Aging disrupts the coordination between mRNA and protein expression in mouse and human midbrain.

Age-related dopamine (DA) neuron loss is a primary feature of Parkinson's disease. However, it remains unclear whether similar biological processes occur during healthy aging, albeit to a lesser degree. We therefore determined whether midbrain DA neurons degenerate during aging in mice and humans. In mice, we identified no changes in midbrain neuron numbers throughout aging. Despite this, we found age-related decreases in midbrain mRNA expression of tyrosine hydroxylase (Th), the rate limiting enzyme of DA synthesis. Among midbrain glutamatergic cells, we similarly identified age-related declines in vesicular glutamate transporter 2 (Vglut2) mRNA expression. In co-transmitting Th +/Vglut2 + neurons, Th and Vglut2 transcripts decreased with aging. Importantly, striatal Th and Vglut2 protein expression remained unchanged. In translating our findings to humans, we found no midbrain neurodegeneration during aging and identified age-related decreases in TH and VGLUT2 mRNA expression similar to mouse. Unlike mice, we discovered diminished density of striatal TH+ dopaminergic terminals in aged human subjects. However, TH and VGLUT2 protein expression were unchanged in the remaining striatal boutons. Finally, in contrast to Th and Vglut2 mRNA, expression of most ribosomal genes in Th + neurons was either maintained or even upregulated during aging. This suggests a homeostatic mechanism where age-related declines in transcriptional efficiency are overcome by ongoing ribosomal translation. Overall, we demonstrate species-conserved transcriptional effects of aging in midbrain dopaminergic and glutamatergic neurons that are not accompanied by marked cell death or lower striatal protein expression. This opens the door to novel therapeutic approaches to maintain neurotransmission and bolster neuronal resilience.

Synaptic alterations in pyramidal cells following genetic manipulation of neuronal excitability in monkey prefrontal cortex.

In schizophrenia, layer 3 pyramidal neurons (L3PNs) in the dorsolateral prefrontal cortex (DLPFC) are thought to receive fewer excitatory synaptic inputs and to have lower expression levels of activity-dependent genes and of genes involved in mitochondrial energy production. In concert, these findings from previous studies suggest that DLPFC L3PNs are hypoactive in schizophrenia, disrupting the patterns of activity that are crucial for working memory, which is impaired in the illness. However, whether lower PN activity produces alterations in inhibitory and/or excitatory synaptic strength has not been tested in the primate DLPFC. Here, we decreased PN excitability in rhesus monkey DLPFC in vivo using adeno-associated viral vectors (AAVs) to produce Cre recombinase-mediated overexpression of Kir2.1 channels, a genetic silencing tool that efficiently decreases neuronal excitability. In acute slices prepared from DLPFC 7-12 weeks post-AAV microinjections, Kir2.1-overexpressing PNs had a significantly reduced excitability largely attributable to highly specific effects of the AAV-encoded Kir2.1 channels. Moreover, recordings of synaptic currents showed that Kir2.1-overexpressing DLPFC PNs had reduced strength of excitatory synapses whereas inhibitory synaptic inputs were not affected. The decrease in excitatory synaptic strength was not associated with changes in dendritic spine number, suggesting that excitatory synapse quantity was unaltered in Kir2.1-overexpressing DLPFC PNs. These findings suggest that, in schizophrenia, the excitatory synapses on hypoactive L3PNs are weaker and thus might represent a substrate for novel therapeutic interventions. Significance Statement: In schizophrenia, dorsolateral prefrontal cortex (DLPFC) pyramidal neurons (PNs) have both transcriptional and structural alterations that suggest they are hypoactive. PN hypoactivity is thought to produce synaptic alterations in schizophrenia, however the effects of lower neuronal activity on synaptic function in primate DLPFC have not been examined. Here, we used, for the first time in primate neocortex, adeno-associated viral vectors (AAVs) to reduce PN excitability with Kir2.1 channel overexpression and tested if this manipulation altered the strength of synaptic inputs onto the Kir2.1-overexpressing PNs. Recordings in DLPFC slices showed that Kir2.1 overexpression depressed excitatory (but not inhibitory), synaptic currents, suggesting that, in schizophrenia, the hypoactivity of PNs might be exacerbated by reduced strength of the excitatory synapses they receive.

