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Alterations in cannabinoid CB1 receptor (CB1R) are implicated in various psychiatric disorders. CB1R participates in both depolarization induced suppression of inhibition (DSI) and depolarization induced suppression of excitation (DSE), suggesting its involvement in regulating excitatory and inhibitory (E/I) balance. Prior studies examining neuronal cell type specific CB1R distribution have been conducted near exclusively within rodents. Identification of these distribution patterns within the human and non-human primate cortex is essential to increase our insight into its function. Using co-labeling immunohistochemistry and fluorescent microscopy, we examined CB1R protein levels within excitatory and inhibitory boutons of male human and non-human primate prefrontal cortex and auditory cortices, regions involved in the behavioral effects of exogenous cannabinoid exposures. We found that CB1R was present in both bouton populations within all brain regions examined in both species. Significantly higher CB1R levels were found within inhibitory than within excitatory boutons across all regions in both species, although the cell type by brain region interactions differed between the two species. Our results support the importance of conducting more in-depth CB1R examinations to understand how cell type and brain region dependent differences contribute to regional E/I balance regulation, and how aberrations in CB1R distribution may contribute to pathology.

Working memory requires efficient excitatory drive to parvalbumin-positive (PV) interneurons in the primate dorsolateral prefrontal cortex (DLPFC). Developmental pruning eliminates superfluous excitatory inputs, suggesting that working memory maturation during adolescence requires pruning of excitatory inputs to PV interneurons. Therefore, we tested the hypothesis that excitatory synapses on PV interneurons are pruned during adolescence. The density of excitatory synapses, defined by overlapping vesicular glutamate transporter 1-positive (VGlut1+) and postsynaptic density 95-positive (PSD95+) puncta, on PV interneurons was lower in postpubertal relative to prepubertal monkeys. In contrast, puncta levels of VGlut1 and PSD95 proteins were higher in postpubertal monkeys and positively predicted activity-dependent PV levels, suggesting a greater strength of the remaining synapses after pruning. Because excitatory synapse number on PV interneurons is regulated by erb-b2 receptor tyrosine kinase 4 (ErbB4), whose function is influenced by alternative splicing, we tested the hypothesis that pruning of excitatory synapses on PV interneurons is associated with developmental shifts in ErbB4 expression and/or splicing. Pan-ErbB4 expression did not change, whereas the minor-to-major splice variant ratios increased with age. In cell culture, the major, but not the minor, variant increased excitatory synapse number on PV interneurons and displayed greater kinase activity than the minor variant, suggesting that the effect of ErbB4 signaling in PV interneurons is mediated by alternative splicing. Supporting this interpretation, in monkey DLPFC, higher minor-to-major variant ratios predicted lower PSD95+ puncta density on PV interneurons. Together, our findings suggest that ErbB4 splicing may regulate the pruning of excitatory synapses on PV interneurons during adolescence.

Recent discoveries of extreme cellular diversity in the brain warrant rapid development of technologies to access specific cell populations within heterogeneous tissue. Available approaches for engineering-targeted technologies for new neuron subtypes are low yield, involving intensive transgenic strain or virus screening. Here, we present Specific Nuclear-Anchored Independent Labeling (SNAIL), an improved virus-based strategy for cell labeling and nuclear isolation from heterogeneous tissue. SNAIL works by leveraging machine learning and other computational approaches to identify DNA sequence features that confer cell type-specific gene activation and then make a probe that drives an affinity purification-compatible reporter gene. As a proof of concept, we designed and validated two novel SNAIL probes that target parvalbumin-expressing (PV+) neurons. Nuclear isolation using SNAIL in wild-type mice is sufficient to capture characteristic open chromatin features of PV+ neurons in the cortex, striatum, and external globus pallidus. The SNAIL framework also has high utility for multispecies cell probe engineering; expression from a mouse PV+ SNAIL enhancer sequence was enriched in PV+ neurons of the macaque cortex. Expansion of this technology has broad applications in cell type-specific observation, manipulation, and therapeutics across species and disease models.

