Sex differences in pre- and post-synaptic glutamate signaling in the nucleus accumbens core.

BACKGROUND: Glutamate signaling within the nucleus accumbens underlies motivated behavior and is involved in psychiatric disease. Although behavioral sex differences in these processes are well-established, the neural mechanisms driving these differences are largely unexplored. In these studies, we examine potential sex differences in synaptic plasticity and excitatory transmission within the nucleus accumbens core. Further understanding of baseline sex differences in reward circuitry will shed light on potential mechanisms driving behavioral differences in motivated behavior and psychiatric disease. METHODS: Behaviorally naïve adult male and female Long-Evans rats, C57Bl/6J mice, and constitutive PKMζ knockout mice were killed and tissue containing the nucleus accumbens core was collected for ex vivo slice electrophysiology experiments. Electrophysiology recordings examined baseline sex differences in synaptic plasticity and transmission within this region and the potential role of PKMζ in long-term depression. RESULTS: Within the nucleus accumbens core, both female mice and rats exhibit higher AMPA/NMDA ratios compared to male animals. Further, female mice have a larger readily releasable pool of glutamate and lower release probability compared to male mice. No significant sex differences were detected in spontaneous excitatory postsynaptic current amplitude or frequency. Finally, the threshold for induction of long-term depression was lower for male animals than females, an effect that appears to be mediated, in part, by PKMζ. CONCLUSIONS: We conclude that there are baseline sex differences in synaptic plasticity and excitatory transmission in the nucleus accumbens core. Our data suggest there are sex differences at multiple levels in this region that should be considered in the development of pharmacotherapies to treat psychiatric illnesses such as depression and substance use disorder.

Understanding normal neural signaling within the nucleus accumbens, a key brain region involved in psychiatric disease including substance use disorder and depression, could provide insight into treatment options for these disorders. Although we know the behaviors regulated by the nucleus accumbens can differ between males and females, we do not understand the underlying differences in brain processing that could contribute to these behavioral differences. Further, even in cases when these behaviors are not different, the underlying brain signaling may exhibit sex-specific mechanisms. The current studies examined excitatory signaling with the nucleus accumbens in both rats and mice at the level of both individual cells and circuits. We found that female rodents (rats and mice) exhibit higher levels of excitatory signaling within the nucleus accumbens than male rodents. Further, procedures that can dampen neural transmission in males are not sufficient to do so in females, suggesting that excitatory signaling in the nucleus accumbens of females is less plastic. Finally, our last set of studies utilized mice missing the protein, PKMζ, and demonstrated that this reversed some of the sex differences seen in normal mice, pointing to a critical role for this protein in maintaining these differences. Our data suggest there are sex differences at multiple levels in this region that should be considered in the development of pharmacotherapies to treat psychiatric illnesses such as depression and substance use disorder.

Sex differences in the medial prefrontal cortical glutamate system.

BACKGROUND: Dysregulation in the prefrontal cortex underlies a variety of psychiatric illnesses, including substance use disorder, depression, and anxiety. Despite the established sex differences in prevalence and presentation of these illnesses, the neural mechanisms driving these differences are largely unexplored. Here, we investigate potential sex differences in glutamatergic transmission within the medial prefrontal cortex (mPFC). The goal of these experiments was to determine if there are baseline sex differences in transmission within this region that may underlie sex differences in diseases that involve dysregulation in the prefrontal cortex. METHODS: Adult male and female C57Bl/6J mice were used for all experiments. Mice were killed and bilateral tissue samples were taken from the medial prefrontal cortex for western blotting. Both synaptosomal and total GluA1 and GluA2 levels were measured. In a second set of experiments, mice were killed and ex vivo slice electrophysiology was performed on prepared tissue from the medial prefrontal cortex. Spontaneous excitatory postsynaptic currents and rectification indices were measured. RESULTS: Females exhibit higher levels of synaptosomal GluA1 and GluA2 in the mPFC compared to males. Despite similar trends, no statistically significant differences are seen in total levels of GluA1 and GluA2. Females also exhibit both a higher amplitude and higher frequency of spontaneous excitatory postsynaptic currents and greater inward rectification in the mPFC compared to males. CONCLUSIONS: Overall, we conclude that there are sex differences in glutamatergic transmission in the mPFC. Our data suggest that females have higher levels of glutamatergic transmission in this region. This provides evidence that the development of sex-specific pharmacotherapies for various psychiatric diseases is important to create more effective treatments.

