AMPA receptors in nucleus accumbens shell mediate extinction of alcohol seeking through interactions with basolateral amygdala

My goal was to study the role of AMPA and NMDA receptors in fear memory using genetic tools. In particular, I aimed to unravel the function of these glutamate receptors in the long-term retrieval of passive avoidance and fear conditioning, both in the brain in general and in the Basolateral Amygdala (BLA). Through the analysis of GluA3 knockout mice, I could show that the AMPA receptor subunit GluA3 is not necessary for the initial phases of cued fear learning, but is required for the normal attenuation of fear to a remote negative event, both in the passive-avoidance and fear-conditioning paradigms. In contrast, GluA1 is essential for the acquisition and short-term retrieval of cue- and context-induced fear. Two gene-targeted mouse lines were used, one with a global GluA1 knockout, and a ”loss of function“ mutant GluA1 (Q586R). Both lines showed impaired acquisition and reduced cue- and context-induced retrieval 24 h and 48 h after fear conditioning, supporting the hypothesis of involvement of GluA1-containing AMPA receptors in learning of emotional associations. Moreover, in a similar way as for GluA3, GluA1 is also required for the normal decrease of fear to remote events in the passive avoidance test, suggesting that both subunits play an important role in the normal destabilization of older memories such as those that no longer provide an advantage in survival. To study AMPA and NMDA receptor function specifically in the retrieval phase of fear conditioning, I used inducible recombinant adeno-associated virus (rAAV)-mediated gene manipulations in the amygdala. The efforts to generate a BLA-specific promoter for rAAV failed. Therefore a doxycycline-inducible neuron-specific system was used for synaptic silencing of neurons in the amygdala, and to inactivate NMDA and GluA1-containing AMPA receptors in the BLA. Altogether, the analysis of rAAV-injected mice provided strong evidence that retrieval of cued fear memory is dependent on the chronic expression of NMDA receptors in the BLA, whereas the contribution of the GluA1-containing AMPA receptors remains to be confirmed. In the second part of this thesis, I generated and analyzed a new rAAV vector for tissue-specific gene delivery. A new endogenous promoter was cloned from the murine lynx2 gene, a member of the Ly-6/neurotoxin superfamily, for overexpression of genes in dentate gyrus granule cells. This virus can be used to further restrict rAAV-mediated targeting to certain groups of cells.

Rationale : The positive reinforcing effects of alcohol (ethanol) drive its repetitive use and contribute to alcohol use disorder (AUD). Ethanol alters the expression of glutamate AMPA receptor (AMPAR) subunits in reward-related brain regions, but the extent to which this effect regulates ethanol's reinforcing properties is unclear. Objective: This study investigates whether ethanol self-administration changes AMPAR subunit expression and synaptic activity in the nucleus accumbens core (AcbC) to regulate ethanol's reinforcing effects in male C57BL/6J mice. Results: Sucrose-sweetened ethanol self-administration (0.81 g/kg/day) increased AMPAR GluA2 protein expression in the AcbC, without effect on GluA1, compared to sucrose-only controls. Infusion of myristoylated Pep2m in the AcbC, which blocks GluA2 binding to N-ethylmaleimide-sensitive fusion protein (NSF) and reduces GluA2-containing AMPAR activity, reduced ethanol-reinforced responding without affecting sucrose-only self-administration or motor activity. Antagonizing GluA2-lacking AMPARs, through AcbC infusion of NASPM, had no effect on ethanol self-administration. AcbC neurons receiving projections from the basolateral amygdala (BLA) showed increased sEPSC area under the curve (a measurement of charge transfer) and slower decay kinetics in ethanol self-administering mice as compared to sucrose. Optogenetic activation of these neurons revealed an ethanol-enhanced AMPA/NMDA ratio and significantly reduced paired-pulse ratio, suggesting elevated GluA2 contributions specifically within the BLA→AcbC pathway. Conclusions: Ethanol use upregulates GluA2 protein expression in the AcbC and AMPAR synaptic activity in AcbC neurons receiving BLA projections and enhances synaptic plasticity directly within the BLA→AcbC circuit. GluA2-containing AMPAR activity in the AcbC regulates the positive reinforcing effects of ethanol through an NSF-dependent mechanism, highlighting a potential therapeutic target in AUD.

