underlies GABAergic Dysregulation in Ethanol Dependence.

Dependence is a hallmark of alcohol use disorder characterized by excessive alcohol intake and withdrawal symptoms. The central nucleus of the amygdala (CeA) is a key brain structure underlying the synaptic and behavioral consequences of ethanol dependence. While accumulating evidence suggests that astrocytes regulate synaptic transmission and behavior, there is a limited understanding of the role astrocytes play in ethanol dependence. The present study used a combination of viral labeling, super resolution confocal microscopy, 3D image analysis, and slice electrophysiology to determine the effects of chronic intermittent ethanol (CIE) exposure on astrocyte plasticity in the CeA. During withdrawal from CIE exposure, we observed increased GABA transmission, an upregulation in astrocytic GAT3 levels, and an increased proximity of astrocyte processes near CeA synapses. Furthermore, GAT3 levels and synaptic proximity were positively associated with voluntary ethanol drinking in dependent rats. Slice electrophysiology confirmed that the upregulation in astrocytic GAT3 levels was functional, as CIE exposure unmasked a GAT3-sensitive tonic GABA current in the CeA. A causal role for astrocytic GAT3 in ethanol dependence was assessed using viral-mediated GAT3 overexpression and knockdown approaches. However, GAT3 knockdown or overexpression had no effect on somatic withdrawal symptoms, dependence-escalated ethanol intake, aversion-resistant drinking, or post-dependent ethanol drinking in male or female rats. Moreover, intra-CeA pharmacological inhibition of GAT3 also did not alter dependent ethanol drinking. Together, these findings indicate that ethanol dependence induces GABAergic dysregulation and astrocyte plasticity in the CeA. However, astrocytic GAT3 does not appear necessary for the drinking related phenotypes associated with dependence.

Constitutive knockout of the membrane cytoskeleton protein beta adducin decreases mushroom spine density in the nucleus accumbens but does not prevent spine remodeling in response to cocaine.

The adducin family of proteins associates with the actin cytoskeleton in a calcium-dependent manner. Beta adducin (ßAdd) is involved in synaptic plasticity in the hippocampus; however, the role of ßAdd in synaptic plasticity in other brain areas is unknown. Using diolistic labeling with the lipophilic dye DiI, we found that the density of mature mushroom-shaped spines was significantly decreased in the nucleus accumbens (NAc) in brain slices from ßAdd-knockout (KO) mice as compared to their wildtype (WT) siblings. The effect of 10 days of daily cocaine (15 mg/kg) administration on NAc spine number and locomotor behavior was also measured in ßAdd WT and KO mice. As expected, there was a significant increase in overall spine density in NAc slices from cocaine-treated WT mice at this time-point; however, there was a greater increase in the density of mushroom spines in ßAdd-KO animals following chronic cocaine administration than in WT. In addition, ßAdd-KO mice showed elevated locomotor activity in response to cocaine treatment compared to WT siblings. These results indicate that ßAdd is required for stabilising mature spines under basal conditions in the NAc, but that lack of this protein does not prevent synaptic remodeling following repeated cocaine administration. In addition, these data are consistent with previous studies suggesting that ßAdd may normally be involved in stabilising spines once drug- or experience-dependent remodeling has occurred.

The glutamatergic component of the mesocortical pathway emanating from different subregions of the ventral midbrain.

The mesocortical pathway projecting from the ventral tegmental area (VTA) to the prefrontal cortex (PFC) plays a critical role in a number of cognitive and emotional processes. While this pathway has been traditionally viewed as dopaminergic, recent data indicate that a considerable proportion of rostromedial VTA neurons possess markers for glutamate transmission. However, the relative density of the glutamatergic projection to the PFC from these rostromedial regions is unknown. In the present study, anterograde tracer injections into 4 ventral midbrain subregions were coupled with immunohistochemical analysis of labeled axons in PFC for markers of dopamine (DA; tyrosine hydroxylase [TH]) and glutamate (vesicular glutamate transporter 2; VGLUT2). We found that while tracer injections into the interfascicular nucleus produced labeled fibers in the PFC that were mainly TH positive, tracer injections into the rostral linear nucleus, rostral VTA, and parabrachial pigmented nucleus produced labeled fibers in PFC that contained mainly VGLUT2-positive rather than TH-positive varicosities. When viewed in the light of the previously documented strong γ-aminobutyric acidergic component, it would seem that the rostromedial mesocortical projection is actually an amino acid pathway that in addition has a DA component.

Involvement of cortical input to the rostromedial tegmental nucleus in aversion to foot shock.

