Proteomic evidence of depression-associated astrocytic dysfunction in the human male olfactory bulb.

The olfactory bulb (OB), a major structure of the limbic system, has been understudied in human investigations of psychopathologies such as depression. To explore more directly the molecular features of the OB in depression, a global comparative proteome analysis was carried out with human post-mortem OB samples from 11 males having suffered from depression and 12 healthy controls. We identified 188 differentially abundant proteins (with adjusted p < 0.05) between depressed cases and controls. Gene ontology and gene enrichment analyses suggested that these proteins are involved in biological processes including the complement and coagulation cascades. Cell type enrichment analysis displayed a significant reduction in several canonical astrocytic proteins in OBs from depressed patients. Furthermore, using RNA-fluorescence in-situ hybridization, we observed a decrease in the percentage of ALDH1L1+ cells expressing canonical astrocytic markers including ALDOC, NFIA, GJA1 (connexin 43) and SLC1A3 (EAAT1). These results are consistent with previous reports of downregulated astrocytic marker expression in other brain regions in depressed patients. We also conducted a comparative phosphoproteomic analysis of OB samples and found a dysregulation of proteins involved in neuronal and astrocytic functions. To determine whether OB astrocytic abnormalities is specific to humans, we also performed proteomics on the OB of socially defeated male mice, a commonly used model of depression. Cell-type specific analysis revealed that in socially defeated animals, the most striking OB protein alterations were associated with oligodendrocyte-lineage cells rather than with astrocytes, highlighting an important species difference. Overall, this study further highlights cerebral astrocytic abnormalities as a consistent feature of depression in humans.

Chondroitin sulfate glycan sulfation patterns influence histochemical labeling of perineuronal nets: a comparative study of interregional distribution in human and mouse brain.

Perineuronal nets (PNNs) are a condensed subtype of extracellular matrix that form a net-like coverings around certain neurons in the brain. PNNs are primarily composed of chondroitin sulfate (CS) proteoglycans from the lectican family that consist of CS-glycosaminoglycan (CS-GAG) side chains attached to a core protein. CS disaccharides can exist in various isoforms with different sulfation patterns. Literature suggests that CS disaccharide sulfation patterns can influence the function of PNNs as well as their labeling. This study was conducted to characterize such interregional CS disaccharide sulfation pattern differences in adult human (N = 81) and mouse (N = 19) brains. Liquid chromatography tandem mass spectrometry was used to quantify five different CS disaccharide sulfation patterns, which were then compared to immunolabeling of PNNs using Wisteria Floribunda Lectin (WFL) to identify CS-GAGs and anti-aggrecan to identify CS proteoglycans. In healthy brains, significant regional and species-specific differences in CS disaccharide sulfation and single versus double-labeling pattern were identified. A secondary analysis to investigate how early-life stress (ELS) impacts these PNN features discovered that although ELS increases WFL+ PNN density, the CS-GAG sulfation code and single versus double PNN-labeling distributions remained unaffected in both species. These results underscore PNN complexity in traditional research, emphasizing the need to consider their heterogeneity in future experiments.

Chondroitin sulfate glycan sulfation patterns influence histochemical labeling of perineuronal nets: a comparative study of interregional distribution in human and mouse brain.

Perineuronal nets (PNNs) are a condensed subtype of extracellular matrix that form a net-like coverings around certain neurons in the brain. PNNs are primarily composed of chondroitin sulfate (CS) proteoglycans from the lectican family that consist of CS-glycosaminoglycan side chains attached to a core protein. CS disaccharides can exist in various isoforms with different sulfation patterns. Literature suggests that CS disaccharide sulfation patterns can influence the function of PNNs as well as their labeling. This study was conducted to characterize such interregional CS disaccharide sulfation pattern differences in adult human (n = 81) and mouse (n = 19) brains. Liquid chromatography tandem mass spectrometry was used to quantify five different CS disaccharide sulfation patterns, which were then compared to immunolabeling of PNNs using Wisteria Floribunda Lectin (WFL) to identify CS-glycosaminoglycans and anti-aggrecan to identify CS proteoglycans. In healthy brains, significant regional and species-specific differences in CS disaccharide sulfation and single versus double-labeling pattern were identified. A secondary analysis to investigate how early-life stress impacts these PNN features discovered that although early-life stress increases WFL+ PNN density, the CS-glycosaminoglycan sulfation code and single versus double PNN-labeling distributions remained unaffected in both species. These results underscore PNN complexity in traditional research, emphasizing the need to consider their heterogeneity in future experiments.