Long-range inhibitory neurons mediate cortical neurovascular coupling.

To meet the high energy demands of brain function, cerebral blood flow (CBF) parallels changes in neuronal activity by a mechanism known as neurovascular coupling (NVC). However, which neurons play a role in mediating NVC is not well understood. Here, we identify in mice and humans a specific population of cortical GABAergic neurons that co-express neuronal nitric oxide synthase and tachykinin receptor 1 (Tacr1). Through whole-tissue clearing, we demonstrate that Tacr1 neurons extend local and long-range projections across functionally connected cortical areas. We show that whisker stimulation elicited Tacr1 neuron activity in the barrel cortex through feedforward excitatory pathways. Additionally, through optogenetic experiments, we demonstrate that Tacr1 neurons are instrumental in mediating CBF through the relaxation of mural cells in a similar fashion to whisker stimulation. Finally, by electron microscopy, we observe that Tacr1 processes contact astrocytic endfeet. These findings suggest that Tacr1 neurons integrate cortical activity to mediate NVC.

Altered Rbfox1-Vamp1 pathway and prefrontal cortical 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.

Scaling of smaller pyramidal neuron size and lower energy production in schizophrenia.

BACKGROUND: Dorsolateral prefrontal cortex (DLPFC) dysfunction in schizophrenia appears to reflect alterations in layer 3 pyramidal neurons (L3PNs), including smaller cell bodies and lower expression of mitochondrial energy production genes. However, prior somal size studies used biased strategies for identifying L3PNs, and somal size and levels of energy production markers have not been assessed in individual L3PNs. STUDY DESIGN: We combined fluorescent in situ hybridization (FISH) of vesicular glutamate transporter 1 (VGLUT1) mRNA and immunohistochemical-labeling of NeuN to determine if the cytoplasmic distribution of VGLUT1 mRNA permits the unbiased identification and somal size quantification of L3PNs. Dual-label FISH for VGLUT1 mRNA and cytochrome C oxidase subunit 4I1 (COX4I1) mRNA, a marker of energy production, was used to assess somal size and COX4I1 transcript levels in individual DLPFC L3PNs from schizophrenia (12 males; 2 females) and unaffected comparison (13 males; 1 female) subjects. STUDY RESULTS: Measures of L3PN somal size with NeuN immunohistochemistry or VGLUT1 mRNA provided nearly identical results (ICC = 0.96, p < 0.0001). Mean somal size of VGLUT1-identified L3PNs was 8.7% smaller (p = 0.004) and mean COX4I1 mRNA levels per L3PN were 16.7% lower (p = 0.01) in schizophrenia. These measures were correlated across individual L3PNs in both subject groups (rrm = 0.81-0.86). CONCLUSIONS: This preliminary study presents a novel method for combining unbiased neuronal identification with quantitative assessments of somal size and mRNA levels. We replicated findings of smaller somal size and lower COX4I1 mRNA levels in DLPFC L3PNs in schizophrenia. The normal scaling of COX4I1 mRNA levels with somal size in schizophrenia suggests that lower markers of energy production are secondary to L3PN morphological alterations in the illness.

Lifetime evaluation of left ventricular structure and function in male ApoE null mice after gamma and space-type radiation exposure.