Adolescent social isolation induced alterations in nucleus accumbens glutamate signalling.

Exposure to adversity during early childhood and adolescence increases an individual's vulnerability to developing substance use disorder. Despite the knowledge of this vulnerability, the mechanisms underlying it are still poorly understood. Excitatory afferents to the nucleus accumbens (NAc) mediate responses to both stressful and rewarding stimuli. Understanding how adolescent social isolation alters these afferents could inform the development of targeted interventions both before and after drug use. Here, we used social isolation rearing as a model of early life adversity which we have previously demonstrated increases vulnerability to cocaine addiction-like behaviour. The current study examined the effect of social isolation rearing on presynaptic glutamatergic transmission in NAc medium spiny neurons in both male and female mice. We show that social isolation rearing alters presynaptic plasticity in the NAc by decreasing the paired-pulse ratio and the size of the readily releasable pool of glutamate. Optogenetically activating the glutamatergic input from the ventral hippocampus to the NAc is sufficient to recapitulate the decreases in paired-pulse ratio and readily releasable pool size seen following electrical stimulation of all NAc afferents. Further, optogenetically inhibiting the ventral hippocampal afferent during electrical stimulation eliminates the effect of early life adversity on the paired-pulse ratio or readily releasable pool size. In summary, we demonstrate that social isolation rearing leads to alterations in glutamate transmission driven by projections from the ventral hippocampus. These data suggest that targeting the circuit from the ventral hippocampus to the nucleus accumbens could provide a means to reverse stress-induced plasticity.

Early life adversity promotes resilience to opioid addiction-related phenotypes in male rats and sex-specific transcriptional changes.

Experiencing some early life adversity can have an "inoculating" effect that promotes resilience in adulthood. However, the mechanisms underlying stress inoculation are unknown, and animal models are lacking. Here we used the limited bedding and nesting (LBN) model of adversity to evaluate stress inoculation of addiction-related phenotypes. In LBN, pups from postnatal days 2 to 9 and their dams were exposed to a low-resource environment. In adulthood, they were tested for addiction-like phenotypes and compared to rats raised in standard housing conditions. High levels of impulsivity are associated with substance abuse, but in males, LBN reduced impulsive choice compared to controls. LBN males also self-administered less morphine and had a lower breakpoint on a progressive ratio reinforcement schedule than controls. These effects of LBN on addiction-related behaviors were not found in females. Because the nucleus accumbens (NAc) mediates these behaviors, we tested whether LBN altered NAc physiology in drug-naïve and morphine-exposed rats. LBN reduced the frequency of spontaneous excitatory postsynaptic currents in males, but a similar effect was not observed in females. Only in males did LBN prevent a morphine-induced increase in the AMPA/NMDA ratio. RNA sequencing was performed to delineate the molecular signature in the NAc associated with LBN-derived phenotypes. LBN produced sex-specific changes in transcription, including in genes related to glutamate transmission. Collectively, these studies reveal that LBN causes a male-specific stress inoculation effect against addiction-related phenotypes. Identifying factors that promote resilience to addiction may reveal novel treatment options for patients.

Sliced Human Cortical Organoids for Modeling Distinct Cortical Layer Formation.