The rostromedial tegmental nucleus (RMTg) encodes negative reward prediction error (RPE) and plays an important role in guiding behavioral responding to aversive stimuli. Previous research has focused on regulation of RMTg activity by the lateral habenula despite studies revealing RMTg afferents from other regions including the frontal cortex. The current study provides a detailed anatomical and functional analysis of cortical input to the RMTg of male rats. Retrograde tracing uncovered dense cortical input to the RMTg spanning the medial prefrontal cortex, the orbitofrontal cortex and anterior insular cortex. Afferents were most dense in the dorsomedial subregion of the PFC (dmPFC), an area that is also implicated in both RPE signaling and aversive responding. RMTg-projecting dmPFC neurons originate in layer V, are glutamatergic, and collateralize to select brain regions. In-situ mRNA hybridization revealed that neurons in this circuit are predominantly D1 receptor-expressing with a high degree of D2 receptor colocalization. Consistent with cFos induction in this neural circuit during exposure to foot shock and shock-predictive cues, optogenetic stimulation of dmPFC terminals in the RMTg drove avoidance. Lastly, acute slice electrophysiology and morphological studies revealed that exposure to repeated foot shock resulted in significant physiological and structural changes consistent with a loss of top-down modulation of RMTg-mediated signaling. Altogether, these data reveal the presence of a prominent cortico-subcortical projection involved in adaptive behavioral responding to aversive stimuli such as foot shock and provide a foundation for future work aimed at exploring alterations in circuit function in diseases characterized by deficits in cognitive control over reward and aversion.

Adolescent Alcohol Exposure Results in Sex-specific Alterations in Conditioned Fear Learning and Memory in Adulthood.

The present study used auditory fear conditioning to assess the impact of repeated binge-like episodes of alcohol exposure during adolescence on conditioned fear in adulthood. Male and female Long-Evans rats were subjected to adolescent intermittent ethanol (AIE) exposure by vapor inhalation between post-natal day 28 and 44. After aging into adulthood, rats then underwent fear conditioning by exposure to a series of tone-shock pairings. This was followed by cued-tone extinction training, and then testing of fear recovery. In male rats, AIE exposure enhanced conditioned freezing but did not alter the time-course of extinction of cued-tone freezing. During subsequent assessment of fear recovery, AIE exposed rats exhibited less freezing during contextual fear renewal, but greater freezing during extinction recall and spontaneous recovery. Compared to males, female rats exhibited significantly lower levels of freezing during fear conditioning, more rapid extinction of freezing behavior, and significantly lower levels of freezing during the tests of fear recovery. Unlike males that were all classified as high conditioners; female rats could be parsed into either a high or low conditioning group. However, irrespective of their level of conditioned freezing, both the high and low conditioning groups of female rats exhibited rapid extinction of conditioned freezing behavior and comparatively low levels of freezing in tests of fear recovery. Regardless of group classification, AIE had no effect on freezing behavior in female rats during acquisition, extinction, or fear recovery. Lastly, exposure of male rats to the mGlu5 positive allosteric modulator CDPPB prevented AIE-induced alterations in freezing. Taken together, these observations demonstrate sex-specific changes in conditioned fear behaviors that are reversible by pharmacological interventions that target mGlu5 receptor activation.

Behavioral and slice electrophysiological assessment of DREADD ligand, deschloroclozapine (DCZ) in rats.

Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) have become a premier neuroscience research tool for enabling reversible manipulations of cellular activity following experimenter-controlled delivery of a DREADD-specific ligand. However, several DREADD ligands, e.g., clozapine-N-oxide (CNO), have metabolic and off-target effects that may confound experimental findings. New DREADD ligands aim to reduce metabolic and potential off-target effects while maintaining strong efficacy for the designer receptors. Recently a novel DREADD ligand, deschloroclozapine (DCZ), was shown to induce chemogenetic-mediated cellular and behavioral effects in mice and monkeys without detectable side effects. The goal of the present study was to examine the effectiveness of systemic DCZ for DREADD-based chemogenetic manipulations in behavioral and slice electrophysiological applications in rats. We demonstrate that a relatively low dose of DCZ (0.1 mg/kg) supports excitatory DREADD-mediated cFos induction, DREADD-mediated inhibition of a central amygdala-dependent behavior, and DREADD-mediated inhibition of neuronal activity in a slice electrophysiology preparation. In addition, we show that this dose of DCZ does not alter gross locomotor activity or induce a place preference/aversion in control rats without DREADD expression. Together, our findings support the use of systemic DCZ for DREADD-based manipulaations in rats, and provide evidence that DCZ is a superior alternative to CNO.

Adolescent alcohol exposure reduces dopamine 1 receptor modulation of prelimbic neurons projecting to the nucleus accumbens and basolateral amygdala.

