Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2024 Jul 15.
Published in final edited form as: Biol Psychiatry. 2022 Dec 8;94(2):142–152. doi: 10.1016/j.biopsych.2022.12.004

Laminar-specific alterations in calbindin-positive boutons in the prefrontal cortex of subjects with schizophrenia

Kenneth N Fish 1, Brad R Rocco 1, James D Wilson 1, David A Lewis 1,2
PMCID: PMC10247897  NIHMSID: NIHMS1856217  PMID: 36868891

Abstract

Background:

Cognitive deficits in schizophrenia are associated with altered GABA neurotransmission in the prefrontal cortex (PFC). GABA neurotransmission requires GABA synthesis by two isoforms of glutamic acid decarboxylase (GAD65 and GAD67) and packaging by the vesicular GABA transporter (vGAT). Current postmortem findings suggest that GAD67 mRNA 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 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 four 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 30–46% 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 differs across cortical layers and bouton classes, suggesting complex contributions to PFC dysfunction and cognitive impairments in the disease.

Keywords: glutamic acid decarboxylase (GAD), vesicular GABA transporter (vGAT), gamma-aminobutyric acid (GABA), quantitative microscopy

Introduction

Certain cognitive deficits in individuals with schizophrenia appear to be associated with altered gamma-aminobutyric acid (GABA) neurotransmission in the prefrontal cortex (PFC) (1). GABA, the major inhibitory neurotransmitter in the neocortex, is mainly synthesized at its site of function (i.e., axon boutons) by the 65 (GAD65) and 67 (GAD67) kDa isoforms of glutamic acid decarboxylase, the products of the GAD2 and GAD1 genes, respectively (2). Studies in rodents suggest that GAD67 accounts for most GABA synthesis in the cortex (3,4), whereas GAD65 contributes to GABA synthesis under conditions of high synaptic demand (4,5).

In schizophrenia, ~30–50% of PFC GABA-expressing (GABAergic) neurons were reported to lack detectable levels of GAD67 mRNA (6,7), whereas the remaining GABAergic neurons expressed normal levels (6,7). We recently reported that a subset of GABA boutons had undetectable or very low levels of GAD67 protein in the PFC of subjects with schizophrenia (8). Together, these cellular GAD67 mRNA and bouton GAD67 protein findings suggest that in schizophrenia only a subset of PFC GABAergic neurons has an impaired capacity to synthesize GABA for synaptic release.

In primate neocortex, ~85% of GABAergic neurons can be differentiated into functionally distinct subtypes based on the expression of one of three calcium-binding proteins—parvalbumin (PV;~20%), calbindin (CB;~20%) or calretinin (CR;~45%) (9–12). In schizophrenia, levels of GAD67 and GAD65 have only been directly assessed in parvalbumin (PV)-expressing neurons. These studies found that GAD67 mRNA expression was markedly lower in ~50% of PV neurons (13), GAD67 protein levels were lower in the boutons of PV basket cells (14,15) but not of PV chandelier cells (8,16), and GAD65 protein levels were lower in parvalbumin basket cell boutons in schizophrenia (15). Given that PV neurons represent only a subset of GABAergic neurons, and that only a subset of PV neurons are affected in schizophrenia, other subtype(s) of GABAergic neurons must also be altered in schizophrenia.

Indirect evidence suggests that the affected GABAergic cell types in schizophrenia might include the CB-expressing subpopulation (16–24). For example, approximately half of CB GABA neurons express the neuropeptide somatostatin (SST) (25–28) and SST mRNA levels have been consistently reported to be lower in the PFC in schizophrenia (20–24). Moreover, the alterations in SST and GAD67 mRNA levels are positively correlated in schizophrenia (21), suggesting that GAD67 protein levels might be lower in CB+/SST+ boutons in schizophrenia. Unfortunately, SST is not a useful marker of boutons in postmortem studies because SST levels decline substantially with age (21), and SST peptides are quickly altered following death (29). Although a subset of cortical pyramidal cells express CB (10,25,30–33), GABAergic CB boutons can be selectively identified by the presence of the GABA neuron-specific marker, vesicular GABA transporter (vGAT), which is required to package GABA for vesicular release (34). As levels of vGAT protein are not altered in axon boutons in the PFC in schizophrenia (8), vGAT immunoreactivity serves as a fiduciary GABAergic bouton marker in human postmortem studies of schizophrenia.

Thus, in the present study we sought to determine if the relative density of CB-containing GABAergic boutons, or the levels of GAD67 and/or GAD65 protein in these boutons, are altered in the PFC in schizophrenia.

Methods and Materials

Human subjects

Brain specimens were recovered during autopsies conducted at the Allegheny County Medical Examiner’s Office (Pittsburgh,PA) after obtaining consent from next of kin. An independent committee of clinicians made consensus DSM-V diagnoses, or confirmed absence of any diagnoses, using results of structured interviews conducted with family members and/or review of medical records (20). A board-certified neuropathologist conducted an exam that involves review of photomicrographs of all surfaces of the brain and all coronal blocks and detailed microscopic examination of standardized brain areas and any suspicious locations. No abnormalities were detected in the PFC of any subjects. To reduce biological variance between groups, each schizophrenia subject was matched to one unaffected comparison (UC) subject for sex, and as closely as possible for age and postmortem interval (PMI) (Supplemental Table S1). Because length of PMI can affect protein integrity (14,35) and aging can differentially affect gene expression (36), we included only subjects with PMI<16 hours and age≤55 years. Groups did not differ in mean age, PMI, or tissue storage time (Table 1). Importantly, although brains were stored for years, correlations between storage time and any of the positive findings of this study were not significant (all r<0.42 and p>0.09). The University of Pittsburgh’s Committee for the Oversight of Research and Clinical Training Involving Decedents and Institutional Review Board for Biomedical Research approved all procedures.

Table 1.

Summary of demographic and postmortem characteristics of human subjects.

Measure Comparison Schizophrenia Statistics
N 20 20 N/A
Sex 14M/6F 14M/6F N/A
Race 17W/3B 18W/2B X2=0.23; p=0.63
Age (years)* 45.05 ± 7.27 45.75 ± 8.69 t38=0.28; p=0.78
Postmortem interval (hours)* 9.53 ± 3.39 9.92 ± 4.04 t38=0.33; p=0.75
Tissue storage time (months)* 140.3 ± 63.5 165.4 ± 63.4 t1,38=0.27; p=0.79
Open in a new tab
*

Values shown are mean ± standard deviation

Immunohistochemistry

The fresh left hemisphere of each human brain was blocked coronally at 1–2 cm intervals, immersed in 4% paraformaldehyde for 48 hours at 4°C and cryoprotected (15). Tissue blocks containing the PFC were sectioned coronally at 40μm and processed for immunohistochemistry. For each human subject, two sections containing PFC area 9 spaced ~500μm apart were used. Antigen retrieval (37) was used to enhance reactivity of antibodies. Sections were permeabilized with 0.3% Triton X-100, blocked with 20% donkey serum, and incubated for ~72 hours at 4°C with primary antibodies that recognize vGAT, GAD65, GAD67, and CB. Sections were then rinsed and incubated for 24 hours with secondary antibodies at 4°C. After rinsing, a tertiary incubation (24 hours) with streptavidin Alexa 405 (CB) was performed. Sections were subsequently rinsed and mounted on slides coded to conceal diagnosis. For detailed information, see Supplemental Information.