The positive reinforcing effects of alcohol (ethanol) drive repetitive use and contribute to alcohol use disorder (AUD). Ethanol alters the expression of glutamate AMPA receptor (AMPAR) subunits in reward-related brain regions, but the extent to which this effect regulates ethanol's reinforcing properties is unclear. This study investigates whether ethanol self-administration changes AMPAR subunit expression and synaptic activity in the nucleus accumbens core (AcbC) to regulate ethanol's reinforcing effects in male C57BL/6J mice. Sucrose-sweetened ethanol self-administration (0.81g/kg/day) increased AMPAR GluA2 protein expression in the AcbC, without effect on GluA1, compared to sucrose-only controls. Infusion of myristoylated Pep2m in the AcbC, which blocks GluA2 binding to N-ethylmaleimide-sensitive fusion protein (NSF) and reduces GluA2-containing AMPAR activity, reduced ethanol-reinforced responding without affecting sucrose-only self-administration or motor activity. Antagonizing GluA2-lacking AMPARs, through AcbC infusion of NASPM, had no effect on ethanol self-administration. AcbC neurons receiving projections from the basolateral amygdala (BLA) showed increased sEPSC area under the curve (a measurement of charge transfer) and slower decay kinetics in ethanol self-administering mice as compared to sucrose. Optogenetic activation of these neurons revealed an ethanol-enhanced AMPA/NMDA ratio and significantly reduced paired-pulse ratio, suggesting elevated GluA2 contributions specifically within the BLA➔AcbC pathway. Ethanol use upregulates GluA2 protein expression in the AcbC and AMPAR synaptic activity in AcbC neurons receiving BLA projections and enhances synaptic plasticity directly within the BLA➔AcbC circuit. GluA2-containing AMPAR activity in the AcbC regulates the positive reinforcing effects of ethanol through an NSF-dependent mechanism, highlighting a potential therapeutic target in AUD.

Neurobiology of Addiction

Anxiety is integrated in the amygdaloid nuclei and involves the interplay of the amygdala and various other areas of the brain. Neuropeptides play a critical role in regulating this process. Neuropeptide Y (NPY), a 36 aa peptide, is highly expressed in the amygdala. It exerts potent anxiolytic effects through cognate postsynaptic Y1 receptors, but augments anxiety through presynaptic Y2 receptors. To identify the precise anatomical site(s) of Y2-mediated anxiogenic action, we investigated the effect of site-specific deletion of the Y2 gene in amygdaloid nuclei on anxiety and depression-related behaviors in mice. Ablating the Y2 gene in the basolateral and central amygdala resulted in an anxiolytic phenotype, whereas deletion in the medial amygdala or in the bed nucleus of the stria terminalis had no obvious effect on emotion-related behavior. Deleting the Y2 receptor gene in the central amygdala, but not in any other amygdaloid nucleus, resulted in an added antidepressant-like effect. It was associated with a reduction of presumably presynaptic Y2 receptors in the stria terminalis/bed nucleus of the stria terminalis, the nucleus accumbens, and the locus ceruleus. Our results are evidence of the highly site-specific nature of the Y2-mediated function of NPY in the modulation of anxiety- and depression-related behavior. The activity of NPY is likely mediated by the presynaptic inhibition of GABA and/or NPY release from interneurons and/or efferent projection neurons of the basolateral and central amygdala.