Binge drinking during adolescence is highly prevalent despite increasing evidence of its long-term impact on behaviors associated with modulation of behavioral flexibility by the medial prefrontal cortex (mPFC). In the present study, male and female rats underwent adolescent intermittent ethanol (AIE) exposure by vapor inhalation. After aging to adulthood, retrograde bead labelling and viral tagging were used to identify populations of neurons in the prelimbic region (PrL) of the mPFC that project to specific subcortical targets. Electrophysiological recording from bead-labelled neurons in PrL slices revealed that AIE did not alter the intrinsic excitability of PrL neurons that projected to either the NAc or the BLA. Similarly, recordings of spontaneous inhibitory and excitatory post-synaptic currents revealed no AIE-induced changes in synaptic drive onto either population of projection neurons. In contrast, AIE exposure was associated with a loss of dopamine receptor 1 (D1), but no change in dopamine receptor 2 (D2), modulation of evoked firing of both populations of projection neurons. Lastly, confocal imaging of proximal and apical dendritic tufts of viral-labelled PrL neurons that projected to the nucleus accumbens (NAc) revealed AIE did not alter the density of dendritic spines. Together, these observations provide evidence that AIE exposure results in disruption of D1 receptor modulation of PrL inputs to at least two major subcortical target regions that have been implicated in AIE-induced long-term changes in behavioral control.

The lateral habenula is not required for ethanol dependence-induced escalation of drinking.

The lateral habenula (LHb) is an epithalamic nuclei that has been shown to signal the aversive properties of ethanol. The present study tested the hypothesis that activity of the LHb is required for the acquisition and/or expression of dependence-induced escalation of ethanol drinking and somatic withdrawal symptoms. Male Sprague-Dawley rats completed 4 weeks of baseline drinking under a standard intermittent access two-bottle choice (2BC) paradigm before undergoing 2 weeks of daily chronic intermittent ethanol (CIE) via vapor inhalation. Following this CIE exposure period, rats resumed 2BC drinking to assess dependence-induced changes in voluntary ethanol consumption. CIE exposed rats exhibited a significant increase in ethanol drinking that was associated with high levels of blood alcohol and a reduction in somatic symptoms of ethanol withdrawal. However, despite robust cFos activation in the LHb during ethanol withdrawal, chemogenetic inhibition of the LHb did not alter either ethanol consumption or somatic signs of ethanol withdrawal. Consistent with this observation, ablating LHb outputs via electrolytic lesions of the fasciculus retroflexus (FR) did not alter the acquisition of somatic withdrawal symptoms or escalation of ethanol drinking in CIE-exposed rats. The LHb controls activity of the rostromedial tegmental nucleus (RMTg), a midbrain nucleus activated by aversive experiences including ethanol withdrawal. During ethanol withdrawal, both FR lesioned and sham control rats exhibited similar cFos activation in the RMTg, suggesting that RMTg activation during ethanol withdrawal does not require LHb input. These data suggest that, at least in male rats, the LHb is not necessary for the acquisition or expression of escalation of ethanol consumption or expression of somatic symptoms of ethanol withdrawal. Overall, our findings provide evidence that the LHb is dispensable for some of the negative consequences of ethanol withdrawal.

Successful choice behavior is associated with distinct and coherent network states in anterior cingulate cortex.

Successful decision making requires an ability to monitor contexts, actions, and outcomes. The anterior cingulate cortex (ACC) is thought to be critical for these functions, monitoring and guiding decisions especially in challenging situations involving conflict and errors. A number of different single-unit correlates have been observed in the ACC that reflect the diverse cognitive components involved. Yet how ACC neurons function as an integrated network is poorly understood. Here we show, using advanced population analysis of multiple single-unit recordings from the rat ACC during performance of an ecologically valid decision-making task, that ensembles of neurons move through different coherent and dissociable states as the cognitive requirements of the task change. This organization into distinct network patterns with respect to both firing-rate changes and correlations among units broke down during trials with numerous behavioral errors, especially at choice points of the task. These results point to an underlying functional organization into cell assemblies in the ACC that may monitor choices, outcomes, and task contexts, thus tracking the animal's progression through "task space."

Impact of sex, strain, and age on blood ethanol concentration and behavioral signs of intoxication during ethanol vapor exposure.

Animal models of alcohol drinking and dependence are a critical resource for understanding the neurobiological mechanisms and development of more effective treatments for alcohol use disorder (AUD). Because most rat strains do not voluntarily consume large enough quantities of alcohol to adequately model heavy drinking, dependence, and withdrawal-related symptoms, researchers frequently turn to experimenter administered methods to investigate how prolonged and repeated exposure to large quantities of alcohol impacts brain and behavior. Vaporized ethanol is a common method used for chronically subjecting rodents to alcohol and has been widely used to model both binge and dependence-inducing heavy drinking patterns observed in humans. Rodent strain, sex, and age during exposure are all well-known to influence outcomes in experiments utilizing intraperitoneal or intragastric methods of repeated ethanol exposure. Yet, despite its frequent use, the impact of these variables on outcomes associated with ethanol vapor exposure has not been widely investigated. The present study analyzed data generated from over 700 rats across an eight-year period to provide a population-level assessment of variables influencing level of intoxication using vapor exposure. Our findings reveal important differences with respect to strain, sex, and age during ethanol exposure in the relationship between blood ethanol concentration and behavioral signs of intoxication. These data provide valuable scientific and practical insight for laboratories utilizing ethanol vapor exposure paradigms to model AUD in rats.