The space radiation (IR) environment contains high charge and energy (HZE) nuclei emitted from galactic cosmic rays with the ability to overcome current shielding strategies, posing increased IR-induced cardiovascular disease risks for astronauts on prolonged space missions. Little is known about the effect of 5-ion simplified galactic cosmic ray simulation (simGCRsim) exposure on left ventricular (LV) function. Three-month-old, age-matched male Apolipoprotein E (ApoE) null mice were irradiated with 137Cs gamma (γ; 100, 200, and 400 cGy) and simGCRsim (50, 100, 150 cGy all at 500 MeV/nucleon (n)). LV function was assessed using transthoracic echocardiography at early/acute (14 and 28 days) and late/degenerative (365, 440, and 660 days) times post-irradiation. As early as 14 and 28-days post IR, LV systolic function was reduced in both IR groups across all doses. At 14 days post-IR, 150 cGy simGCRsim-IR mice had decreased diastolic wall strain (DWS), suggesting increased myocardial stiffness. This was also observed later in 100 cGy γ-IR mice at 28 days. At later stages, a significant decrease in LV systolic function was observed in the 400 cGy γ-IR mice. Otherwise, there was no difference in the LV systolic function or structure at the remaining time points across the IR groups. We evaluated the expression of genes involved in hemodynamic stress, cardiac remodeling, inflammation, and calcium handling in LVs harvested 28 days post-IR. At 28 days post-IR, there is increased expression of Bnp and Ncx in both IR groups at the lowest doses, suggesting impaired function contributes to hemodynamic stress and altered calcium handling. The expression of Gals3 and ß-Mhc were increased in simGCRsim and γ-IR mice respectively, suggesting there may be IR-specific cardiac remodeling. IR groups were modeled to calculate the Relative Biological Effectiveness (RBE) and Radiation Effects Ratio (RER). No lower threshold was determined using the observed dose-response curves. These findings do not exclude the possibility of the existence of a lower IR threshold or the presence of IR-induced cardiovascular disease (CVD) when combined with additional space travel stressors, e.g., microgravity.

Sexually dimorphic mechanisms of VGLUT-mediated protection from dopaminergic neurodegeneration.

Parkinson's disease (PD) targets some dopamine (DA) neurons more than others. Sex differences offer insights, with females more protected from DA neurodegeneration. The mammalian vesicular glutamate transporter VGLUT2 and Drosophila ortholog dVGLUT have been implicated as modulators of DA neuron resilience. However, the mechanisms by which VGLUT2/dVGLUT protects DA neurons remain unknown. We discovered DA neuron dVGLUT knockdown increased mitochondrial reactive oxygen species in a sexually dimorphic manner in response to depolarization or paraquat-induced stress, males being especially affected. DA neuron dVGLUT also reduced ATP biosynthetic burden during depolarization. RNA sequencing of VGLUT+ DA neurons in mice and flies identified candidate genes that we functionally screened to further dissect VGLUT-mediated DA neuron resilience across PD models. We discovered transcription factors modulating dVGLUT-dependent DA neuroprotection and identified dj-1ß as a regulator of sex-specific DA neuron dVGLUT expression. Overall, VGLUT protects DA neurons from PD-associated degeneration by maintaining mitochondrial health.

Diagnostic Specificity and Association With Cognition of Molecular Alterations in Prefrontal Somatostatin Neurons in Schizophrenia.