Striatal projection neurons coexpressing dopamine D1 and D2 receptors modulate the motor function of D1- and D2-SPNs.

The role of the striatum in motor control is commonly assumed to be mediated by the two striatal efferent pathways characterized by striatal projection neurons (SPNs) expressing dopamine (DA) D1 receptors or D2 receptors (D1-SPNs and D2-SPNs, respectively), without regard to SPNs coexpressing both receptors (D1/D2-SPNs). Here we developed an approach to target these hybrid SPNs in mice and demonstrate that, although these SPNs are less abundant, they have a major role in guiding the motor function of the other two populations. D1/D2-SPNs project exclusively to the external globus pallidus and have specific electrophysiological features with distinctive integration of DA signals. Gain- and loss-of-function experiments indicate that D1/D2-SPNs potentiate the prokinetic and antikinetic functions of D1-SPNs and D2-SPNs, respectively, and restrain the integrated motor response to psychostimulants. Overall, our findings demonstrate the essential role of this population of D1/D2-coexpressing neurons in orchestrating the fine-tuning of DA regulation in thalamo-cortico-striatal loops.

Nucleus accumbens D1- and D2-expressing neurons control the balance between feeding and activity-mediated energy expenditure.

Accumulating evidence points to dysregulations of the Nucleus Accumbens (NAc) in eating disorders (ED), however its precise contribution to ED symptomatic dimensions remains unclear. Using chemogenetic manipulations in male mice, we found that activity of dopamine D1 receptor-expressing neurons of the NAc core subregion facilitated effort for a food reward as well as voluntary exercise, but decreased food intake, while D2-expressing neurons have opposite effects. These effects are congruent with D2-neurons being more active than D1-neurons during feeding while it is the opposite during running. Chronic manipulations of each subpopulations had limited effects on energy balance. However, repeated activation of D1-neurons combined with inhibition of D2-neurons biased behavior toward activity-related energy expenditure, whilst the opposite manipulations favored energy intake. Strikingly, concomitant activation of D1-neurons and inhibition of D2-neurons precipitated weight loss in anorexia models. These results suggest that dysregulations of NAc dopaminoceptive neurons might be at the core of EDs.

The neurons that drive infradian sleep-wake and mania-like behavioral rhythms.

Infradian mood and sleep-wake rhythms with periods of 48 hr and beyond have been observed in bipolar disorder (BD) subjects that even persist in time isolation, indicating an endogenous origin. Here we show that mice exposed to methamphetamine (Meth) in drinking water develop infradian locomotor rhythms with periods of 48 hr and beyond which extend to sleep length and mania-like behaviors in support of a model for cycling in BD. This cycling capacity is abrogated upon genetic disruption of DA production in DA neurons of the ventral tegmental area (VTA) or ablation of nucleus accumbens (NAc) projecting, dopamine (DA) neurons. Chemogenetic activation of NAc-projecting DA neurons leads to locomotor period lengthening in clock deficient mice, while cytosolic calcium in DA processes of the NAc was found fluctuating synchronously with locomotor behavior. Together, our findings argue that BD cycling relies on infradian rhythm generation that depends on NAc-projecting DA neurons.

Persistent extrasynaptic hyperdopaminergia in the mouse hippocampus induces plasticity and recognition memory deficits reversed by the atypical antipsychotic sulpiride.