Human brain organoids provide unique platforms for modeling development and diseases by recapitulating the architecture of the embryonic brain. However, current organoid methods are limited by interior hypoxia and cell death due to insufficient surface diffusion, preventing generation of architecture resembling late developmental stages. Here, we report the sliced neocortical organoid (SNO) system, which bypasses the diffusion limit to prevent cell death over long-term cultures. This method leads to sustained neurogenesis and formation of an expanded cortical plate that establishes distinct upper and deep cortical layers for neurons and astrocytes, resembling the third trimester embryonic human neocortex. Using the SNO system, we further identify a critical role of WNT/ß-catenin signaling in regulating human cortical neuron subtype fate specification, which is disrupted by a psychiatric-disorder-associated genetic mutation in patient induced pluripotent stem cell (iPSC)-derived SNOs. These results demonstrate the utility of SNOs for investigating previously inaccessible human-specific, late-stage cortical development and disease-relevant mechanisms.

Reversing Cocaine-Induced Plasticity with Zeta Inhibitory Peptide.

Cocaine-induced plasticity persists during abstinence and is thought to underlie cue-evoked craving. Reversing this plasticity could provide an opportunity for therapeutic intervention. Converging evidence suggest that zeta inhibitory peptide (ZIP) eliminates memories for experience-dependent behaviors, including conditioned drug associations. However, the effect of ZIP on reward seeking and drug-induced plasticity is unknown. The current study examined the effect of ZIP administration in the nucleus accumbens on reinstatement (RI) of cocaine seeking, a rodent model of relapse. We demonstrate that intra-accumbal ZIP administration blocks cocaine-primed RI in rats when administered 24 h or 1 week before testing. These effects of ZIP on drug seeking are specific, as we did not see any effect of ZIP on RI of sucrose seeking. ZIP is a synthetic compound designed to inhibit the atypical PKC, PKMζ, a protein implicated in learning and memory. However, recent evidence from PKMζ-knock-out (KO) mice suggests that ZIP may function through alternative mechanisms. In support of this, we found that ZIP was able to block cue-induced RI in PKMζ-KO mice. One possible mechanism underlying addictive phenotypes is the ability of cocaine to block further plasticity. We hypothesized that ZIP may be working to reverse this anaplasticity. Although ZIP has no effect on accumbal LTD in slices from naive or yoked saline mice, it is able to restore both NMDA-dependent and mGluR5-dependent LTD in animals after cocaine self-administration and withdrawal. These findings demonstrate that intra-accumbal ZIP persistently reverses cocaine-induced behavioral and synaptic plasticity in male and female rodents.SIGNIFICANCE STATEMENT Zeta-inhibitory peptide (ZIP) has been shown to disrupt memory maintenance for experience-dependent behaviors. We examined the effect of ZIP infused into the nucleus accumbens on the reinstatement (RI) of cocaine seeking. We found that intra-accumbal ZIP blocked RI of cocaine seeking 24 h and 1 week later. This effect was specific to RI of cocaine seeking as ZIP did not disrupt RI of food seeking. In conjunction with these behavioral studies we examined the ability of ZIP to reverse cocaine-induced deficits in LTD. We found that ZIP was able to rescue two forms of LTD in cocaine-experienced mice. These studies demonstrate that ZIP is able to reverse cocaine-induced behavioral and synaptic plasticity in a persistent manner.

Glutamate receptor interacting protein acts within the prefrontal cortex to blunt cocaine seeking.

Glutamate receptor interacting protein (GRIP) is a neuronal scaffolding protein that anchors GluA2-containing AMPA receptors to the cell membrane. GRIP plays a critical role in activity-dependent synaptic plasticity, including that which occurs after drug exposure. Given that cocaine administration alters glutamate receptor trafficking within the prefrontal cortex (PFC), a better understanding of the role of receptor trafficking proteins could lead to a more complete understanding of addictive phenotypes. AMPA receptor trafficking in general, and GRIP specifically, is known to play a role in cocaine seeking and conditioned reward in the nucleus accumbens, but its role in the PFC has not been characterized. The current study demonstrates that conditional deletion of GRIP1 in the medial prefrontal cortex increases the motivation for cocaine and potentiates cue-induced reinstatement of cocaine seeking in male and female mice. As no effects of PFC GRIP1 deletion were seen in reinstatement of food seeking, strategy set-shifting, or reversal learning the effects on cocaine seeking are not related to generalized alterations in cognitive function. While disrupting GRIP1 might be expected to lead to decreased AMPA transmission, our electrophysiological data indicate an increase in sEPSC amplitude in the prefrontal cortex and a corresponding decrease in paired pulse facilitation in the nucleus accumbens. Taken together this suggests a strengthening of the PFC to NAc input following prefrontal GRIP1 deletion that may mediate the enhanced drug seeking behavior.