Importance: Individuals with schizophrenia (SZ) exhibit pronounced deficits in somatostatin (SST) messenger RNA (mRNA) levels in the dorsolateral prefrontal cortex (DLPFC). Molecularly distinct subtypes of SST neurons, located in the superficial and deep zones of the DLPFC, are thought to contribute to different functional processes of this region; understanding the specificity of SST alterations in SZ across these zones could inform the functional consequences of those alterations, including cognitive impairments characteristic of SZ. Objective: To quantify mRNA levels of SST and related neuropeptides in the DLPFC in individuals with SZ, bipolar disorder (BPD), or major depressive disorder (MDD) and unaffected comparison individuals. Design, Setting, and Participants: This case-control study, conducted from January 20, 2020, to March 30, 2022, used postmortem brain tissue specimens previously obtained from individuals with SZ, MDD, or BPD and unaffected individuals from a community population through 2 medical examiners' offices. Demographic, clinical, and educational information was ascertained through psychological autopsies. Exposures: Diagnosis of SZ, BPD, or MDD. Main Outcome and Measures: The main outcome was levels of SST and related neuropeptide mRNA in 2 DLPFC zones, examined using laser microdissection and quantitative polymerase chain reaction or fluorescent in situ hybridization (FISH). Findings were compared using educational attainment as a proxy measure of premorbid cognition. Results: A total of 200 postmortem brain specimens were studied, including 65 from unaffected comparison individuals (42 [65%] male; mean [SD] age, 49.2 [14.1] years); 54 from individuals with SZ (37 [69%] male; mean [SD] age, 47.5 [13.3] years); 42 from individuals with MDD (24 [57%] male; mean [SD] age, 45.6 [12.1] years); and 39 from individuals with BPD (23 [59%] male; mean (SD) age, 46.2 [12.5] years). Compared with unaffected individuals, levels of SST mRNA were lower in both superficial (Cohen d, 0.68; 95% CI, 0.23-1.13; P = .004) and deep (Cohen d, 0.60; 95% CI, 0.16-1.04; P = .02) DLPFC zones in individuals with SZ; findings were confirmed using FISH. Levels of SST were lower only in the superficial zone in the group with MDD (Cohen d, 0.58; 95% CI, 0.14-1.02; P = .12), but the difference was not significant; SST levels were not lower in either zone in the BPD group. Levels of neuropeptide Y and tachykinin 1 showed similar patterns. Neuropeptide alterations in the superficial, but not deep, zone were associated with lower educational attainment only in the group with SZ (superficial: adjusted odds ratio, 1.71 [95% CI, 1.11-2.69]; P = .02; deep: adjusted odds ratio, 1.08 [95% CI, 0.64-1.84]; P = .77). Conclusions and Relevance: The findings revealed diagnosis-specific patterns of molecular alterations in SST neurons in the DLPFC, suggesting that distinct disease processes are reflected in the differential vulnerability of SST neurons in individuals with SZ, MDD, and BPD. In SZ, alterations specifically in the superficial zone may be associated with cognitive dysfunction.

Terminal type-specific cannabinoid CB1 receptor alterations in patients with schizophrenia: A pilot study.

BACKGROUND: Individuals with schizophrenia are at elevated genetic risks for comorbid cannabis use, and often experience exacerbations of cognitive and psychotic symptoms when exposed to cannabis. These findings have led a number of investigators to examine cannabinoid CB1 receptor (CB1R) alterations in schizophrenia, though with conflicting results. We recently demonstrated the presence of CB1R in both excitatory and inhibitory boutons in the human prefrontal cortex, with differential levels of the receptor between bouton types. We hypothesized that the differential enrichment of CB1R between bouton types - a factor previously unaccounted for when examining CB1R changes in schizophrenia - may resolve prior discrepant reports and increase our insight into the effects of CB1R alterations on the pathophysiology of schizophrenia. METHODS: Using co-labeling immunohistochemistry and fluorescent microscopy, we examined total CB1R levels and CB1R levels within excitatory (vGlut1-positive) and inhibitory (vGAT-positive) boutons of prefrontal cortex samples from ten pairs of individuals (nine male pairs and one female pair) diagnosed with schizophrenia and non-psychiatric comparisons. RESULTS: Significantly higher total CB1R levels were found within samples from individuals with schizophrenia. Terminal type-specific analyses identified significantly higher CB1R levels within excitatory boutons in samples from individuals with schizophrenia relative to comparisons. In contrast, CB1R levels within the subset of inhibitory boutons that normally express high CB1R levels (presumptive cholecystokinin neuron boutons) were lower in samples from individuals with schizophrenia relative to comparison samples. CONCLUSION: Given CB1R's role in suppressing neurotransmission upon activation, these results suggest an overall shift in excitatory and inhibitory balance regulation toward a net reduction of excitatory activity in schizophrenia.

Altered Rbfox1-Vamp1 pathway and prefrontal cortical 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.