Evidence suggests that subcortical hyperdopaminergia alters cognitive function in schizophrenia and antipsychotic drugs (APD) fail at rescuing cognitive deficits in patients. In a previous study, we showed that blocking D2 dopamine receptors (D2R), a core action of APD, led to profound reshaping of mesohippocampal fibers, deficits in synaptic transmission and impairments in learning and memory in the mouse hippocampus (HP). However, it is currently unknown how excessive dopamine affects HP-related cognitive functions, and how APD would impact HP functions in such a state. After verifying the presence of DAT-positive neuronal projections in the ventral (temporal), but not in the dorsal (septal), part of the HP, GBR12935, a blocker of dopamine transporter (DAT), was infused in the CA1 of adult C57Bl/6 mice to produce local hyperdopaminergia. Chronic GBR12935 infusion in temporal CA1 induced a mild learning impairment in the Morris Water Maze and abolished long-term recognition memory in novel-object (NORT) and object-place recognition tasks (OPRT). Deficits were accompanied by a significant decrease in DAT+ mesohippocampal fibers. Intrahippocampal or systemic treatment with sulpiride during GBR infusions improved the NORT deficit but not that of OPRT. In vitro application of GBR on hippocampal slices abolished long-term depression (LTD) of fEPSP in temporal CA1. LTD was rescued by co-application with sulpiride. In conclusion, chronic DAT blockade in temporal CA1 profoundly altered mesohippocampal modulation of hippocampal functions. Contrary to previous observations in normodopaminergic mice, antagonising D2Rs was beneficial for cognitive functions in the context of hippocampal hyperdopaminergia.

Disruption of Astrocyte-Dependent Dopamine Control in the Developing Medial Prefrontal Cortex Leads to Excessive Grooming in Mice.

BACKGROUND: Astrocytes control synaptic activity by modulating perisynaptic concentrations of ions and neurotransmitters including dopamine (DA) and, as such, could be involved in the modulating aspects of mammalian behavior. METHODS: We produced a conditional deletion of the vesicular monoamine transporter 2 (VMAT2) specifically in astrocytes (aVMTA2cKO mice) and studied the effects of the lack of VMAT2 in prefrontal cortex (PFC) astrocytes on the regulation of DA levels, PFC circuit functions, and behavioral processes. RESULTS: We found a significant reduction of medial PFC (mPFC) DA levels and excessive grooming and compulsive repetitive behaviors in aVMAT2cKO mice. The mice also developed a synaptic pathology, expressed through increased relative AMPA versus NMDA receptor currents in synapses of the dorsal striatum receiving inputs from the mPFC. Importantly, behavioral and synaptic phenotypes were rescued by re-expression of mPFC VMAT2 and L-DOPA treatment, showing that the deficits were driven by mPFC astrocytes that are critically involved in developmental DA homeostasis. By analyzing human tissue samples, we found that VMAT2 is expressed in human PFC astrocytes, corroborating the potential translational relevance of our observations in mice. CONCLUSIONS: Our study shows that impairment of the astrocytic control of DA in the mPFC leads to symptoms resembling obsessive-compulsive spectrum disorders such as trichotillomania and has a profound impact on circuit function and behaviors.

Behavioral and Transcriptomic Changes Following Brain-Specific Loss of Noradrenergic Transmission.

Noradrenaline (NE) plays an integral role in shaping behavioral outcomes including anxiety/depression, fear, learning and memory, attention and shifting behavior, sleep-wake state, pain, and addiction. However, it is unclear whether dysregulation of NE release is a cause or a consequence of maladaptive orientations of these behaviors, many of which associated with psychiatric disorders. To address this question, we used a unique genetic model in which the brain-specific vesicular monoamine transporter-2 (VMAT2) gene expression was removed in NE-positive neurons disabling NE release in the entire brain. We engineered VMAT2 gene splicing and NE depletion by crossing floxed VMAT2 mice with mice expressing the Cre-recombinase under the dopamine ß-hydroxylase (DBH) gene promotor. In this study, we performed a comprehensive behavioral and transcriptomic characterization of the VMAT2DBHcre KO mice to evaluate the role of central NE in behavioral modulations. We demonstrated that NE depletion induces anxiolytic and antidepressant-like effects, improves contextual fear memory, alters shifting behavior, decreases the locomotor response to amphetamine, and induces deeper sleep during the non-rapid eye movement (NREM) phase. In contrast, NE depletion did not affect spatial learning and memory, working memory, response to cocaine, and the architecture of the sleep-wake cycle. Finally, we used this model to identify genes that could be up- or down-regulated in the absence of NE release. We found an up-regulation of the synaptic vesicle glycoprotein 2c (SV2c) gene expression in several brain regions, including the locus coeruleus (LC), and were able to validate this up-regulation as a marker of vulnerability to chronic social defeat. The NE system is a complex and challenging system involved in many behavioral orientations given it brain wide distribution. In our study, we unraveled specific role of NE neurotransmission in multiple behavior and link it to molecular underpinning, opening future direction to understand NE role in health and disease.