In human prefrontal cortex (PFC), ~85% of γ-aminobutyric acid (GABA)-expressing neurons can be subdivided into non-overlapping groups by the presence of calbindin (CB), calretinin (CR) or parvalbumin (PV). Substantial research has focused on the differences in the laminar locations of the cells bodies of these neurons, with limited attention to the distribution of their axon terminals, their sites of action. We previously reported that in non-human primates subtypes of these cells are distinguishable by differences in terminal protein levels of the GABA synthesizing enzymes glutamic acid decarboxylase 65 (GAD65) and GAD67. Here we used multi-label fluorescence microscopy in human PFC to assess: (1) the laminar distributions of axon terminals containing CB, CR, or PV; and (2) the relative protein levels of GAD65, GAD67 and vesicular GABA transporter (vGAT) in CB, CR and PV terminals. The densities of the different CB, CR and PV terminal subpopulations differed across layers of the PFC. PV terminals comprised two subsets based on the presence of only GAD67 (GAD67+) or both GADs (GAD65/GAD67+), whereas CB and CR terminals comprised three subsets (GAD65+, GAD67+, or GAD65/GAD67+). The densities of the different CB, CR and PV GAD terminal subpopulations also differed across layers. Finally, within each of the three calcium-binding protein subpopulations intra-terminal protein levels of GAD and vGAT differed by GAD subpopulation. These findings are discussed in the context of the laminar distributions of CB, CR and PV cell bodies and the synaptic targets of their axons.

Alterations in cortical parvalbumin (PV)-containing neurons, including a reduced density of detectable neurons and lower PV levels, have frequently been reported in the dorsolateral prefrontal cortex (DLPFC) of schizophrenia subjects. Most PV neurons are surrounded by perineuronal nets (PNNs) and the density of PNNs, as detected by Wisteria floribunda agglutinin (WFA) labeling, has been reported to be lower in schizophrenia. However, the nature of these PNN alterations, and their relationship to disease-related changes in PV neurons, has not been assessed. Using confocal microscopy, we quantified the densities and fluorescence intensities of PV neurons and PNNs labeled with WFA or immunoreactive for the major PNN protein, aggrecan, in the DLPFC from schizophrenia and matched comparison subjects. In schizophrenia, the densities of PV cells and of PNNs were not altered; however, the fluorescence intensities of PV immunoreactivity in cell bodies and of WFA labeling and aggrecan immunoreactivity in individual PNNs around PV cells were lower. These findings indicate that the normal complements of PV cells and PNNs are preserved in schizophrenia, but the levels of PV protein and of individual PNN components, especially the carbohydrate moieties on proteoglycans to which WFA binds, are lower. Given the roles of PV neurons in regulating DLPFC microcircuits and of PNNs in regulating PV cellular physiology, the identified alterations in PV neurons and their PNNs could contribute to DLPFC dysfunction in schizophrenia.

Deficient gamma oscillations in prefrontal cortex (PFC) of individuals with schizophrenia appear to involve impaired inhibitory drive from parvalbumin-expressing interneurons (PVIs). Inhibitory drive from PVIs is regulated, in part, by RNA binding fox-1 homolog 1 (Rbfox1). Rbfox1 is spliced into nuclear or cytoplasmic isoforms, which regulate alternative splicing or stability of their target transcripts, respectively. One major target of cytoplasmic Rbfox1 is vesicle associated membrane protein 1 (Vamp1). Vamp1 mediates GABA release probability from PVIs, and the loss of Rbfox1 reduces Vamp1 levels which in turn impairs cortical inhibition. In this study, we investigated if the Rbfox1-Vamp1 pathway is altered in PVIs in PFC of individuals with schizophrenia by utilizing a novel strategy that combines multi-label in situ hybridization and immunohistochemistry. In the PFC of 20 matched pairs of schizophrenia and comparison subjects, cytoplasmic Rbfox1 protein levels were significantly lower in PVIs in schizophrenia and this deficit was not attributable to potential methodological confounds or schizophrenia-associated co-occurring factors. In a subset of this cohort, Vamp1 mRNA levels in PVIs were also significantly lower in schizophrenia and were predicted by lower cytoplasmic Rbfox1 protein levels across individual PVIs. To investigate the functional impact of Rbfox1-Vamp1 alterations in schizophrenia, we simulated the effect of lower GABA release probability from PVIs on gamma power in a computational model network of pyramidal neurons and PVIs. Our simulations showed that lower GABA release probability reduces gamma power by disrupting network synchrony while minimally affecting network activity. Finally, lower GABA release probability synergistically interacted with lower strength of inhibition from PVIs in schizophrenia to reduce gamma power non-linearly. Together, our findings suggest that the Rbfox1-Vamp1 pathway in PVIs is impaired in schizophrenia and that this alteration likely contributes to deficient PFC gamma power in the illness.