Distorted neurocomputation by a small number of extra-large spines in psychiatric disorders.

Human genetics strongly support the involvement of synaptopathy in psychiatric disorders. However, trans-scale causality linking synapse pathology to behavioral changes is lacking. To address this question, we examined the effects of synaptic inputs on dendrites, cells, and behaviors of mice with knockdown of SETD1A and DISC1, which are validated animal models of schizophrenia. Both models exhibited an overrepresentation of extra-large (XL) synapses, which evoked supralinear dendritic and somatic integration, resulting in increased neuronal firing. The probability of XL spines correlated negatively with working memory, and the optical prevention of XL spine generation restored working memory impairment. Furthermore, XL synapses were more abundant in the postmortem brains of patients with schizophrenia than in those of matched controls. Our findings suggest that working memory performance, a pivotal aspect of psychiatric symptoms, is shaped by distorted dendritic and somatic integration via XL spines.

The Relationship Between Serum Angiotensin Converting Enzyme Level and the Decision to Escalate Treatment of Sarcoidosis.

PURPOSE: We performed a retrospective analysis of a sarcoidosis cohort who had sACE obtained at their initial clinic visit, but the treating physician was blinded to the results. We examined the relationship between sACE and the treating physician's decision to escalate sarcoidosis treatment. METHODS: Treatment was considered escalated if the prednisone dose was increased or if the prednisone dose was not changed but an additional anti-sarcoidosis drug was added or the dose was increased. RESULTS: 561 sarcoidosis patients were analyzed. The most common target organ was the lung (84%). Using a cut-off of > 82 units/L for an elevated sACE, 31/82 (38%) with an elevated sACE had treatment escalation whereas 91/497 (18%) had treatment escalation with a normal sACE (p < 0.0001). For the need of treatment escalation, a sACE (cut-off of > 82) had sensitivity 0.25, specificity 0.89, positive predictive value 0.38, negative predictive value 0.81. These results were not appreciably different using other sACE cut-off values such as 70, 80, 90, or 100. A multivariable logistic regression model that included demographics, the target organ, spirometry results estimated that sACE level and lower FVC were significantly associated with the likelihood of treatment escalation. These findings held when sACE > 82 replaced sACE level in the multivariable logistic regression model. CONCLUSIONS: Although there was a strong correlation between sACE at the initial sarcoidosis clinic visit and subsequent treatment escalation of sarcoidosis, the predictive power was such that sACE is not adequately reliable to be used in isolation to make this determination.

Vesicular Glutamate Transporter Changes in the Cortical Default Mode Network During the Clinical and Pathological Progression of Alzheimer's Disease.

BACKGROUND: Altered glutamatergic neurotransmission may contribute to impaired default mode network (DMN) function in Alzheimer's disease (AD). Among the DMN hub regions, frontal cortex (FC) was suggested to undergo a glutamatergic plasticity response in prodromal AD, while the status of glutamatergic synapses in the precuneus (PreC) during clinical-neuropathological AD progression is not known. OBJECTIVE: To quantify vesicular glutamate transporter VGluT1- and VGluT2-containing synaptic terminals in PreC and FC across clinical stages of AD. METHODS: Unbiased sampling and quantitative confocal immunofluorescence of cortical VGluT1- and VGluT2-immunoreactive profiles and spinophilin-labeled dendritic spines were performed in cases with no cognitive impairment (NCI), mild cognitive impairment (MCI), mild-moderate AD (mAD), or moderate-severe AD (sAD). RESULTS: In both regions, loss of VGluT1-positive profile density was seen in sAD compared to NCI, MCI, and mAD. VGluT1-positive profile intensity in PreC did not differ across groups, while in FC it was greater in MCI, mAD, and sAD compared to NCI. VGluT2 measures were stable in PreC while FC had greater VGluT2-positive profile density in MCI compared to sAD, but not NCI or mAD. Spinophilin measures in PreC were lower in mAD and sAD compared to NCI, while in FC they were stable across groups. Lower VGluT1 and spinophilin measures in PreC, but not FC, correlated with greater neuropathology. CONCLUSION: Frank loss of VGluT1 in advanced AD relative to NCI occurs in both DMN regions. In FC, an upregulation of VGluT1 protein content in remaining glutamatergic terminals may contribute to this region's plasticity response in AD.