The Orphan GPCR Receptor, GPR88, Interacts with Nuclear Protein Partners in the Cerebral Cortex.

GPR88 is an orphan G-protein-coupled receptor (GPCR) highly expressed in striatal medium spiny neurons (MSN), also found in cortical neurons at low level. In MSN, GPR88 has a canonical GPCR plasma membrane/cytoplasmic expression, whereas in cortical neurons, we previously reported an atypical intranuclear localization. Molecular size analysis suggests that GPR88, expressed in plasma membrane of MSN or in nuclear compartment of cortical neurons, corresponds to the full-length protein. By transfection of cortical neurons, we showed that GPR88 fluorescent chimeras exhibit a nuclear localization. This localization is contingent on the third intracytoplasmic loop and C-terminus domains, even though these domains do not contain any known nuclear localization signals (NLS). Using yeast two-hybrid screening with these domains, we identified the nuclear proteins ATRX, TOP2B, and BAZ2B, all involved in chromatin remodeling, as potential protein partners of GPR88. We also validated the interaction of GPR88 with these nuclear proteins by proximity ligation assay on cortical neurons in culture and coimmunoprecipitation experiments on cortical extracts from GPR88 wild-type (WT) and knockout (KO) mice. The identification of GPR88 subcellular partners may provide novel functional insights for nonclassical modes of GPCR action that could be relevant in the maturating process of neocortical neurons.

Mice with dopaminergic neuron-specific deletion of DTNBP-1 gene show blunted nucleus accumbens dopamine release and associated behaviors.

Reduced expression of a schizophrenia-associated gene Dystrobrevin Binding Protein 1 (DTNBP1) and its protein product dysbindin-1, has been reported in the brains of schizophrenia patients. DTNBP1-null mutant Sdy (Sandy) mice exhibit several behavioral features relevant to schizophrenia. Changes in dopaminergic as well as glutamatergic and GABAergic neurotransmission in cortico-limbic regions have been reported in Sdy mice. Since dysbindin-1 is expressed in multiple brain regions, it is not known whether dopamine (DA) changes observed in Sdy null mutants are due to dysbindin-1 deficiency in DAergic neurons specifically. Here, using a mouse line with conditional knockout (cKO) of DTNBP1 in DA neurons, we studied the effects of dysbindin-1 deficiency on DA release and DA-dependent behaviors. Spontaneous locomotor activity of cKO mice in novel environment was significantly reduced initially but was comparable at later time points with littermate controls. However, the locomotion-enhancing effect of a low dose of d-amphetamine (d-AMPH; 2.5 mg/kg, ip) was significantly attenuated in the cKO mice suggesting a dampened mesolimbic DA transmission. Similarly, the prepulse inhibition disrupting effect of d-AMPH was found to be significantly reduced in the mutant mice. No significant differences between the cKO and control mice were observed in tests of anxiety, spatial learning and memory and social interaction. In- vivo microdialysis in the nucleus accumbens (NAc) showed a decrease in d-AMPH-induced extracellular DA release in the cKO mice. No significant alterations in protein levels of DA transporter, phosphorylated CaM kinase-II or Akt308 in the NAc were observed in the cKO mice. Taken together, our data suggest an important role of dysbindin-1 in maintaining mesolimbic DA tone and call for further investigations identifying mechanisms linking dysbindin-1, DA and schizophrenia.

Regulation of GluA1 phosphorylation by d-amphetamine and methylphenidate in the cerebellum.

Prescription stimulants, such as d-amphetamine or methylphenidate are used to treat suffering from attention-deficit hyperactivity disorder (ADHD). They potently release dopamine (DA) and norepinephrine (NE) and cause phosphorylation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluA1 in the striatum. Whether other brain regions are also affected remains elusive. Here, we demonstrate that d-amphetamine and methylphenidate increase phosphorylation at Ser845 (pS845-GluA1) in the membrane fraction of mouse cerebellum homogenate. We identify Bergmann glial cells as the source of pS845-GluA1 and demonstrate a requirement for intact NE release. Consequently, d-amphetamine-induced pS845-GluA1 was prevented by ß1-adenoreceptor antagonist, whereas the blockade of DA D1 receptor had no effect. Together, these results indicate that NE regulates GluA1 phosphorylation in Bergmann glial cells in response to prescription stimulants.