Microscopy

Data were collected on an Olympus IX81 inverted microscope equipped with an Olympus spinning disk confocal (Supplemental Information). 3D image stacks (512×512 pixels;~137×137 μm) were acquired over 50% of the total tissue section thickness starting at the coverslip. Imaging the same percentage of tissue rather than a fixed depth controls for the potential confound of storage and/or mounting related volume differences.

Sampling

As determined by measurements made in Nissl-stained sections (38), the boundaries of the six layers (L) of the PFC can be estimated based on the distance from the pial surface to the white matter as follows: L1 (pia-9%), L2 (10–19%), L3 (20–49%), L4 (50–59%), L5 (60–79%), and L6 (80%-gray/white matter border). Previous studies in schizophrenia of GABAergic markers in cell bodies (6,7,21) or axonal boutons (8,38) divided the superficial and middle cortical layers into 2/superficial 3 (L2/3s;10–35%) and deep 3/4 (L3d/4;35–60%). The cell bodies and targets of CB+ GABAergic neuron subtypes are concentrated in and have synaptic targets in these layers as well as the other cortical layers (25,39–44). Thus, for the present study, the cortical gray matter was similarly divided as follows: L1, L2/3s, L3d/4, L5, and L6. Systematic randomly sampled image stacks were taken within each layer using a sampling grid of 180×180 μm. The same investigator, who was blind to subject and diagnosis, collected a total of 4,000 image stacks.

Image processing

Each fluorescent channel was deconvolved using the AutoQuant adaptive blind deconvolution algorithm (Media Cybernetics, Rockville, MD). For data segmentation, a Gaussian channel was made for each deconvolved channel by calculating a difference of Gaussians using sigma values of 0.7 and 2. Data segmentation was performed as described (Supplemental Information) (8). Final object masks (Fig 1) were then used to collect information on the deconvolved channels.

Figure 1. CB bouton subtypes are distinguishable by GAD65 and GAD67 protein content.

Figure 1.

Open in a new tab

(A-D) Projection image (3 z-planes separated by 0.25 μm) of a human PFC tissue section from an unaffected comparison (UC) subject immunolabeled for CB, vGAT, GAD65, and GAD67. (E) Lipofuscin fluorescence. Merged (F) CB, GAD65, and GAD67 channels and (G) vGAT, GAD65, and GAD67 channels. (H) and (I) are masks of the channels in (G) and (F), respectively. The open arrowhead indicates vGAT+/CB+/GAD65+ boutons that are negative for GAD67, solid arrowhead indicates vGAT+/CB+/GAD67+ boutons that are negative for GAD65, and the solid arrow indicates a vGAT+/CB+/GAD65+/GAD67+ bouton. Thus, in UC subjects, all 3 vGAT+/CB+/GAD+ bouton subpopulations were present. Bar = 5 μm.

Definitions of vGAT+/CB+/GAD+ boutons

All vGAT-immunoreactive puncta within a size range of 0.05–0.7μm3 were considered axonal boutons (vGAT+ boutons) (34,45). vGAT+ boutons were classified as containing CB, GAD65, or GAD67 as follows. Briefly, mask operations, which assess the degree of overlap between voxels of different object masks, were used to identify vGAT object masks and CB, GAD65, or GAD67 object masks that overlapped each other’s center. For each site the mean fluorescence intensity of GAD67 for all boutons identified in the first step were used as seeds in a K-means cluster analysis to classify vGAT+ boutons as being CB+, GAD65+, or GAD67+ (8,46,47). Examples of classified boutons are shown in Figure 1.

Lipofuscin autofluorescence detection

In human cortex, lipofuscin accumulates with age in lysosomes and autofluoresces (48) with a broad excitation and emission spectra (49), which can potentially confound the detection of specific fluorochrome labeled signal (Supplemental Figs S1–S2). To eliminate the potential for confounding, we modified a previously described approach (50) to capture and eliminate lipofuscin signal (Supplemental Information) (8,16).

Statistics

A linear mixture model was used to compare CB bouton density across layers in UC subjects to account for the within-subject correlation among layers and to characterize the within- and between-subject variability (51). To test for interaction effects among cortical layer and bouton type, the mixed model included the density of CB boutons as the dependent variable, layer, bouton type, and layer-by-bouton type interaction as the fixed effects, and sex, age, PMI, and storage time as covariates.

To assess if CB bouton density and protein levels were altered in schizophrenia within each PFC layer, analysis of covariance (ANCOVA) models were used. Each ANCOVA model included CB bouton density or protein level (GAD65, GAD67, or CB) as the dependent variable, diagnostic group as the main effect, and sex, age, PMI, and tissue storage time as covariates. Because bouton density and GAD levels differed between bouton type and across layers, analyses across diagnostic groups were restricted to assessments within layers. Within each layer, 10 dependent measures were assessed: density (3 bouton types), GAD65 (2 bouton types), GAD67 (2 bouton types), and CB (3 bouton types).

The Benjamini and Hochberg Step-Up Correction method (52) (q = 0.05) was used to control the false discovery rate for Type I errors due to multiple comparisons. Although we previously found GABA bouton vGAT levels are unaltered in schizophrenia (8,15), potential differences in bouton vGAT levels were assessed in secondary tests. Denominator degrees of freedom (df) were computed using Satterthwaite approximation and effect sizes (Cohen’s ds) are included. In addition, Bayesian statistics were used to further assess the strength of the null hypothesis for all results (53–56). Bayes factors for all tests are provided in Supplemental Table S2–S4. The potential effects of sex, suicide, diagnosis of schizoaffective disorder, or use of nicotine, antidepressants, benzodiazepines/sodium valproate and/or antipsychotic at time of death were examined using t-tests (15) (Supplemental Table S5). All data are presented as mean±standard deviation.

