Stephen M. Eggan

Gene Expression Deficits in a Subclass of GABA Neurons in the Prefrontal Cortex of Subjects with Schizophrenia

Markers of inhibitory neurotransmission are altered in the prefrontal cortex (PFC) of subjects with schizophrenia, and several lines of evidence suggest that these alterations may be most prominent in the subset of GABA-containing neurons that express the calcium-binding protein, parvalbumin (PV). To test this hypothesis, we evaluated the expression of mRNAs for PV, another calcium-binding protein, calretinin (CR), and glutamic acid decarboxylase (GAD67) in postmortem brain specimens from 15 pairs of subjects with schizophrenia and matched control subjects using single- and dual-label in situ hybridization. Signal intensity for PV mRNA expression in PFC area 9 was significantly decreased in the subjects with schizophrenia, predominately in layers III and IV. Analysis at the cellular level revealed that this decrease was attributable principally to a reduction in PV mRNA expression per neuron rather than by a decreased density of PV mRNA-positive neurons. In contrast, the same measures of CR mRNA expression were not altered in schizophrenia. These findings were confirmed by findings from cDNA microarray studies using different probes. Across the subjects with schizophrenia, the decrease in neuronal PV mRNA expression was highly associated (r = 0.84) with the decrease in the density of neurons containing detectable levels of GAD67 mRNA. Furthermore, simultaneous detection of PV and GAD67 mRNAs revealed that in subjects with schizophrenia only 55% of PV mRNA-positive neurons had detectable levels of GAD67 mRNA. Given the critical role that PV-containing GABA neurons appear to play in regulating the cognitive functions mediated by the PFC, the selective alterations in gene expression in these neurons may contribute to the cognitive deficits characteristic of schizophrenia.

Reduced Cortical Cannabinoid 1 Receptor Messenger RNA and Protein Expression in Schizophrenia

CONTEXT Cannabis use is associated with both impaired cognitive functions, including working memory, and an increased risk of schizophrenia. Schizophrenia is characterized by impairments in working memory that are associated with reduced gamma-aminobutyric acid (GABA) neurotransmission in the dorsolateral prefrontal cortex. The cannabinoid 1 receptor (CB1R) is highly expressed in the dorsolateral prefrontal cortex, is contained in the axon terminals of a subpopulation of perisomatic-targeting GABA neurons, and, when activated, suppresses the release of GABA. OBJECTIVE To determine the potential relationship between CB1R signaling and altered GABA neurotransmission in schizophrenia by evaluating CB1R messenger RNA (mRNA) and protein expression in the dorsolateral prefrontal cortex. DESIGN In situ hybridization and immunocytochemistry techniques were used to examine the cortical levels of CB1R mRNA and protein, respectively. SETTING Brain specimens were obtained from autopsies conducted at the Allegheny County Medical Examiners Office, Pittsburgh, Pennsylvania. PARTICIPANTS Postmortem brain specimens from 23 pairs of subjects with schizophrenia and age-, sex-, and postmortem interval-matched comparison subjects, as well as brain specimens from 18 macaque monkeys with long-term exposure to haloperidol, olanzapine, or placebo. MAIN OUTCOME MEASURES Optical density measures of CB1R mRNA expression and protein levels and correlations with previously reported glutamic acid decarboxylase 67 and cholecystokinin mRNA measures. RESULTS Levels of CB1R mRNA were significantly lower by 14.8% in the subjects with schizophrenia. Similarly, CB1R protein levels, assessed by radioimmunocytochemistry and standard immunocytochemistry, were significantly decreased by 11.6% and 13.9%, respectively. Group differences in CB1R mRNA levels were significantly correlated with those in glutamic acid decarboxylase 67 and cholecystokinin mRNA levels. Expression of CB1R mRNA was not changed in antipsychotic-exposed monkeys, and neither CB1R mRNA levels nor protein levels were affected by potential confounding factors in the subjects with schizophrenia. CONCLUSIONS This combination of findings suggests the testable hypothesis that reduced CB1R mRNA and protein levels in schizophrenia represent a compensatory mechanism to increase GABA transmission from perisomatic-targeting cholecystokinin interneurons with impaired GABA synthesis.