In this PhD thesis some molecular mechanisms underlying fear conditioning are addressed by genetic manipulation of a key determinant of synaptic plasticity, namely the AMPA receptor subunit GluR-A. GluR-A is critically involved in longterm potentiation at hippocampal CA3-to-CA1 synapses and is necessary for the formation of spatial working memory. To elucidate whether GluR-A, within the lateral nucleus of the amygdala (LA), is required for the acquisition of fear memories, procedures to generate LA-specific GluR-A depletion, either by generating amygdala-specific transgenic mice, or by employing stereotactic virus delivery, were implemented. First, transgenic mouse lines were generated by expressing enhanced green fluorescent protein (EGFP) under control of the promoter for the Lypdc1 gene, for which in situ hybridization studies showed specific activity in the basolateral amygdala. Unfortunately, in all transgenic lines the Lypdc1-promoter driven EGFP expression was not restricted to the amygdala but was also detected in additional brain regions. Therefore, the Lypdc1-promoter is not useful for manipulating the GluR-A gene specifically in the amygdala. As an alternative strategy, recombinant adeno-associated-Cre virus (rAAVhSyn- Cre/IRESven) was stereotactically delivered into the LA of mice with loxPflanked exon 11 of the Gria1 gene (GluR-A2lox/2lox) prior to fear conditioning. In twothirds of the injected animals, Cre recombinase expression, which was accompanied by loss of GluR-A signal, could be detected in 10-30% of the LAneurons. This level of ablation had been previously shown by others to be sufficient to evoke a phenotype in fear conditioning. As an essential step, different paradigms for fear behavior in wildtype (WT) and global GluR-A knockout (KO) mice were established. The GluR-A KO mice showed a prominent impairment during the acquisition of conditioned fear, demonstrated by the absence of tone-shock induced freezing behavior. Since the sensory systems of GluR-A KO mice were not impaired, this observation suggested that the short-term association of tone and shock is GluR-A dependent. When challenged 24 hours later, the GluR-A KO mice exhibited reduced, although still detectable, memory of the conditioned tone. Thus, it is possible that efficient shortterm association of tone and shock is not necessary for the formation of long-term memory of the aversive stimulus. It seems that GluR-A dependent plasticity mechanisms are operative during the acquisition phase and that GluR-A independent mechanisms can be used for long-term fear memory formation. GluR-A in the LA might be necessary for the immediate tone-shock association during fear acquisition, since the rAAV-Cre mediated LA-specific GluRA KO mice showed a trend to exhibit less freezing during the acquisition phase than uninjected control animals. However, in order to substantiate this finding it would be necessary to optimize virus injection to achieve more efficient Cre targeting within the LA.

Cocaine Experience Enhances Thalamo-Accumbens N-Methyl-D-Aspartate Receptor Function

Peripuberty, comprising childhood and adolescence, constitutes an important time window in the brain development and maturation. Exposure to prolonged stress during this period has been shown to increase the risk for the development of psychopathologies later in life. However, the mechanisms leading to the pathology are still poorly understood. Our laboratory has established a rat model of psychopathology based on unpredictable exposure to different stressors at peripuberty. Previous work on this model has revealed psychopathology-like behaviors in adult rats including pathological aggression and altered sociability. Interestingly, implication of the glucocorticoids, end product of the hypothalamic-pituitary-adrenal (HPA) axis, in the social deficits has been suggested. The glucocorticoids exert their functions through binding to two receptors, the mineralocorticoid (MR) and glucocorticoid receptor (GR). This thesis focused on the GR, as it is the major mediator of the neurobehavioral actions of stress, and its role in the programming and modulation of social behaviors. In the first study, we examined whether blocking glucocorticoid action prior to peripubertal stress (PPS) prevents the subsequent behavioral deficits. We observed that administration of a GR antagonist prior to each stress exposure reversed the alterations in social but not emotional domain at adulthood. Furthermore, the treatment normalized the increased expression of GR in the central amygdala (CeA) found in PPS rats. The CeA is strongly implicated in the regulation of social behaviors. Next, we evaluated the role of GR expression in this brain region in naive rats. We showed that viral downregulation of this receptor had an anxiolytic effect and reduced aggression. We then examined whether administration of the same treatment as described above also reverses PPS-induced behavioral dysfunctions when given at adulthood. Injections of the GR antagonist normalized the pathological aggression observed in adult PPS rats, but not the anxiety or sociability deficits. The third study focused on play fighting, a behavior essential for the development of social skills. We investigated the effects of PPS on play fighting at adolescence and analyzed the expression of genes related to the endocannabinoid, dopamine and opioid systems in the nucleus accumbens (NAc) and basolateral amygdala (BLA). These systems were reported as pivotal modulators of play fighting. We showed that PPS protocol led to enhanced play fighting, together with differential expression of the genes linked to endocannabinoid signaling in the NAc shell. The mitochondrial respiration in the amygdala was also increased in PPS animals following play fighting. Furthermore, play levels predicted some behavioral dysfunctions observed in adult PPS rats. Finally, we studied the role of GR expression in the NAc in the attainment of social rank. We showed that viral downregulation of GR in this nucleus had an anxiolytic effect and enhanced dominance. Rats infused with the virus also exhibited reduced mitochondrial respiration in the NAc. In summary, we have highlighted GR as an important modulator of social behaviors both under normal conditions and after exposure to peripubertal stress. Expression of this receptor in the CeA is tightly linked with aggressive phenotype, whereas in the NAc, GR expression plays a role in social dominance.