Terminal type-specific cannabinoid CB1 receptor alterations in patients with schizophrenia: a pilot study.

Background: Individuals with schizophrenia are at elevated genetic risks for comorbid cannabis use, and often experience exacerbations of cognitive and psychotic symptoms when exposed to cannabis. These findings have led a number of investigators to examine cannabinoid CB1 receptor (CB1R) alterations in schizophrenia, though with conflicting results. We recently demonstrated the presence of CB1R in both excitatory and inhibitory boutons in the human prefrontal cortex, with differential levels of the receptor between bouton types. We hypothesized that the differential enrichment of CB1R between bouton types - a factor previously unaccounted for when examining CB1R changes in schizophrenia - may resolve prior discrepant reports and increase our insight into the effects of CB1R alterations on the pathophysiology of schizophrenia. Methods: Using co-labeling immunohistochemistry and fluorescent microscopy, we examined total CB1R levels and CB1R levels within excitatory (vGlut1-positive) and inhibitory (vGAT-positive) boutons of prefrontal cortex samples from ten pairs of individuals diagnosed with schizophrenia and non-psychiatric comparisons. Results: Significantly higher total CB1R levels were found within samples from individuals with schizophrenia. Terminal type-specific analyses identified significantly higher CB1R levels within excitatory boutons in samples from individuals with schizophrenia relative to comparisons. In contrast, CB1R levels within the subset of inhibitory boutons that normally express high CB1R levels (presumptive cholecystokinin neuron boutons) were lower in samples from individuals with schizophrenia relative to comparison samples. Conclusion: Given CB1R's role in suppressing neurotransmission upon activation, these results suggest an overall shift in excitatory and inhibitory balance regulation toward a net reduction of excitatory activity in schizophrenia.

The Nature of Prefrontal Cortical GABA Neuron Alterations in Schizophrenia: Markedly Lower Somatostatin and Parvalbumin Gene Expression Without Missing Neurons.

OBJECTIVE: In schizophrenia, somatostatin (SST) and parvalbumin (PV) mRNA levels are lower in the dorsolateral prefrontal cortex (DLPFC), but it remains unclear whether these findings reflect lower transcript levels per neuron, fewer neurons, or both. Distinguishing among these alternatives has implications for understanding the pathogenesis of, and developing new treatments for, DLPFC dysfunction in schizophrenia. METHODS: To identify SST and PV neurons in postmortem human DLPFC, the authors used fluorescent in situ hybridization to label cells expressing two transcripts not altered in schizophrenia: vesicular GABA transporter (VGAT; a marker of all GABA neurons) and SOX6 (a marker of only SST and PV neurons). In cortical layers 2 and 4, where SST and PV neurons, respectively, are differentially enriched, levels of SST and PV mRNA per neuron and the relative densities of SST-, PV-, and VGAT/SOX6-positive neurons were quantified. RESULTS: In individuals with schizophrenia, mRNA levels per positive neuron were markedly and significantly lower for SST in both layers (effect sizes >1.48) and for PV only in layer 4 (effect size=1.14) relative to matched unaffected individuals. In contrast, the relative densities of all SST-, PV-, or VGAT/SOX6-positive neurons were unaltered in schizophrenia. CONCLUSIONS: Novel multiplex fluorescent in situ hybridization techniques permit definitive distinction between cellular levels of transcripts and the presence of neurons expressing those transcripts. In schizophrenia, pronounced SST and PV mRNA deficits are attributable to lower levels of each transcript per neuron, not fewer neurons, arguing against death or abnormal migration of these neurons. Instead, these neurons appear to be functionally altered and thus amenable to therapeutic interventions.