Postnatal development of prefrontal inhibitory circuits and the pathophysiology of cognitive dysfunction in schizophrenia

Abstract: The typical appearance of the clinical features of schizophrenia during late adolescence or early adulthood suggests that adolescence‐related neurodevelopmental events may contribute to the pathophysiology of this disorder. Here the role that GABA‐mediated inhibition in the dorsal lateral prefrontal cortex (DLPFC) plays in regulating working memory, a core cognitive process that matures late and that is disturbed in schizophrenia, is reviewed. Recent studies are summarized that demonstrate (1) that certain pre‐ and postsynaptic markers of GABA neurotransmission in the monkey DLPFC exhibit striking changes during adolescence, and (2) that these same markers are markedly altered in the DLPFC of subjects with schizophrenia. The implications of these findings for treatment and prevention strategies are discussed.

Postnatal development of pre- and postsynaptic GABA markers at chandelier cell connections with pyramidal neurons in monkey prefrontal cortex

The protracted postnatal maturation of the primate prefrontal cortex (PFC) is associated with substantial changes in the number of excitatory synapses on pyramidal neurons, whereas the total number of inhibitory synapses appears to remain constant. In this study, we sought to determine whether the developmental changes in excitatory input to pyramidal cells are paralleled by changes in functional markers of inhibitory inputs to pyramidal neurons. The chandelier subclass of γ‐aminobutyric acid (GABA) neurons provides potent inhibitory control over pyramidal neurons by virtue of their axon terminals, which form distinct vertical structures (termed cartridges) that synapse at the axon initial segment (AIS) of pyramidal neurons. Thus, we examined the relative densities, laminar distributions, and lengths of presynaptic chandelier axon cartridges immunoreactive for the GABA membrane transporter 1 (GAT1) or the calcium‐binding protein parvalbumin (PV) and of postsynaptic pyramidal neuron AIS immunoreactive for the GABAA receptor α2 subunit (GABAA α2) in PFC area 46 of 38 rhesus monkeys (Macaca mulatta). From birth through 2 years of age, the relative densities and laminar distributions of these three markers exhibited different trajectories, suggesting developmental shifts in the weighting of at least some factors that determine inhibition at the AIS. In contrast, from 2 to 4 years of age, all three markers exhibited similar declines in density and length that paralleled the periadolescent pruning of excitatory synapses to pyramidal neurons. Across development, the predominant laminar location of PV‐labeled cartridges and GABAA α2‐immunoreactive AIS shifted from the middle to superficial layers, whereas the laminar distribution of GAT1‐positive cartridges did not change. Together, these findings suggest that the maturation of inhibitory inputs to the AIS of PFC pyramidal neurons is a complex process that may differentially affect the firing patterns of subpopulations of pyramidal neurons at specific postnatal time points. J. Comp. Neurol. 465:385–400, 2003.

Cannabinoid CB1 Receptor Immunoreactivity in the Prefrontal Cortex: Comparison of Schizophrenia and Major Depressive Disorder

We recently showed that measures of cannabinoid 1 receptor (CB1R) mRNA and protein were significantly reduced in dorsolateral prefrontal cortex (DLPFC) area 9 in schizophrenia subjects relative to matched normal comparison subjects. However, other studies have reported unaltered or higher measures of CB1R levels in schizophrenia. To determine whether these discrepancies reflect differences across brain regions or across subject groups (eg, presence of depression, cannabis exposure, etc), we used immunocytochemical techniques to determine whether lower levels of CB1R immunoreactivity are (1) present in another DLPFC region, area 46, in the same subjects with schizophrenia, (2) present in area 46 in a new cohort of schizophrenia subjects, (3) present in major depressive disorder (MDD) subjects, or (4) attributable to factors other than a diagnosis of schizophrenia, including prior cannabis use. CB1R immunoreactivity levels in area 46 were significantly 19% lower in schizophrenia subjects relative to matched normal comparison subjects, a deficit similar to that observed in area 9 in the same subjects. In a new cohort of subjects, CB1R immunoreactivity levels were significantly 20 and 23% lower in schizophrenia subjects relative to matched comparison and MDD subjects, respectively. The lower levels of CB1R immunoreactivity in schizophrenia subjects were not explained by other factors such as cannabis use, suicide, or pharmacological treatment. In addition, CB1R immunoreactivity levels were not altered in monkeys chronically exposed to haloperidol. Thus, the lower levels of CB1R immunoreactivity may be common in schizophrenia, conserved across DLPFC regions, not present in MDD, and not attributable to other factors, and thus a reflection of the underlying disease process.