Results

GAD expression in vGAT+/CB+ boutons in the PFC of UC subjects

GABAergic CB boutons can be selectively identified by the presence of vGAT, a GABA neuron-specific marker required to package GABA for vesicular release (34). We previously found that patterns of GAD expression distinguished 3 subpopulations of vGAT+/CB+ boutons in monkey PFC: GAD65+/GAD67+, GAD65+ only, and GAD67+ only (46). Thus, we determined if the same was true in human PFC. In UC subjects, all 3 vGAT+/CB+/GAD+ bouton subpopulations were present (Fig 1 and Supplemental Fig S3). Accordingly, we quantified vGAT, CB, GAD65 and/or GAD67 protein levels in these subpopulations of boutons: 1) vGAT+/CB+/GAD65+/GAD67+; 2) vGAT+/CB+/GAD65+; and 3) vGAT+/CB+/GAD67+. In UC subjects, the densities of detectable boutons differed by bouton type (F2,284=28.66;p<0.0005) and layer (F4,284=37.51;p<0.0005). In addition, the interaction between bouton type and layer significantly differed (F8,284=28.48;p<0.0005; Supplemental Fig S4). Indeed, the rank ordering (bouton density) of the layers was different for each bouton type (Supplemental Fig S4). For example, the density of vGAT+/CB+/GAD67+ boutons was 8.5 times greater in L1 than in L6, and in L1 the density of vGAT+/CB+/GAD67+ boutons was 2 times greater than either of the other two bouton types. In UC subjects, GAD65 levels in vGAT+/CB+/GAD65+/GAD67+ and vGAT+/CB+/GAD65+ boutons differed by bouton type (F1,181=79.26;p<0.0005) and layer (F4,188=2.44;p=0.048); however, the interaction between bouton type and layer was not significant (F4,188=0.81; p=0.52), suggesting that the difference in GAD65 levels between the two bouton types was similar in each layer (Supplemental Fig S5A). GAD67 levels in vGAT+/CB+/GAD65+/GAD67+ and vGAT+/CB+/GAD67+ boutons differed by bouton type (F1,187=269.53;p<0.0005) and layer (F4,187=26.45;p<0.0005). In addition, the interaction between bouton type and layer was significant (F4,187=8.06;p<0.0005), suggesting that the difference in GAD67 levels between the two boutons differed between layers (Supplemental Fig S5B).

vGAT+/CB+ bouton density in schizophrenia

The same tissue volume was sampled for all subjects and an average of 15,910±5,307 and 15,226±6,665 vGAT+/CB+/GAD65+/GAD67+ boutons were assessed for each UC and schizophrenia subject, respectively. The density of vGAT+/CB+/GAD65+/GAD67+ boutons did not differ between groups in any layer (Fig 2A). An average of 10,694±4,865 and 13,852±4,340 vGAT+/CB+/GAD65+ boutons were assessed for each UC and schizophrenia subject, respectively. Notably, the mean density of vGAT+/CB+/GAD65+ boutons was 86% greater in L2/3s in schizophrenia subjects relative to UC subjects (Fig 2B), with the density being higher in the schizophrenia subject for 15 of the 20 pairs (Supplemental Fig S6A). An average of 19,846±5,127 and 17,113±6,150 vGAT+/CB+/GAD67+ boutons were assessed for each UC and schizophrenia subject, respectively. The density of vGAT+/CB+/GAD67+ boutons was 36% lower in L5 and L6 in schizophrenia subjects relative to UC subjects (Fig 2C) with the density lower in the schizophrenia subject for 14 of the 20 pairs (Supplemental Fig S6B–C).

Figure 2. vGAT+/CB+ bouton density in schizophrenia.

Figure 2.

Open in a new tab

(A-C) The density (boutons/μm3) of vGAT+/CB+/GAD65+/GAD67+ (A), vGAT+/CB+/GAD65+ (B), and vGAT+/CB+/GAD67+ (C) boutons in unaffected comparison (UC) and schizophrenia subjects. The data points per layer represent an UC (black) and schizophrenia (gray) subject. Asterisks indicate a statistical difference (p < 0.05) between groups within a layer and ds = effect size (Cohen’s ds). F statistics and p values are provided in Supplemental Table S2–S4. The densities of the 3 vGAT+/CB+/GAD+ bouton subpopulations were differentially affected in schizophrenia.

vGAT+/CB+/GAD65+/GAD67+ and vGAT+/CB+/GAD65+ bouton GAD65 levels in schizophrenia

vGAT+/CB+/GAD65+/GAD67+ bouton levels of GAD65 were 17% lower in L2/3s, L5, and L6 but did not differ between groups in L1 or L3 (Fig 3A). Within L2/3s, L5, and L6, GAD65 levels were lower in 70–80% of schizophrenia subjects relative to their matched UC subjects (Supplemental Fig S7A–C). vGAT+/CB+/GAD65+ bouton levels of GAD65 were 16–21% lower across L2/3s-5 in schizophrenia relative to UC subjects but did not differ between groups in L1 and L6 (Fig 3B). Across L2/3s-5, GAD65 was lower in 70–75% of schizophrenia subjects relative to their matched UC subjects (Supplemental Fig S7D–F).

Figure 3. vGAT+/CB+/GAD65+/GAD67+ and vGAT+/CB+/GAD65+ bouton GAD65 protein levels and vGAT+/CB+/GAD65+/GAD67+ and vGAT+/CB+/GAD67+ bouton GAD67 protein levels in schizophrenia.

Figure 3.

Open in a new tab

vGAT+/CB+/GAD65+/GAD67+ (A) and vGAT+/CB+/GAD65+ (B) bouton GAD65 protein levels within each layer. vGAT+/CB+/GAD65+/GAD67+ (C) and vGAT+/CB+/GAD67+ (D) bouton GAD67 protein levels within each layer. The data points per layer represent an unaffected comparison (UC) (black) and schizophrenia (gray) subject. Asterisks indicate a statistical difference (p < 0.05) between groups, a.u. = arbitrary units, and ds = effect size (Cohen’s ds). F statistics and p values are provided in Supplemental Table S2–S4. GAD65 and GAD67 terminal levels were differentially affected across the 3 vGAT+/CB+/GAD+ bouton subpopulations in schizophrenia.

vGAT+/CB+/GAD65+/GAD67+ and vGAT+/CB+/GAD67+ bouton GAD67 levels in schizophrenia

vGAT+/CB+/GAD65+/GAD67+ bouton GAD67 protein levels were 14–20% lower within L2/3s-L6 in schizophrenia relative to UC subjects, and unchanged in L1 (Fig 3C). GAD67 levels were lower in 70–90% of schizophrenia subjects relative to their matched UC subjects in L2/3s-L6 (Supplemental Fig S8A–D). vGAT+/CB+/GAD67+ bouton GAD67 levels were 17–20% lower across L2/3s-3d/4 in schizophrenia relative to UC subjects (Fig 3D). Within these layers, GAD67 levels were lower in 70–75% of schizophrenia subjects relative to their matched UC subjects (Supplemental Fig S8E–F).

vGAT+/CB+ bouton CB and vGAT levels in schizophrenia

vGAT+/CB+/GAD65+/GAD67+ bouton CB protein levels were 17–18% lower in L2/3s-3d/4 (Fig 4A). Within these layers, CB levels were lower in 70–75% of schizophrenia subjects relative to their matched UC subjects. vGAT+/CB+/GAD65+ bouton CB levels were unchanged in schizophrenia (Fig 4B), while vGAT+/CB+/GAD67+ bouton CB levels per bouton were 16–23% lower across L1-6 in schizophrenia relative to UC subjects (Fig 4C). Within these layers, CB was lower in 70–80% of the schizophrenia subjects relative to their matched UC subjects. vGAT bouton levels were unaltered across all bouton types and layers (Supplemental Fig S9A–C).