GPR56/ADGRG1 is associated with response to antidepressant treatment.

It remains unclear why many patients with depression do not respond to antidepressant treatment. In three cohorts of individuals with depression and treated with serotonin-norepinephrine reuptake inhibitor (N = 424) we show that responders, but not non-responders, display an increase of GPR56 mRNA in the blood. In a small group of subjects we also show that GPR56 is downregulated in the PFC of individuals with depression that died by suicide. In mice, we show that chronic stress-induced Gpr56 downregulation in the blood and prefrontal cortex (PFC), which is accompanied by depression-like behavior, and can be reversed by antidepressant treatment. Gpr56 knockdown in mouse PFC is associated with depressive-like behaviors, executive dysfunction and poor response to antidepressant treatment. GPR56 peptide agonists have antidepressant-like effects and upregulated AKT/GSK3/EIF4 pathways. Our findings uncover a potential role of GPR56 in antidepressant response.

LSP5-2157 a new inhibitor of vesicular glutamate transporters.

Vesicular glutamate transporters (VGLUT1-3) mediate the uptake of glutamate into synaptic vesicles. VGLUTs are pivotal actors of excitatory transmission and of almost all brain functions. Their implication in various pathologies has been clearly documented. Despite their functional importance, the pharmacology of VGLUTs is limited to a few dyes such as Trypan Blue, Rose Bengal or Brilliant Yellow type. Here, we report the design and evaluation of new potent analogs based on Trypan Blue scaffold. Our best compound, named LSP5-2157, has an EC50 of 50 nM on glutamate vesicular uptake. Using a 3D homology model of VGLUT1 and docking experiments, we determined its putative binding subdomains within vesicular glutamate transporters and validated the structural requirement for VGLUT inhibition. To better estimate the specificity and potency of LSP5-2157, we also investigated its ability to block glutamatergic transmission in autaptic hippocampal cells. Neither glutamate receptors nor GABAergic transmission or transmission machinery were affected by LSP5-2157. Low doses of compound reversibly reduce glutamatergic neurotransmission in hippocampal autpases. LSP5-2157 had a low and depressing effect on synaptic efficacy in hippocampal slice. Furthermore, LSP5-2157 had no effect on NMDA-R- mediated fEPSP but reduce synaptic plasticity induced by 3 trains of 100 Hz. Finally, LSP5-2157 had the capacity to inhibit VGLUT3-dependent auditory synaptic transmission in the guinea pig cochlea. In this model, it abolished the compound action potential of auditory nerve at high concentration showing the limited permeation of LSP5-2157 in an in-vivo model. In summary, the new ligand LSP5-2157, has a high affinity and specificity for VGLUTs and shows some permeability in isolated neuron, tissue preparations or in vivo in the auditory system. These findings broaden the field of VGLUTs inhibitors and open the way to their use to assess glutamatergic functions in vitro and in vivo.

Dysfunction of homeostatic control of dopamine by astrocytes in the developing prefrontal cortex leads to cognitive impairments.

Astrocytes orchestrate neural development by powerfully coordinating synapse formation and function and, as such, may be critically involved in the pathogenesis of neurodevelopmental abnormalities and cognitive deficits commonly observed in psychiatric disorders. Here, we report the identification of a subset of cortical astrocytes that are competent for regulating dopamine (DA) homeostasis during postnatal development of the prefrontal cortex (PFC), allowing for optimal DA-mediated maturation of excitatory circuits. Such control of DA homeostasis occurs through the coordinated activity of astroglial vesicular monoamine transporter 2 (VMAT2) together with organic cation transporter 3 and monoamine oxidase type B, two key proteins for DA uptake and metabolism. Conditional deletion of VMAT2 in astrocytes postnatally produces loss of PFC DA homeostasis, leading to defective synaptic transmission and plasticity as well as impaired executive functions. Our findings show a novel role for PFC astrocytes in the DA modulation of cognitive performances with relevance to psychiatric disorders.