Relationship of cannabinoid CB1 receptor and cholecystokinin immunoreactivity in monkey dorsolateral prefrontal cortex.

Exposure to cannabis impairs cognitive functions reliant on the circuitry of the dorsolateral prefrontal cortex (DLPFC) and increases the risk of schizophrenia. The actions of cannabis are mediated via the brain cannabinoid 1 receptor (CB1R), which in rodents is heavily localized to the axon terminals of cortical GABA basket neurons that contain cholecystokinin (CCK). Differences in the laminar distribution of CB1R-immunoreactive (IR) axons have been reported between rodent and monkey neocortex, suggesting that the cell type(s) containing CB1Rs, and the synaptic targets of CB1R-IR axon terminals, may differ across species; however, neither the relationship of CB1Rs to CCK-containing interneurons, nor the postsynaptic targets of CB1R and CCK axon terminals, have been examined in primate DLPFC. Consequently, we compared the distribution patterns of CB1R- and CCK-IR structures, determined the proportions of CB1R and CCK neurons that were dual-labeled, and identified the synaptic types and postsynaptic targets of CB1R- and CCK-IR axon terminals in macaque monkey DLPFC. By light microscopy, CB1R- and CCK-IR axons exhibited a similar laminar distribution, with their greatest densities in layer 4. Dual-label fluorescence experiments demonstrated that 91% of CB1R-IR neurons were immunopositive for CCK, whereas only 51% of CCK-IR neurons were immunopositive for CB1R. By electron microscopy, all synapses formed by CB1R-IR axon terminals were symmetric, whereas CCK-IR axon terminals formed both symmetric (88%) and asymmetric (12%) synapses. The primary postsynaptic target of both CB1R- and CCK-IR axon terminals forming symmetric synapses was dendritic shafts (81-88%), with the remainder targeting cell bodies or dendritic spines. Thus, despite species differences in laminar distribution, CB1Rs are principally localized to CCK basket neuron axons in both rodent neocortex and monkey DLPFC. These axons target the perisomatic region of pyramidal neurons, providing a potential anatomical substrate for the impaired function of the DLPFC associated with cannabis use and schizophrenia.

Synaptic targets of calretinin-containing axon terminals in macaque monkey prefrontal cortex

The coordinated activity of specific populations of pyramidal cells and GABA-containing, local circuit neurons in the primate prefrontal cortex (PFC) appears to be critical for working memory. Different subclasses of GABA-containing neurons can be distinguished by their content of the calcium-binding proteins parvalbumin (PV) and calretinin (CR). The postsynaptic targets of PV-containing cells have been well characterized in the primate PFC, but the postsynaptic targets of CR-containing neurons in this cortical region remain unknown. In the present study, we used immuno-electron microscopy to examine the synaptic type and postsynaptic targets of CR-immunoreactive (IR) axon terminals in the superficial and deep layers of macaque monkey PFC. Labeled axon terminals formed both symmetric and asymmetric synapses. Within the superficial layers, 93% of the synapses formed by CR-IR were symmetric, whereas in the deep layers the labeled axon terminals forming synapses were more evenly divided between symmetric (57%) and asymmetric (43%). The primary postsynaptic target of these two populations of CR-IR axon terminals also differed; unlabeled dendritic shafts were the predominant target of the symmetric synapses, whereas dendritic spines were the most common target of the asymmetric synapses. In addition, the mean cross-sectional area of the terminals forming asymmetric synapses was significantly larger than that of the terminals forming symmetric synapses. The presence of CR-IR asymmetric synapses suggested that they might arise from neurons that do not utilize GABA; indeed, dual-label fluorescent immunocytochemistry revealed that a subpopulation (23%) of CR-containing neurons in monkey PFC were not GABA-IR. These findings indicate that the synaptology of CR-containing neurons is more heterogeneous than that of PV-containing cells and suggests that the contributions of CR-containing neurons to cognitive processes mediated by the PFC may be more diverse.