Figure 4. vGAT+/CB+ bouton CB levels in schizophrenia.

Figure 4.

Open in a new tab

vGAT+/CB+/GAD65+/GAD67+ (A), vGAT+/CB+/GAD65+ (B), and vGAT+/CB+/GAD67+ (C) bouton CB protein levels. The data points per layer represent an unaffected comparison (UC) (black) and schizophrenia (gray) subject. Asterisks indicate a statistical difference (p < 0.05) between groups, a.u. = arbitrary units, and ds = effect size (Cohen’s ds). F statistics and p values are provided in Supplemental Table S2–S4. CB bouton levels were differentially altered across bouton types and layers in schizophrenia.

Diagnostic differences in total GAD levels in vGAT+/CB+/GAD65+/GAD67+, vGAT+/CB+/GAD65+, and vGAT+/CB+/GAD67+ boutons

To estimate the capacity of CB+ GABAergic neurons for GABA synthesis within a cortical layer, a composite measure was generated for each layer. Specifically, the total amount of GAD protein (GAD65 and/or GAD67) within each bouton was summed for each layer. Total GAD in vGAT+/CB+/GAD65+/GAD67+ boutons was 36% lower in L6 of schizophrenia subjects (Fig 5A), with this measure being lower in 85% of schizophrenia subjects relative to their matched UC subjects. In contrast, total GAD65 protein was 51% higher in L2 vGAT+/CB+/GAD65+ boutons from schizophrenia subjects relative to UC subjects (Fig 5B), with this finding present in 70% of schizophrenia subjects relative to their matched UC subjects. Analysis of vGAT+/CB+/GAD67+ boutons found that the composite measure in subjects with schizophrenia was 30–46% lower in layers 2/3s-6 than in UC subjects (Fig 5C). Within these layers, the amount of GAD in vGAT+/CB+/GAD67+ boutons was lower in 75–80% of the schizophrenia subjects relative to their matched UC subjects.

Figure 5. One index of vGAT+/CB+/GAD65+/GAD67+, vGAT+/CB+/GAD65+, and vGAT+/CB+/GAD67+ boutons to synthesize GABA.

Figure 5.

Open in a new tab

To estimate the capacity of CB+ GABAergic neurons for GABA synthesis within a cortical layer, a composite measure was generated. Specifically, the total amount of GAD protein (GAD65 and/or GAD67) within each bouton was summed for each layer. An estimate of vGAT+/CB+/GAD65+/GAD67+ (A), vGAT+/CB+/GAD65+ (B), and vGAT+/CB+/GAD67+ (C) boutons to synthesize GABA is plotted for each subject within each layer. The data points per layer represent an unaffected comparison (UC) (black) and schizophrenia (gray) subject. Asterisks indicate a statistical difference (p < 0.01) between groups, a.u. = arbitrary units, and ds = effect size (Cohen’s ds). F statistics and p values are provided in Supplemental Table S2–S4. These findings indicate that schizophrenia-associated alterations in the ability of CB+ GABA neurons to synthesize GABA in the PFC differs across cortical layers and bouton classes.

CB bouton alterations in schizophrenia identified here are not attributable to comorbid factors

An assessment of comorbid factors found that in schizophrenia subjects, altered bouton density and protein levels in vGAT+/CB+/GAD65+/GAD67+, vGAT+/CB+/GAD65+, and vGAT+/CB+/GAD67 boutons did not appear to differ as a function of sex, nicotine use at time of death (ATOD), benzodiazepines and/or sodium valproate ATOD, antidepressants ATOD, antipsychotics ATOD, diagnosis of schizoaffective disorder, or suicide (Supplemental Table S5).

Discussion

Altered GABA function in the PFC in schizophrenia is thought to contribute to impairments in certain cognitive functions, such as working memory (57). Our findings provide the first direct evidence that the ability of subtypes of CB+ GABA neurons, which are likely involved in working memory processes, to synthesize GABA is differentially altered in people with schizophrenia. For example, the density of vGAT+/CB+/GAD65+ boutons was 86% higher in L2/3s of schizophrenia subjects, whereas the densities of vGAT+/CB+/GAD65+/GAD67+ and vGAT+/CB+/GAD67+ boutons did not differ from UC subjects. Although the cause of the higher vGAT+/CB+/GAD65+ bouton density in L2/3s cannot be determined from our data, previous studies suggest that our finding might reflect a deficit in developmental pruning. For example, we recently reported that 1) the mean density of boutons arising from a subpopulation of chandelier neurons that co-express CB is higher exclusively in L2 in schizophrenia relative to UC subjects (16) and 2) chandelier cell synaptic inputs are pruned over development in monkeys (58). Other studies suggest that some developmental processes are arrested in schizophrenia (59,60). Thus, because cortical L2/3 matures later relative to deeper cortical layers (61,62), it is possible that environmental insults at a specific developmental time point could arrest or blunt the pruning process of vGAT+/CB+/GAD65+ boutons specifically in L2/3s. Alternatively, it is possible that collateral sprouting, albeit via an unknown mechanism, from some CB+ GABA neurons is responsible for the higher density. Future experiments are needed to differentiate between these and other possible mechanisms, such as genetic liabilities.

In contrast to the layer 2/3s vGAT+/CB+/GAD65+ bouton density finding, the density of vGAT+/CB+/GAD67+ boutons was 36% lower in L5-6 of schizophrenia subjects, whereas the densities of the other CB+ boutons in these layers were not altered. We previously reported the density of vGAT+ boutons is unaltered in schizophrenia (8). In addition, most chandelier cell boutons, which contain GAD67 but not GAD65 (50,63), in layers 5–6 express CB and we recently showed there is no alteration in CB+ chandelier cell bouton density in L5-6 in schizophrenia (16). Together, these findings suggest that in L5-6 of schizophrenia subjects 1) the lower density of vGAT+/CB+/GAD67+ boutons could represent some terminals no longer being detectable by CB and/or GAD67 immunoreactivity rather than fewer boutons; (2) a subpopulation(s) of vGAT+/CB+/GAD67+ boutons other than those from chandelier cells is affected; and 3) the magnitude of reported vGAT+/CB+/GAD67+ bouton alterations might be underestimated for the affected subpopulation(s). However, we cannot exclude the possibility that lower vGAT+/CB+/GAD67+ bouton density reflects fewer vGAT+/CB+/GAD67+ boutons in the presence of an increase in boutons from other GABA neuron subtypes.