Antidepressant efficacy of a selective organic cation transporter blocker in a mouse model of depression.

Current antidepressants act principally by blocking monoamine reuptake by high-affinity transporters in the brain. However, these antidepressants show important shortcomings such as slow action onset and limited efficacy in nearly a third of patients with major depression disorder. Here, we report the development of a prodrug targeting organic cation transporters (OCT), atypical monoamine transporters recently implicated in the regulation of mood. Using molecular modeling, we designed a selective OCT2 blocker, which was modified to increase brain penetration. This compound, H2-cyanome, was tested in a rodent model of chronic depression induced by 7-week corticosterone exposure. In male mice, prolonged administration of H2-cyanome induced positive effects on several behaviors mimicking symptoms of depression, including anhedonia, anxiety, social withdrawal, and memory impairment. Importantly, in this validated model, H2-cyanome compared favorably with the classical antidepressant fluoxetine, with a faster action on anhedonia and better anxiolytic effects. Integrated Z-scoring across these depression-like variables revealed a lower depression score for mice treated with H2-cyanome than for mice treated with fluoxetine for 3 weeks. Repeated H2-cyanome administration increased ventral tegmental area dopaminergic neuron firing, which may underlie its rapid action on anhedonia. H2-cyanome, like fluoxetine, also modulated several intracellular signaling pathways previously involved in antidepressant response. Our findings provide proof-of-concept of antidepressant efficacy of an OCT blocker, and a mechanistic framework for the development of new classes of antidepressants and therapeutic alternatives for resistant depression and other psychiatric disturbances such as anxiety.

Early reduced dopaminergic tone mediated by D3 receptor and dopamine transporter in absence epileptogenesis.

OBJECTIVE: In Genetic Absence Epilepsy Rats From Strasbourg (GAERSs), epileptogenesis takes place during brain maturation and correlates with increased mRNA expression of D3 dopamine receptors (D3R). Whether these alterations are the consequence of seizure repetition or contribute to the development of epilepsy remains to be clarified. Here, we addressed the involvement of the dopaminergic system in epilepsy onset in GAERSs. METHODS: Experiments were performed using rats at different stages of brain maturation from three strains according to their increasing propensity to develop absence seizures: nonepileptic control rats (NECs), Wistar Hannover rats, and GAERSs. Changes in dopaminergic neurotransmission were investigated using different behavioral and neurochemical approaches: autoradiography of D3R and dopamine transporter, single photon emission computed tomographic imaging, acute and chronic drug effects on seizure recordings (dopaminergic agonists and antagonists), quinpirole-induced yawns and dopamine synaptosomal uptake, microdialysis, brain tissue monoamines, and brain-derived neurotrophic factor quantification. RESULTS: Autoradiography revealed an increased expression of D3R in 14-day-old GAERSs, before absence seizure onset, that persists in adulthood, as compared to age-matched NECs. This was confirmed by increased yawns, a marker of D3R activity, and increased seizures when animals were injected with quinpirole at low doses to activate D3R. We also observed a concomitant increase in the expression and activity of the dopamine transporter in GAERSs before seizure onset, consistent with both lowered dopamine basal level and increased phasic responses. SIGNIFICANCE: Our data show that the dopaminergic system is persistently altered in GAERSs, which may contribute not only to behavioral comorbidities but also as an etiopathogenic factor in the development of epilepsy. The data suggest that an imbalanced dopaminergic tone may contribute to absence epilepsy development and seizure onset, as its reversion by a chronic treatment with a dopamine stabilizer significantly suppressed epileptogenesis. Our data suggest a potential new target for antiepileptic therapies and/or improvement of quality of life of epileptic patients.