The dose-dependent effect of the D2R agonist quinpirole microinjected into the ventral pallidum on information flow in the limbic system

Regional differences in the kinetics and pharmacological inhibition of dopamine uptake were investigated with fast-scan cyclic voltammetry in both the intact rat brain and a brain slice preparation. The regions compared were the basolateral amygdaloid nucleus, caudate-putamen, and nucleus accumbens. The frequency dependence of dopamine efflux evoked in vivo by electrical stimulation of the medial forebrain bundle was evaluated by nonlinear curve fitting with a Michaelis-Menten-based kinetic model. The Km for dopamine uptake was found to be significantly higher in the basolateral amygdala (0.6 microM) than in the other two regions (0.2 microM), whereas the Vmax value for dopamine uptake in the basolateral amygdala was significantly lower (0.49 microM/s vs. 3.8 and 2.4 microM/s in the caudate and accumbens, respectively). Similar kinetics were also obtained in brain slices. Addition of a dopamine uptake inhibitor, cocaine or nomifensine (10 microM), to the perfusion buffer increased the apparent Km value > 25-fold in slices of both the caudate-putamen and nucleus accumbens. In contrast, neither uptake inhibitor had an observable effect in the basolateral amygdaloid nucleus. Thus, dopamine uptake in the rat brain is regionally distinct with regard to rate, affinity, and sensitivity to competitive inhibition.

The development and progression of alcohol use disorder (AUD) are widely viewed as maladaptive neuroplasticity. The transmembrane alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) regulatory protein γ8 (TARP γ-8) is a molecular mechanism of neuroplasticity that has not been evaluated in AUD or other addictions. To address this gap in knowledge, we evaluated the mechanistic role of TARP γ-8 bound AMPAR activity in the basolateral amygdala (BLA) and ventral hippocampus (vHPC) in the positive reinforcing effects of alcohol, which drive repetitive alcohol use throughout the course of AUD, in male C57BL/6J mice. These brain regions were selected because they exhibit high levels of TARP γ-8 expression and send glutamate projections to the nucleus accumbens (NAc), which is a key nucleus in the brain reward pathway. Site-specific pharmacological inhibition of AMPARs bound to TARP γ-8 in the BLA via bilateral infusion of the selective negative modulator JNJ-55511118 (0-2µg/µl/side) significantly decreased operant alcohol self-administration with no effect on sucrose self-administration in behavior-matched controls. Temporal analysis showed that reductions in alcohol-reinforced response rate occurred > 25min after the onset of responding, consistent with a blunting of the positive reinforcing effects of alcohol in the absence of nonspecific behavioral effects. In contrast, inhibition of TARP γ-8 bound AMPARs in the vHPC selectively decreased sucrose self-administration with no effect on alcohol. This study reveals a novel brain region-specific role of TARP γ-8 bound AMPARs as a molecular mechanism of the positive reinforcing effects of alcohol and non-drug rewards.