In addition to the CB+ bouton density findings, bouton GAD levels were differentially altered across bouton types and cortical layers. GAD levels were not altered in any of the bouton types in L1. GAD65 was lower in vGAT+/CB+/GAD65+/GAD67+ boutons in L2/3s and in L5-6, as well as lower in vGAT+/CB+/GAD65+ boutons in L2/3s-5. GAD67 was lower in vGAT+/CB+/GAD65+/GAD67+ boutons in all layers but L1, while it was lower in vGAT+/CB+/GAD67+ boutons in L2/3s-3d/4. The lower levels of GAD in CB+ boutons might be a compensatory response to changes in network activity. GAD1 and GAD2 gene expression are highly regulated processes governed, in part, by excitatory input from neighboring pyramidal neurons and projections from other brain regions. Subtypes of CB+ neurons in specific layers have been shown to be differentially recruited into local circuits upon increased activity (64). Pyramidal neurons in L3, which are thought to be impaired in schizophrenia (65–67), innervate pyramidal neurons in L5-6. Thus, the superficial and deep layer findings here could represent a compensatory response to lower pyramidal neuron activity in these layers. Interestingly, there were no differences between groups in L1. The main dendritic target of Martinotti cells, which co-express somatostatin and CB, are in L1. The soma of Martinotti cells are found in L2/3s-6 (68–70), suggesting that the excitatory drive onto these Martinotti cells might be normal in schizophrenia.

There are multiple potential functional consequences of the findings reported here. For example, in L2/3s we found a higher density of vGAT+/CB+/GAD65+ boutons and lower GAD67 levels in vGAT+/CB+/GAD65+/GAD67+ and vGAT+/CB+/GAD67+ boutons in schizophrenia. More vGAT+/CB+/GAD65+ boutons than normal in L2/3s might result in more activity-dependent GABA synthesis in response to increases in neuronal activity. This is because GAD65 activity is regulated by a cycle of activation and inactivation, which is determined by the binding and release, respectively, of its co-factor, pyridoxal 5’-phosphate (PLP). During periods of low activity in PFC networks, GAD65 is largely inactive (unbound to PLP) (71). As neuronal activity increases, more GAD65 binds PLP leading to an increase in GABA synthesis (71). This would suggest that having more vGAT+/CB+/GAD65+ boutons would result in more inhibition during periods of increased network activity. However, this might be counter-balanced by there being lower GAD67 levels in vGAT+/CB+/GAD65+/GAD67+ and vGAT+/CB+/GAD67+ boutons in schizophrenia. It is interesting to speculate that in L2/3s of subjects with schizophrenia lower GAD67 in some CB+ boutons results in increased resting-state activity, which is kept in check due to GAD65 activity-dependent GABA synthesis in other boutons. However, under high demand network activity might be dysfunctional because not enough GABA can be made, not enough GABA is released at some synapses (e.g., vGAT+/CB+/GAD67+ synapses), or too much GABA is released onto specific cells due to there being more vGAT+/CB+/GAD65+ boutons. Interestingly, layer 2/3s pyramidal neurons control the gain of cortical output via L5 neurons (72). Thus, if the net outcome of the CB+ bouton findings in L2/3s is there being more inhibition onto pyramidal neurons PFC output in schizophrenia might be reduced; however, if there is less inhibition output could be higher.

The L5-6 findings here suggest the capacity of CB+ boutons to synthesize GABA is lower in schizophrenia. Subpopulations of pyramidal neurons that reside in deep layers have distinct roles in working memory and information processing (73,74). GABA neuron subpopulations are believed to play specific roles in these processes (75). Several lines of evidence suggest CB GABA neurons connect to deep layer pyramidal neurons with some specificity. For example, a subset of CB neurons synapse onto the distal dendrites of thick-tufted layer 5 neurons through preferential/reciprocal connectivity (76). Thus, a significant reduction in the capacity to synthesize GABA by CB+ neurons that innervate deep PFC pyramidal neurons could result in alterations of these processes, contributing to schizophrenia associated cognitive deficits. Interestingly, our current and previous (16) findings support the idea that only a subset of CB neurons that form synaptic connections onto layer 5–6 pyramidal neurons are altered in schizophrenia suggesting that only specific microcircuits are altered.

Importantly, although the power to detect differences due to comorbid factors was limited, our findings do not appear to be attributable to such factors, consistent with our prior studies of measure of GAD67 and GAD65 (14–16,47). Specifically, the potential effects of 7 comorbid factors on 29 dependent measures (all measures that achieved statistical significance after correcting for multiple comparisons; Supplemental Table S5) were assessed here. Of the resulting 203 tests, which were not corrected for multiple comparisons, only 8 tests resulted in a p-value<0.05 (range was 0.01–0.04), an expected rate of positive findings given the number of tests performed. These findings support the interpretation that the CB bouton alterations in schizophrenia identified here are unlikely to be attributable to comorbid factors. Furthermore, our previous findings in monkeys treated long-term with haloperidol decanoate (7) or oral haloperidol or olanzapine (77) suggest that neither bouton density nor GAD levels are altered in response to antipsychotic administration. However, it is important to acknowledge that we cannot completely exclude the possibility of an effect of comorbid factors on our results.

Together, the vGAT+/CB+/GAD65+/GAD67+, vGAT+/CB+/GAD65+, and vGAT+/CB+/GAD67+ bouton findings presented here suggest the presence of CB cell type- and layer-specific alterations in the PFC of schizophrenia subjects. As such, there does not appear to be a general deficit in CB+ GABA neurons in schizophrenia but cell type- and layer-specific alterations in the disease. Future experiments should identify the affected CB neuron subtypes using cell type-specific CB neuron markers and the laminar location of their cell bodies in schizophrenia.

Supplementary Material

2

KEY RESOURCES TABLE.

Resource Type Specific Reagent or Resource Source or Reference Identifiers Additional Information
Antibody vGAT (mouse host; 1:500) Synaptic Systems, Goettingen, Germany product # 131011 Lots 131011/41 and 131011/42
Antibody CB (rabbit host; 1:1000) Swant, Switzerland product # CB-38a Lot 9.03
Antibody GAD67 (goat host; 1:100 ) R&D Systems, Minneapolis, MN, USA product # AF2086 Lot KRD0110031
Antibody GAD65 (guinea pig host; 1:500) Synaptic Systems product # 198104 Lot 3
Open in a new tab

ACKNOWLEDGEMENTS

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Mental Health, the National Institutes of Health, or the United States Government.