Development of Cannabinoid 1 Receptor Protein and Messenger RNA in Monkey Dorsolateral Prefrontal Cortex

Adolescent cannabis use is associated with an increased risk of schizophrenia and with impairments in cognitive processes reliant on the circuitry of the dorsolateral prefrontal cortex (DLPFC). Additionally, maternal cannabis use is associated with cognitive dysfunction in offspring. The effects of cannabis are mediated by the cannabinoid 1 receptor (CB1R), which is present in high density in the primate DLPFC. In order to determine how developmental changes in CB1Rs might render DLPFC circuitry vulnerable to cannabis exposure, we examined the density and innervation patterns of CB1R-immunoreactive (IR) axons and the expression of CB1R mRNA in the DLPFC from 81 macaque monkeys, ranging in age from embryonic 82 days to 18 years. Overall CB1R immunoreactivity in the gray matter robustly increased during the perinatal period and achieved adult levels by 1 week postnatal. However, laminar analyses revealed that CB1R-IR axon density significantly decreased with age in layers 1-2 but significantly increased in layer 4, especially during adolescence. In contrast, CB1R mRNA levels were highest 1 week postnatal, declined over the next 2 months, and then remained unchanged into adulthood. These findings provide a potential substrate for discrete, age-dependent effects of cannabis exposure on the maturation of primate DLPFC circuitry.

Role of glutamic acid decarboxylase 67 in regulating cortical parvalbumin and GABA membrane transporter 1 expression: Implications for schizophrenia

Markers of GABA neurotransmission are altered in multiple regions of the neocortex in individuals with schizophrenia. Lower levels of glutamic acid decarboxylase 67 (GAD67) mRNA and protein, which is responsible for most cortical GABA synthesis, are accompanied by lower levels of GABA membrane transporter 1 (GAT1) mRNA. These alterations are thought to be most prominent in the parvalbumin (PV)-containing subclass of interneurons, which also contain lower levels of PV mRNA. Since GAT1 and PV each reduce the availability of GABA at postsynaptic receptors, lower levels of GAT1 and PV mRNAs have been hypothesized to represent compensatory responses to an upstream reduction in cortical GABA synthesis in schizophrenia. However, such cause-and-effect hypotheses cannot be directly tested in a human illness. Consequently, we used two mouse models with reduced GAD67 expression specifically in PV neurons (PV(GAD67+/-)) or in all interneurons (GABA(GAD67+/-)) and quantified GAD67, GAT1 and PV mRNA levels using methods identical to those employed in studies of schizophrenia. Cortical levels of PV or GAT1 mRNAs were not altered in PV(GAD67+/-) mice during postnatal development or in adulthood. Furthermore, cellular analyses confirmed the predicted reduction in GAD67 mRNA, but failed to show a deficit in PV mRNA in these animals. Levels of PV and GAT1 mRNAs were also unaltered in GABA(GAD67+/-) mice. Thus, mouse lines with cortical reductions in GAD67 mRNA that match or exceed those present in schizophrenia, and that differ in the developmental timing and cell type-specificity of the GAD67 deficit, failed to provide proof-of-concept evidence that lower PV and GAT1 expression in schizophrenia are a consequence of lower GAD67 expression. Together, these findings suggest that the correlated decrements in cortical GAD67, PV and GAT1 mRNAs in schizophrenia may be a common consequence of some other upstream factor.

Reciprocal alterations in cortical cannabinoid receptor 1 binding relative to protein immunoreactivity and transcript levels in schizophrenia

The deleterious effects of cannabis use in schizophrenia have been linked, in part, to underlying disturbances in endogenous cannabinoid signaling in the prefrontal cortex. However, while receptor autoradiography studies of the primary cannabinoid receptor (CB1R) have consistently found higher CB1R binding in the prefrontal cortex in schizophrenia, deficits in CB1R mRNA levels and protein immunoreactivity have also been reported in the illness. To investigate this apparent discrepancy, we quantified CB1R binding using receptor autoradiography with the selective CB1R ligand [(3)H]-OMAR in the prefrontal cortex of 21 subjects with schizophrenia who were previously found to have lower levels of both CB1R mRNA using in situ hybridization and CB1R protein using radioimmunocytochemistry relative to matched healthy comparison subjects. We observed higher levels of [(3)H]-OMAR binding in the prefrontal cortex of schizophrenia subjects that did not appear to be attributable to psychotropic medications or substance abuse. The combination of lower levels of CB1R mRNA and immunoreactivity with higher CB1R receptor binding may reflect 1) altered trafficking of the receptor resulting in higher levels of membrane-bound CB1R or 2) higher CB1R affinity. In either case, greater CB1R receptor availability may contribute to the increased susceptibility of schizophrenia subjects to the deleterious effects of cannabis use.