Increased vulnerability of nigral dopamine neurons after expansion of their axonal arborization size through D2 dopamine receptor conditional knockout.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). Rare genetic mutations in genes such as Parkin, Pink1, DJ-1, α-synuclein, LRRK2 and GBA are found to be responsible for the disease in about 15% of the cases. A key unanswered question in PD pathophysiology is why would these mutations, impacting basic cellular processes such as mitochondrial function and neurotransmission, lead to selective degeneration of SNc DA neurons? We previously showed in vitro that SNc DA neurons have an extremely high rate of mitochondrial oxidative phosphorylation and ATP production, characteristics that appear to be the result of their highly complex axonal arborization. To test the hypothesis in vivo that axon arborization size is a key determinant of vulnerability, we selectively labeled SNc or VTA DA neurons using floxed YFP viral injections in DAT-cre mice and showed that SNc DA neurons have a much more arborized axon than those of the VTA. To further enhance this difference, which may represent a limiting factor in the basal vulnerability of these neurons, we selectively deleted in mice the DA D2 receptor (D2-cKO), a key negative regulator of the axonal arbour of DA neurons. In these mice, SNc DA neurons have a 2-fold larger axonal arborization, release less DA and are more vulnerable to a 6-OHDA lesion, but not to α-synuclein overexpression when compared to control SNc DA neurons. This work adds to the accumulating evidence that the axonal arborization size of SNc DA neurons plays a key role in their vulnerability in the context of PD.

The NeuroD6 Subtype of VTA Neurons Contributes to Psychostimulant Sensitization and Behavioral Reinforcement.

Reward-related behavior is complex and its dysfunction correlated with neuropsychiatric illness. Dopamine (DA) neurons of the ventral tegmental area (VTA) have long been associated with different aspects of reward function, but it remains to be disentangled how distinct VTA DA neurons contribute to the full range of behaviors ascribed to the VTA. Here, a recently identified subtype of VTA neurons molecularly defined by NeuroD6 (NEX1M) was addressed. Among all VTA DA neurons, less than 15% were identified as positive for NeuroD6. In addition to dopaminergic markers, sparse NeuroD6 neurons expressed the vesicular glutamate transporter 2 (Vglut2) gene. To achieve manipulation of NeuroD6 VTA neurons, NeuroD6(NEX)-Cre-driven mouse genetics and optogenetics were implemented. First, expression of vesicular monoamine transporter 2 (VMAT2) was ablated to disrupt dopaminergic function in NeuroD6 VTA neurons. Comparing Vmat2lox/lox;NEX-Cre conditional knock-out (cKO) mice with littermate controls, it was evident that baseline locomotion, preference for sugar and ethanol, and place preference upon amphetamine-induced and cocaine-induced conditioning were similar between genotypes. However, locomotion upon repeated psychostimulant administration was significantly elevated above control levels in cKO mice. Second, optogenetic activation of NEX-Cre VTA neurons was shown to induce DA release and glutamatergic postsynaptic currents within the nucleus accumbens. Third, optogenetic stimulation of NEX-Cre VTA neurons in vivo induced significant place preference behavior, while stimulation of VTA neurons defined by Calretinin failed to cause a similar response. The results show that NeuroD6 VTA neurons exert distinct regulation over specific aspects of reward-related behavior, findings that contribute to the current understanding of VTA neurocircuitry.

Role of D3 dopamine receptors in modulating neuroanatomical changes in response to antipsychotic administration.

Clinical research has shown that chronic antipsychotic drug (APD) treatment further decreases cortical gray matter and hippocampus volume, and increases striatal and ventricular volume in patients with schizophrenia. D2-like receptor blockade is necessary for clinical efficacy of the drugs, and may be responsible for inducing these volume changes. However, the role of other D2-like receptors, such as D3, remains unclear. Following our previous work, we undertook a longitudinal study to examine the effects of chronic (9-week) typical (haloperidol (HAL)) and atypical (clozapine (CLZ)) APDs on the neuroanatomy of wild-type (WT) and dopamine D3-knockout (D3KO) mice using magnetic resonance imaging (MRI) and histological assessments in a sub-region of the anterior cingulate cortex (the prelimbic [PL] area) and striatum. D3KO mice had larger striatal volume prior to APD administration, coupled with increased glial and neuronal cell density. Chronic HAL administration increased striatal volume in both WT and D3KO mice, and reduced PL area volume in D3KO mice both at trend level. CLZ increased volume of the PL area of WT mice at trend level, but decreased D3KO PL area glial cell density. Both typical and atypical APD administration induced neuroanatomical remodeling of regions rich in D3 receptor expression, and typically altered in schizophrenia. Our findings provide novel insights on the role of D3 receptors in structural changes observed following APD administration in clinical populations.