Laminar-Specific Alterations in Calbindin-Positive Boutons in the Prefrontal Cortex of Subjects With Schizophrenia.

BACKGROUND: Cognitive deficits in schizophrenia are associated with altered GABA (gamma-aminobutyric acid) neurotransmission in the prefrontal cortex (PFC). GABA neurotransmission requires GABA synthesis by 2 isoforms of glutamic acid decarboxylase (GAD65 and GAD67) and packaging by the vesicular GABA transporter (vGAT). Current postmortem findings suggest that GAD67 messenger RNA is lower in a subset of the calbindin-expressing (CB+) class of GABA neurons in schizophrenia. Hence, we assessed if CB+ GABA neuron boutons are affected in schizophrenia. METHODS: For 20 matched pairs of subjects with schizophrenia and unaffected comparison subjects, PFC tissue sections were immunolabeled for vGAT, CB, GAD67, and GAD65. The density of CB+ GABA boutons and levels of the 4 proteins per bouton were quantified. RESULTS: Some CB+ GABA boutons contained both GAD65 and GAD67 (GAD65+/GAD67+), whereas others contained only GAD65 (GAD65+) or GAD67 (GAD67+). In schizophrenia, vGAT+/CB+/GAD65+/GAD67+ bouton density was not altered, vGAT+/CB+/GAD65+ bouton density was 86% higher in layers 2/superficial 3 (L2/3s), and vGAT+/CB+/GAD67+ bouton density was 36% lower in L5-6. Bouton GAD levels were differentially altered across bouton types and layers. In schizophrenia, the sum of GAD65 and GAD67 levels in vGAT+/CB+/GAD65+/GAD67+ boutons was 36% lower in L6, GAD65 levels were 51% higher in vGAT+/CB+/GAD65+ boutons in L2, and GAD67 levels in vGAT+/CB+/GAD67+ boutons were 30% to 46% lower in L2/3s-6. CONCLUSIONS: These findings indicate that schizophrenia-associated alterations in the strength of inhibition from CB+ GABA neurons in the PFC differ across cortical layers and bouton classes, suggesting complex contributions to PFC dysfunction and cognitive impairments in schizophrenia.

Lifetime Evaluation of Left Ventricular Structure and Function in Male C57BL/6J Mice after Gamma and Space-Type Radiation Exposure.

The lifetime effects of space irradiation (IR) on left ventricular (LV) function are unknown. The cardiac effects induced by space-type IR, specifically 5-ion simplified galactic cosmic ray simulation (simGCRsim), are yet to be discovered. Three-month-old, age-matched, male C57BL/6J mice were irradiated with 137Cs gamma (γ; 100, 200 cGy) and simGCRsim (50 and 100 cGy). LV function was assessed via transthoracic echocardiography at 14 and 28 days (early), and at 365, 440, and 660 (late) days post IR. We measured the endothelial function marker brain natriuretic peptide in plasma at three late timepoints. We assessed the mRNA expression of the genes involved in cardiac remodeling, fibrosis, inflammation, and calcium handling in LVs harvested at 660 days post IR. All IR groups had impaired global LV systolic function at 14, 28, and 365 days. At 660 days, 50 cGy simGCRsim-IR mice exhibited preserved LV systolic function with altered LV size and mass. At this timepoint, the simGCRsim-IR mice had elevated levels of cardiac fibrosis, inflammation, and hypertrophy markers Tgfß1, Mcp1, Mmp9, and ßmhc, suggesting that space-type IR may induce the cardiac remodeling processes that are commonly associated with diastolic dysfunction. IR groups showing statistical significance were modeled to calculate the Relative Biological Effectiveness (RBE) and Radiation Effects Ratio (RER). The observed dose-response shape did not indicate a lower threshold at these IR doses. A single full-body IR at doses of 100-200 cGy for γ-IR, and 50-100 cGy for simGCRsim-IR decreases the global LV systolic function in WT mice as early as 14 and 28 days after exposure, and at 660 days post IR. Interestingly, there is an intermediate time point (365 days) where the impairment in LV function is observed. These findings do not exclude the possibility of increased acute or degenerative cardiovascular disease risks at lower doses of space-type IR, and/or when combined with other space travel-associated stressors such as microgravity.