Support:

This work was supported by the NSF (DGE-0549352 to BRR) and NIH (MH043784 to DAL; MH096985 to KNF).

Footnotes

Financial Disclosures

David A. Lewis currently receives investigator-initiated research support from Merck. Drs. Rocco, Wilson, and Fish have no biomedical financial interests or potential conflicts of interest to report.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  • 1.Dienel SJ, Lewis DA (2019): Alterations in cortical interneurons and cognitive function in schizophrenia. Neurobiol Dis. 131:104208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Erlander MG, Tillakaratne NJ, Feldblum S, Patel N, Tobin AJ (1991): Two genes encode distinct glutamate decarboxylases. Neuron. 7:91–100. [DOI] [PubMed] [Google Scholar]
  • 3.Mason GF, Martin DL, Martin SB, Manor D, Sibson NR, Patel A, et al. (2001): Decrease in GABA synthesis rate in rat cortex following GABA-transaminase inhibition correlates with the decrease in GAD(67) protein. Brain Res. 914:81–91. [DOI] [PubMed] [Google Scholar]
  • 4.Battaglioli G, Liu H, Martin DL (2003): Kinetic differences between the isoforms of glutamate decarboxylase: implications for the regulation of GABA synthesis. J Neurochem. 86:879–887. [DOI] [PubMed] [Google Scholar]
  • 5.Patel AB, de Graaf RA, Martin DL, Battaglioli G, Behar KL (2006): Evidence that GAD65 mediates increased GABA synthesis during intense neuronal activity in vivo. J Neurochem. 97:385–396. [DOI] [PubMed] [Google Scholar]
  • 6.Akbarian S, Kim JJ, Potkin SG, Hagman JO, Tafazzoli A, Bunney WE Jr, et al. (1995): Gene expression for glutamic acid decarboxylase is reduced without loss of neurons in prefrontal cortex of schizophrenics. Arch Gen Psychiatry. 52:258–266. [DOI] [PubMed] [Google Scholar]
  • 7.Volk DW, Austin MC, Pierri JN, Sampson AR, Lewis DA (2000): Decreased glutamic acid decarboxylase67 messenger RNA expression in a subset of prefrontal cortical gamma-aminobutyric acid neurons in subjects with schizophrenia. Arch Gen Psychiatry. 57:237–245. [DOI] [PubMed] [Google Scholar]
  • 8.Rocco BR, Lewis DA, Fish KN (2016): Markedly Lower Glutamic Acid Decarboxylase 67 Protein Levels in a Subset of Boutons in Schizophrenia. Biol Psychiatry. 79:1006–1015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Conde F, Lund JS, Jacobowitz DM, Baimbridge KG, Lewis DA (1994): Local circuit neurons immunoreactive for calretinin, calbindin D-28k or parvalbumin in monkey prefrontal cortex: distribution and morphology. J Comp Neurol. 341:95–116. [DOI] [PubMed] [Google Scholar]
  • 10.del Rio MR, DeFelipe J (1996): Colocalization of calbindin D-28k, calretinin, and GABA immunoreactivities in neurons of the human temporal cortex. J Comp Neurol. 369:472–482. [DOI] [PubMed] [Google Scholar]
  • 11.Barinka F, Druga R (2010): Calretinin expression in the mammalian neocortex: a review. Physiological research / Academia Scientiarum Bohemoslovaca. 59:665–677. [DOI] [PubMed] [Google Scholar]
  • 12.Gabbott PL, Bacon SJ (1996): Local circuit neurons in the medial prefrontal cortex (areas 24a,b,c, 25 and 32) in the monkey: II. Quantitative areal and laminar distributions. J Comp Neurol. 364:609–636. [DOI] [PubMed] [Google Scholar]
  • 13.Hashimoto T, Volk DW, Eggan SM, Mirnics K, Pierri JN, Sun Z, et al. (2003): Gene expression deficits in a subclass of GABA neurons in the prefrontal cortex of subjects with schizophrenia. J Neurosci. 23:6315–6326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Curley AA, Arion D, Volk DW, Asafu-Adjei JK, Sampson AR, Fish KN, et al. (2011): Cortical deficits of glutamic acid decarboxylase 67 expression in schizophrenia: clinical, protein, and cell type-specific features. Am J Psychiatry. 168:921–929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Fish KN, Rocco BR, DeDionisio AM, Dienel SJ, Sweet RA, Lewis DA (2021): Altered Parvalbumin Basket Cell Terminals in the Cortical Visuospatial Working Memory Network in Schizophrenia. Biol Psychiatry. 90:47–57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Rocco BR, DeDionisio AM, Lewis DA, Fish KN (2017): Alterations in a Unique Class of Cortical Chandelier Cell Axon Cartridges in Schizophrenia. Biol Psychiatry. 82:40–48. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Beasley CL, Zhang ZJ, Patten I, Reynolds GP (2002): Selective deficits in prefrontal cortical GABAergic neurons in schizophrenia defined by the presence of calcium-binding proteins. Biol Psychiatry. 52:708–715. [DOI] [PubMed] [Google Scholar]
  • 18.Sakai T, Oshima A, Nozaki Y, Ida I, Haga C, Akiyama H, et al. (2008): Changes in density of calcium-binding-protein-immunoreactive GABAergic neurons in prefrontal cortex in schizophrenia and bipolar disorder. Neuropathology. 28:143–150. [DOI] [PubMed] [Google Scholar]
  • 19.Reynolds GP, Zhang ZJ, Beasley CL (2001): Neurochemical correlates of cortical GABAergic deficits in schizophrenia: selective losses of calcium binding protein immunoreactivity. Brain Res Bull. 55:579–584. [DOI] [PubMed] [Google Scholar]
  • 20.Hashimoto T, Bazmi HH, Mirnics K, Wu Q, Sampson AR, Lewis DA (2008): Conserved regional patterns of GABA-related transcript expression in the neocortex of subjects with schizophrenia. Am J Psychiatry. 165:479–489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Morris HM, Hashimoto T, Lewis DA (2008): Alterations in somatostatin mRNA expression in the dorsolateral prefrontal cortex of subjects with schizophrenia or schizoaffective disorder. Cereb Cortex. 18:1575–1587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Mellios N, Huang HS, Baker SP, Galdzicka M, Ginns E, Akbarian S (2009): Molecular determinants of dysregulated GABAergic gene expression in the prefrontal cortex of subjects with schizophrenia. Biol Psychiatry. 65:1006–1014. [DOI] [PubMed] [Google Scholar]
  • 23.Fung SJ, Webster MJ, Sivagnanasundaram S, Duncan C, Elashoff M, Weickert CS (2010): Expression of interneuron markers in the dorsolateral prefrontal cortex of the developing human and in schizophrenia. Am J Psychiatry. 167:1479–1488. [DOI] [PubMed] [Google Scholar]