Pathway-Specific Trafficking of Native AMPARs by In Vivo Experience

Fast excitatory synaptic responses in basolateral amygdala (BLA) neurons are mainly mediated by ionotropic glutamate receptors of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) subtype. AMPA receptors containing an edited GluR2 subunit are calcium impermeable, whereas those that lack this subunit are calcium permeable and also inwardly rectifying. Here, we sought to determine the extent to which synapses in the rat BLA have AMPA receptors with GluR2 subunits. We assessed GluR2 protein expression in the BLA by immunocytochemistry with a GluR2 subunit-specific antiserum at the light and electron microscopic level; for comparison, a parallel examination was carried out in the hippocampus. We also recorded from amygdala brain slices to examine the voltage-dependent properties of AMPA receptor- mediated evoked synaptic currents in BLA principal neurons. At the light microscopic level, GluR2 immunoreactivity was localized to the perikarya and proximal dendrites of BLA neurons; dense labeling was also present over the pyramidal cell layer of hippocampal subfields CA1 and CA3. In electron micrographs from the BLA, most of the synapses were asymmetrical with pronounced postsynaptic densities (PSD). They contained clear, spherical vesicles apposed to the PSD and were predominantly onto spines (86%), indicating that they are mainly with BLA principal neurons. Only 11% of morphological synapses in the BLA were onto postsynaptic elements that showed GluR2 immunoreactivity, in contrast to hippocampal subfields CA1 and CA3 in which 76% and 71% of postsynaptic elements were labeled (p < 0.001). Synaptic staining in the BLA and hippocampus, when it occurred, was exclusively postsynaptic, and particularly heavy over the PSD. In whole-cell voltage clamp recordings, 72% of BLA principal neurons exhibited AMPA receptor-mediated synaptic currents evoked by external capsule stimulation that were inwardly rectifying. Although BLA principal neurons express perikaryal and proximal dendritic GluR2 immunoreactivity, few synapses onto these neurons express GluR2, and a preponderance of principal neurons have inwardly rectifying AMPA-mediated synaptic currents, suggesting that targeting of GluR2 to synapses is restricted. Many BLA synaptic AMPA receptors are likely to be calcium permeable and could play roles in synaptic plasticity, epileptogenesis and excitoxicity.

Neuropeptide Y (NPY) is a 36-amino acid neuropeptide that is widely expressed in the central nervous system and the nucleus accumbens (NAc) is one of the NPY abundant regions as well as arcuate and paraventricular hypothalamic nuclei. NPY in the hypothalamus is well known to enhance food intake. NAc is a hub region of reward and dependence of alcohol and drug intake. We previously suggested that NPY- expressing neurons in the NAc modulate anxiety-behavior using NPY-Cre mice and Cre-dependent AAV. To investigate the projection sites of NAc NPY neurons, we injected AAV(DJ)-FLEX-mCherry into the NAc of NPY-Cre mice. We found mCherry-positive fibers in the lateral hypothalamus (LH), the feeding center. We confirmed this projection to the LH using retrograde tracer. Many NPY nerve fibers are also observed in the NAc. To investigate the origin (s) of NPY fibers in the NAc, we injected AAV(rg)-FLEX- mCherry, permits retrograde access and expression of mCherry to projection neurons, into the NAc in NPY-Cre mice. We found the existence of mCherry-positive cell bodies in the basolateral amygdala (BLA) as well as NAc. Next, we investigated the role of accumbal NPY in the palatable food (high fat diet) intake. We found NPY and NPY Y1 agonist treatment in the NAc induced increase in high fat diet. Then we investigated the involvement of NPY neurons in the BLA projecting to the NAc in high fat diet consumption. Mice stably consume HFD in 1-h during light cycle. To perform projection-specific activation or inactivation of NPY neurons in the BLA, we employed a two-virus strategy, injecting a AAV(rg)-FLEX-Flippase (FLPo) into the NAc and AAV expressing FLPo-dependent hM3Dq-mCherry or - hM4Di-mCherry into the BLA of NPY-Cre mice. CNO-induced activation of BLA NPY neurons projecting to the NAc did not affect to the 1-h HFD intake in the model. On the other hand, CNO-induced inactivation of BLA NPY neurons projecting to the NAc significantly decreased 1-h HFD consumption. These results indicate that NPY neurons in the NAc and those in the BLA that project to the NAc are involved in palatable food consumption in mice.

SummaryMonoamine neurotransmitters are released by specialized neurons regulating behavioral, motor, and cognitive functions. Although the localization of monoaminergic neurons in the brain is well known, the distribution and kinetics of monoamines remain unclear. Here, we generated a murine brain atlas of serotonin (5-HT), dopamine (DA), and norepinephrine (NE) levels using mass spectrometry imaging (MSI). We found several nuclei rich in both 5-HT and a catecholamine (DA or NE) and identified the paraventricular nucleus of the thalamus (PVT), where 5-HT and NE are co-localized. The analysis of 5-HT fluctuations in response to acute tryptophan depletion and infusion of isotope-labeled tryptophan in vivo revealed a close kinetic association between the raphe nuclei, PVT, and amygdala but not the other nuclei. Our findings imply the existence of a highly dynamic 5-HT-mediated raphe to PVT pathway that likely plays a role in the brain monoamine system.