Sphingosine 1-phosphate lyase ablation disrupts presynaptic architecture and function via an ubiquitin- proteasome mediated mechanism.

The bioactive lipid sphingosine 1-phosphate (S1P) is a degradation product of sphingolipids that are particularly abundant in neurons. We have shown previously that neuronal S1P accumulation is toxic leading to ER-stress and an increase in intracellular calcium. To clarify the neuronal function of S1P, we generated brain-specific knockout mouse models in which S1P-lyase (SPL), the enzyme responsible for irreversible S1P cleavage was inactivated. Constitutive ablation of SPL in the brain (SPLfl/fl/Nes) but not postnatal neuronal forebrain-restricted SPL deletion (SPLfl/fl/CaMK) caused marked accumulation of S1P. Hence, altered presynaptic architecture including a significant decrease in number and density of synaptic vesicles, decreased expression of several presynaptic proteins, and impaired synaptic short term plasticity were observed in hippocampal neurons from SPLfl/fl/Nes mice. Accordingly, these mice displayed cognitive deficits. At the molecular level, an activation of the ubiquitin-proteasome system (UPS) was detected which resulted in a decreased expression of the deubiquitinating enzyme USP14 and several presynaptic proteins. Upon inhibition of proteasomal activity, USP14 levels, expression of presynaptic proteins and synaptic function were restored. These findings identify S1P metabolism as a novel player in modulating synaptic architecture and plasticity.

Disrupting GluA2 phosphorylation potentiates reinstatement of cocaine seeking.

Addiction is associated with changes in synaptic plasticity mediated, in part, by alterations in the trafficking and stabilization of AMPA receptors at synapses within the nucleus accumbens. Exposure to cocaine can lead to protein kinase C-mediated phosphorylation of GluA2 AMPA subunits and this phosphorylation event leads to the internalization of GluA2-containing AMPARs, which are calcium-impermeable. However, it is not clear whether this internalization is necessary for the expression of addictive phenotypes. Utilizing a mouse with a point mutation within the GluA2 subunit c-terminus, the current study demonstrates that disrupting PKC-mediated GluA2 phosphorylation potentiates reinstatement of both cue-induced cocaine seeking and cocaine conditioned reward without affecting operant learning, food self-administration or cocaine sensitization. Electrophysiological recordings revealed increased GluA2-mediated AMPA transmission as evidenced by increased sEPSC amplitude without any changes in sEPSC frequency or rectification. In support of this increase in GluA2 activity mediating the augmented cocaine reinstatement, we found that accumbal overexpression of GluA2 recapitulated this behavioral effect in wildtype mice while not altering reinstatement behavior in the GluA2 K882A knock-in mice. In addition, disrupting GluA2 phosphorylation was associated with blunted long-term depression in the nucleus accumbens, mimicking the anaplasticity seen following cocaine self-administration. Taken together these results indicate that disrupting GluA2 phosphorylation and increasing GluA2-mediated transmission in the nucleus accumbens leads to increased vulnerability to cocaine relapse. Further, these results indicate that modulating GluA2-containing AMPAR trafficking can contribute to addictive phenotypes in the absence of alterations in GluA2-lacking receptors. These results highlight the GluA2 phosphorylation site as a novel target for the development of cocaine addiction therapeutics.