  • 24.Volk DW, Matsubara T, Li S, Sengupta EJ, Georgiev D, Minabe Y, et al. (2012): Deficits in transcriptional regulators of cortical parvalbumin neurons in schizophrenia. Am J Psychiatry. 169:1082–1091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Gonzalez-Albo MC, Elston GN, DeFelipe J (2001): The human temporal cortex: characterization of neurons expressing nitric oxide synthase, neuropeptides and calcium-binding proteins, and their glutamate receptor subunit profiles. Cereb Cortex. 11:1170–1181. [DOI] [PubMed] [Google Scholar]
  • 26.Gonchar Y, Burkhalter A (1997): Three distinct families of GABAergic neurons in rat visual cortex. Cereb Cortex. 7:347–358. [DOI] [PubMed] [Google Scholar]
  • 27.Kubota Y, Hattori R, Yui Y (1994): Three distinct subpopulations of GABAergic neurons in rat frontal agranular cortex. Brain Res. 649:159–173. [DOI] [PubMed] [Google Scholar]
  • 28.Rogers JH (1992): Immunohistochemical markers in rat cortex: co-localization of calretinin and calbindin-D28k with neuropeptides and GABA. Brain Res. 587:147–157. [DOI] [PubMed] [Google Scholar]
  • 29.Hayes TL, Cameron JL, Fernstrom JD, Lewis DA (1991): A comparative analysis of the distribution of prosomatostatin-derived peptides in human and monkey neocortex. J Comp Neurol. 303:584–599. [DOI] [PubMed] [Google Scholar]
  • 30.Hayes TL, Lewis DA (1992): Nonphosphorylated neurofilament protein and calbindin immunoreactivity in layer III pyramidal neurons of human neocortex. Cereb Cortex. 2:56–67. [DOI] [PubMed] [Google Scholar]
  • 31.Hof PR, Morrison JH (1991): Neocortical neuronal subpopulations labeled by a monoclonal antibody to calbindin exhibit differential vulnerability in Alzheimer’s disease. Experimental Neurology. 111:293–301. [DOI] [PubMed] [Google Scholar]
  • 32.Kobayashi K, Emson PC, Mountjoy CQ, Thornton SN, Lawson DE, Mann DM (1990): Cerebral cortical calbindin D28K and parvalbumin neurones in Down’s syndrome. Neurosci Lett. 113:17–22. [DOI] [PubMed] [Google Scholar]
  • 33.Daviss SR, Lewis DA (1995): Local circuit neurons of the prefrontal cortex in schizophrenia: selective increase in the density of calbindin-immunoreactive neurons. Psychiatry Res. 59:81–96. [DOI] [PubMed] [Google Scholar]
  • 34.Chaudhry FA, Reimer RJ, Bellocchio EE, Danbolt NC, Osen KK, Edwards RH, et al. (1998): The vesicular GABA transporter, VGAT, localizes to synaptic vesicles in sets of glycinergic as well as GABAergic neurons. Journal of Neuroscience. 18:9733–9750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Beneyto M, Sibille E, Lewis D (2008): Human postmortem brain research in mental illness syndromes, in Neurobiology of Mental Illness. Edited by Charney DS, Nestler E. New York, Oxford University Press.202–214. [Google Scholar]
  • 36.Hoftman GD, Volk DW, Bazmi HH, Li S, Sampson AR, Lewis DA (2013): Altered Cortical Expression of GABA-Related Genes in Schizophrenia: Illness Progression vs Developmental Disturbance. Schizophr Bull. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Jiao Y, Sun Z, Lee T, Fusco FR, Kimble TD, Meade CA, et al. (1999): A simple and sensitive antigen retrieval method for free-floating and slide-mounted tissue sections. JNeurosciMeth. 93:149–162. [DOI] [PubMed] [Google Scholar]
  • 38.Pierri JN, Chaudry AS, Woo TU, Lewis DA (1999): Alterations in chandelier neuron axon terminals in the prefrontal cortex of schizophrenic subjects. Am J Psychiatry. 156:1709–1719. [DOI] [PubMed] [Google Scholar]
  • 39.Kubota Y, Shigematsu N, Karube F, Sekigawa A, Kato S, Yamaguchi N, et al. (2011): Selective coexpression of multiple chemical markers defines discrete populations of neocortical GABAergic neurons. Cereb Cortex. 21:1803–1817. [DOI] [PubMed] [Google Scholar]
  • 40.Hendry SH, Jones EG, Emson PC (1984): Morphology, distribution, and synaptic relations of somatostatin- and neuropeptide Y-immunoreactive neurons in rat and monkey neocortex. J Neurosci. 4:2497–2517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Lewis DA, Campbell MJ, Morrison JH (1986): An immunohistochemical characterization of somatostatin-28 and somatostatin-28 (1–12) in monkey prefrontal cortex. The Journal of Comparative Neurology. 248:1–18. [DOI] [PubMed] [Google Scholar]
  • 42.Kawaguchi Y, Kubota Y (1997): GABAergic cell subtypes and their synaptic connections in rat frontal cortex. Cerebral Cortex. 7:476–486. [DOI] [PubMed] [Google Scholar]
  • 43.Pesold C, Liu WS, Guidotti A, Costa E, Caruncho HJ (1999): Cortical bitufted, horizontal, and Martinotti cells preferentially express and secrete reelin into perineuronal nets, nonsynaptically modulating gene expression. Proc Natl Acad Sci U S A. 96:3217–3222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Ma Y, Hu H, Berrebi AS, Mathers PH, Agmon A (2006): Distinct subtypes of somatostatin-containing neocortical interneurons revealed in transgenic mice. J Neurosci. 26:5069–5082. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.McIntire SL, Reimer RJ, Schuske K, Edwards RH, Jorgensen EM (1997): Identification and characterization of the vesicular GABA transporter. Nature. 389:870–876. [DOI] [PubMed] [Google Scholar]
  • 46.Rocco BR, Sweet RA, Lewis DA, Fish KN (2016): GABA-Synthesizing Enzymes in Calbindin and Calretinin Neurons in Monkey Prefrontal Cortex. Cereb Cortex. 26:2191–2204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Fish KN, Rocco BR, Lewis DA (2018): Laminar Distribution of Subsets of GABAergic Axon Terminals in Human Prefrontal Cortex. Front Neuroanat. 12:9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Terman A, Brunk UT (2004): Lipofuscin. Int J Biochem Cell Biol. 36:1400–1404. [DOI] [PubMed] [Google Scholar]
  • 49.Seehafer SS, Pearce DA (2006): You say lipofuscin, we say ceroid: defining autofluorescent storage material. Neurobiol Aging. 27:576–588. [DOI] [PubMed] [Google Scholar]