Previously, we demonstrated that dendritic spine density (DSD) in deep layer 3 of the primary auditory cortex (A1) is lower, due to having fewer small spines, in subjects with schizophrenia (SZ) than non-psychiatric control (NPC) subjects. We also previously demonstrated that microtubule-associated-protein-2 immunoreactivity (MAP2-IR) in A1 deep layer 3 is lower, and positively correlated with DSD, in SZ subjects. Here, we first sought to confirm these findings in an independent cohort of 25 SZ-NPC subject pairs (cohort 1). We used immunohistochemistry and confocal microscopy to measure DSD and MAP2-IR in A1 deep layer 3. Consistent with previous studies, both DSD and MAP2-IR were lower in SZ subjects. We then tested the hypothesis that MAP2-IR mediates the effect of SZ on DSD in a cohort of 45 SZ-NPC subject pairs (combined cohort) that included all subjects from cohort 1 and two previously studied cohorts. Based on the distribution of MAP2-IR values in NPC subjects, we categorized each SZ subject as having either low MAP2-IR (SZ MAP2-IR(low)) or normal MAP2-IR (SZ MAP2-IR(normal)). Among SZ MAP-IR(low) subjects, mean DSD was significantly lower than in NPC subjects. However, mean DSD did not differ between SZ MAP2-IR(normal) and NPC subjects. Moreover, MAP2-IR statistically mediated small spine differences, with lower MAP2-IR values associated with fewer small spines. Our findings confirm that low density of small spines and low MAP2-IR are robust SZ phenotypes and suggest that MAP2-IR mediates the effect of SZ on DSD.

Converging lines of evidence indicate that schizophrenia is characterized by impairments of synaptic machinery within cerebral cortical circuits. Efforts to localize these alterations in brain tissue from subjects with schizophrenia have frequently been limited to the quantification of structures that are non-selectively identified (e.g., dendritic spines labeled in Golgi preparations, axon boutons labeled with synaptophysin), or to quantification of proteins using methods unable to resolve relevant cellular compartments. Multiple label fluorescence confocal microscopy represents a means to circumvent many of these limitations, by concurrently extracting information regarding the number, morphology, and relative protein content of synaptic structures. An important adaptation required for studies of human disease is coupling this approach to stereologic methods for systematic random sampling of relevant brain regions. In this review article we consider the application of multiple label fluorescence confocal microscopy to the mapping of synaptic alterations in subjects with schizophrenia and describe the application of a novel, readily automated, iterative intensity/morphological segmentation algorithm for the extraction of information regarding synaptic structure number, size, and relative protein level from tissue sections obtained using unbiased stereological principles of sampling. In this context, we provide examples of the examination of pre- and post-synaptic structures within excitatory and inhibitory circuits of the cerebral cortex.

Age is the greatest risk factor for Parkinson's disease (PD) which causes progressive loss of dopamine (DA) neurons, with males at greater risk than females. Intriguingly, some DA neurons are more resilient to degeneration than others. Increasing evidence suggests that vesicular glutamate transporter (VGLUT) expression in DA neurons plays a role in this selective vulnerability. We investigated the role of DA neuron VGLUT in sex‐ and age‐related differences in DA neuron vulnerability using the genetically tractable Drosophila model. We found sex differences in age‐related DA neurodegeneration and its associated locomotor behavior, where males exhibit significantly greater decreases in both DA neuron number and locomotion during aging compared with females. We discovered that dynamic changes in DA neuron VGLUT expression mediate these age‐ and sex‐related differences, as a potential compensatory mechanism for diminished DA neurotransmission during aging. Importantly, female Drosophila possess higher levels of VGLUT expression in DA neurons compared with males, and this finding is conserved across flies, rodents, and humans. Moreover, we showed that diminishing VGLUT expression in DA neurons eliminates females' greater resilience to DA neuron loss across aging. This offers a new mechanism for sex differences in selective DA neuron vulnerability to age‐related DA neurodegeneration. Finally, in mice, we showed that the ability of DA neurons to achieve optimal control over VGLUT expression is essential for DA neuron survival. These findings lay the groundwork for the manipulation of DA neuron VGLUT expression as a novel therapeutic strategy to boost DA neuron resilience to age‐ and PD‐related neurodegeneration. Dopamine neuron VGLUT mediates age‐ and sex‐related differences in dopamine neurodegeneration during aging. Female Drosophila, rats and humans express more VGLUT in dopamine neurons than males, and VGLUT knockdown in dopamine neurons diminishes sex differences in vulnerability to age‐related degeneration. The ability to finely tune dopamine neuron VGLUT expression boosts resilience while either too little or too much VGLUT leaves dopamine neurons more vulnerable to neurodegeneration.