Outcomes of prednisone-tapering regimens for cardiac sarcoidosis: A retrospective analysis demonstrating a benefit of infliximab.

BACKGROUND: The optimal treatment strategy for cardiac sarcoidosis has not been standardized. We examined the effectiveness of three prednisone-tapering treatment regimens for cardiac sarcoidosis. METHODS: We retrospectively reviewed prednisone-tapering treatment regimens for cardiac sarcoidosis that contained prednisone alone (P), prednisone plus methotrexate (P-M), and prednisone plus infliximab containing regimens (P-I). We defined the success of each regimen as the ability to lower the daily prednisone dose to 7.5 mg or less for 6 or more months without developing an adverse cardiac event. We also examined the lowest effective daily prednisone dose achieved without developing an adverse cardiac event. RESULTS: We identified 61 treatment regimens in 33 cardiac sarcoidosis patients that were analyzed. The success rate of prednisone-tapering regimens was significantly different P: 8/30, 27%; P-M: 3/23, 13%; P-I: 6/8, 75%., p = 0.04. The lowest effective daily prednisone dose for the regimens was also significantly different: P: 14.1 ± 10.1 mg; P-M: 16.9 ± 9.4 mg; infliximab: 7.8 ± 4.9 mg, (p = 0.03); by both measures the success was greatest with the P-I regimen. CONCLUSIONS: For the treatment of cardiac sarcoidosis, prednisone-tapering regimens containing infliximab are superior to those containing prednisone alone or prednisone plus methotrexate in terms of reaching 7.5 mg/day of prednisone for more than 6 months and achieving the lowest effective prednisone.

Targeting prefrontal cortex GABAergic microcircuits for the treatment of alcohol use disorder.

Developing novel treatments for alcohol use disorders (AUDs) is of paramount importance for improving patient outcomes and alleviating the suffering related to the disease. A better understanding of the molecular and neurocircuit mechanisms through which alcohol alters brain function will be instrumental in the rational development of new efficacious treatments. Clinical studies have consistently associated the prefrontal cortex (PFC) function with symptoms of AUDs. Population-level analyses have linked the PFC structure and function with heavy drinking and/or AUD diagnosis. Thus, targeting specific PFC cell types and neural circuits holds promise for the development of new treatments. Here, we overview the tremendous diversity in the form and function of inhibitory neuron subtypes within PFC and describe their therapeutic potential. We then summarize AUD population genetics studies, clinical neurophysiology findings, and translational neuroscience discoveries. This study collectively suggests that changes in fast transmission through PFC inhibitory microcircuits are a central component of the neurobiological effects of ethanol and the core symptoms of AUDs. Finally, we submit that there is a significant and timely need to examine sex as a biological variable and human postmortem brain tissue to maximize the efforts in translating findings to new clinical treatments.

Total Internal Reflection Fluorescence (TIRF) Microscopy.

Total internal reflection fluorescence (TIRF) microscopy (TIRFM) is an elegant optical technique that provides for the excitation of fluorophores in an extremely thin axial region ("optical section"). The method is based on the principle that when excitation light is completely internally reflected in a transparent solid (e.g., coverglass) at its interface with liquid, an electromagnetic field, called the evanescent wave, is generated in the liquid at the solid-liquid interface and is the same frequency as the excitation light. Since the intensity of the evanescent wave exponentially decays with distance from the surface of the solid, only fluorescent molecules within a few hundred nanometers of the solid are efficiently excited. This overview will review the history, optical theory, and hardware configurations used in TIRFM. In addition, it will provide experimental details and methodological considerations for studying receptors at the plasma membrane in neurons. © 2022 Wiley Periodicals LLC.