  • 50.Glausier JR, Fish KN, Lewis DA (2014): Altered parvalbumin basket cell inputs in the dorsolateral prefrontal cortex of schizophrenia subjects. Mol Psychiatry. 19:30–36. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Tsubomoto M, Kawabata R, Zhu X, Minabe Y, Chen K, Lewis DA, et al. (2019): Expression of Transcripts Selective for GABA Neuron Subpopulations across the Cortical Visuospatial Working Memory Network in the Healthy State and Schizophrenia. Cereb Cortex. 29:3540–3550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Benjamini Y, Hochberg Y (1995): Controlling the False Discovery Rate - a Practical and Powerful Approach to Multiple Testing. J R Stat Soc B. 57:289–300. [Google Scholar]
  • 53.Schmalz X, Biurrun Manresa J, Zhang L (2021): What is a Bayes factor? Psychol Methods. [DOI] [PubMed] [Google Scholar]
  • 54.Dienes Z (2011): Bayesian Versus Orthodox Statistics: Which Side Are You On? Perspect Psychol Sci. 6:274–290. [DOI] [PubMed] [Google Scholar]
  • 55.Rouder JN, Speckman PL, Sun D, Morey RD, Iverson G (2009): Bayesian t tests for accepting and rejecting the null hypothesis. Psychon Bull Rev. 16:225–237. [DOI] [PubMed] [Google Scholar]
  • 56.Wagenmakers EJ (2007): A practical solution to the pervasive problems of p values. Psychon Bull Rev. 14:779–804. [DOI] [PubMed] [Google Scholar]
  • 57.Senkowski D, Gallinat J (2015): Dysfunctional prefrontal gamma-band oscillations reflect working memory and other cognitive deficits in schizophrenia. Biol Psychiatry. 77:1010–1019. [DOI] [PubMed] [Google Scholar]
  • 58.Fish KN, Hoftman GD, Sheikh W, Kitchens M, Lewis DA (2013): Parvalbumin-containing chandelier and basket cell boutons have distinctive modes of maturation in monkey prefrontal cortex. J Neurosci. 33:8352–8358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59.Hyde TM, Lipska BK, Ali T, Mathew SV, Law AJ, Metitiri OE, et al. (2011): Expression of GABA signaling molecules KCC2, NKCC1, and GAD1 in cortical development and schizophrenia. J Neurosci. 31:11088–11095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 60.Hoftman GD, Volk DW, Bazmi HH, Li S, Sampson AR, Lewis DA (2015): Altered cortical expression of GABA-related genes in schizophrenia: illness progression vs developmental disturbance. Schizophr Bull. 41:180–191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 61.Angevine JB Jr, Sidman RL (1961): Autoradiographic study of cell migration during histogenesis of cerebral cortex in the mouse. Nature. 192:766–768. [DOI] [PubMed] [Google Scholar]
  • 62.Rakic P (1974): Neurons in rhesus monkey visual cortex: systematic relation between time of origin and eventual disposition. Science. 183:425–427. [DOI] [PubMed] [Google Scholar]
  • 63.Fish KN, Sweet RA, Lewis DA (2011): Differential distribution of proteins regulating GABA synthesis and reuptake in axon boutons of subpopulations of cortical interneurons. Cereb Cortex. 21:2450–2460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 64.Naka A, Veit J, Shababo B, Chance RK, Risso D, Stafford D, et al. (2019): Complementary networks of cortical somatostatin interneurons enforce layer specific control. Elife. 8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 65.Hoftman GD, Datta D, Lewis DA (2017): Layer 3 Excitatory and Inhibitory Circuitry in the Prefrontal Cortex: Developmental Trajectories and Alterations in Schizophrenia. Biol Psychiatry. 81:862–873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 66.Lewis DA, Curley AA, Glausier JR, Volk DW (2012): Cortical parvalbumin interneurons and cognitive dysfunction in schizophrenia. Trends Neurosci. 35:57–67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 67.Arion D, Corradi JP, Tang S, Datta D, Boothe F, He A, et al. (2015): Distinctive transcriptome alterations of prefrontal pyramidal neurons in schizophrenia and schizoaffective disorder. Mol Psychiatry. 20:1397–1405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 68.Ferrer I, Fabregues I, Condom E (1986): A Golgi study of the sixth layer of the cerebral cortex. II. The gyrencephalic brain of Carnivora, Artiodactyla and Primates. J Anat. 146:87–104. [PMC free article] [PubMed] [Google Scholar]
  • 69.Markram H, Toledo-Rodriguez M, Wang Y, Gupta A, Silberberg G, Wu C (2004): Interneurons of the neocortical inhibitory system. Nat Rev Neurosci. 5:793–807. [DOI] [PubMed] [Google Scholar]
  • 70.Xu X, Callaway EM (2009): Laminar specificity of functional input to distinct types of inhibitory cortical neurons. J Neurosci. 29:70–85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 71.Kleppner SR, Tobin AJ (2002): GABA. In: Ramachandran VS, editor. Encyclopedia of the Human Brain: Academic Press, pp 353–367. [Google Scholar]
  • 72.Quiquempoix M, Fayad SL, Boutourlinsky K, Leresche N, Lambert RC, Bessaih T (2018): Layer 2/3 Pyramidal Neurons Control the Gain of Cortical Output. Cell reports. 24:2799–2807 e2794. [DOI] [PubMed] [Google Scholar]
  • 73.Bae JW, Jeong H, Yoon YJ, Bae CM, Lee H, Paik SB, et al. (2021): Parallel processing of working memory and temporal information by distinct types of cortical projection neurons. Nature communications. 12:4352. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 74.Garcia AF, Crummy EA, Webb IG, Nooney MN, Ferguson SM (2021): Distinct populations of cortical pyramidal neurons mediate drug reward and aversion. Nature communications. 12:182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 75.Wang XJ, Tegner J, Constantinidis C, Goldman-Rakic PS (2004): Division of labor among distinct subtypes of inhibitory neurons in a cortical microcircuit of working memory. Proc Natl Acad Sci U S A. 101:1368–1373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 76.Hilscher MM, Leao RN, Edwards SJ, Leao KE, Kullander K (2017): Chrna2-Martinotti Cells Synchronize Layer 5 Type A Pyramidal Cells via Rebound Excitation. PLoS Biol. 15:e2001392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 77.Hashimoto T, Arion D, Unger T, Maldonado-Aviles JG, Morris HM, Volk DW, et al. (2008): Alterations in GABA-related transcriptome in the dorsolateral prefrontal cortex of subjects with schizophrenia. Mol Psychiatry. 13:147–161. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

2
NIHMS1856217-supplement-2.pdf (2.9MB, pdf)

RESOURCES