Joseph N. Pierri

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.

Relationship of Brain-Derived Neurotrophic Factor and Its Receptor TrkB to Altered Inhibitory Prefrontal Circuitry in Schizophrenia

Dysfunction of inhibitory neurons in the prefrontal cortex (PFC), represented by decreased expression of GABA-related genes such as the 67 kDa isoform of glutamate decarboxylase (GAD67) and parvalbumin (PV), appears to contribute to cognitive deficits in subjects with schizophrenia. We investigated the involvement of signaling mediated by brain-derived neurotrophic factor (BDNF) and its receptor tyrosine kinase TrkB in producing the altered GABA-related gene expression in schizophrenia. In 15 pairs of subjects with schizophrenia and matched control subjects, both BDNF and TrkB mRNA levels, as assessed by in situ hybridization, were significantly decreased in the PFC of the subjects with schizophrenia, whereas the levels of mRNA encoding the receptor tyrosine kinase for neurotrophin-3, TrkC, were unchanged. In this cohort, within-pair changes in TrkB mRNA levels were significantly correlated with those in both GAD67 and PV mRNA levels. Decreased BDNF, TrkB, and GAD67 mRNA levels were replicated in a second cohort of 12 subject pairs. In the combined cohorts, the correlation between within-pair changes in TrkB and GAD67 mRNA levels was significantly stronger than the correlation between the changes in BDNF and GAD67 mRNA levels. Neither BDNF nor TrkB mRNA levels were changed in the PFC of monkeys after a long-term exposure to haloperidol. Genetically introduced decreases in TrkB expression, but not in BDNF expression, also resulted in decreased GAD67 and PV mRNA levels in the PFC of adult mice; in addition, the cellular pattern of altered GAD67 mRNA expression paralleled that present in schizophrenia. Decreased TrkB signaling appears to underlie the dysfunction of inhibitory neurons in the PFC of subjects with schizophrenia.

The influence of chronic exposure to antipsychotic medications on brain size before and after tissue fixation: a comparison of haloperidol and olanzapine in macaque monkeys.

It is unclear to what degree antipsychotic therapy confounds longitudinal imaging studies and post-mortem studies of subjects with schizophrenia. To investigate this problem, we developed a non-human primate model of chronic antipsychotic exposure. Three groups of six macaque monkeys each were exposed to oral haloperidol, olanzapine or sham for a 17–27 month period. The resulting plasma drug levels were comparable to those seen in subjects with schizophrenia treated with these medications. After the exposure, we observed an 8–11% reduction in mean fresh brain weights as well as left cerebrum fresh weights and volumes in both drug-treated groups compared to sham animals. The differences were observed across all major brain regions (frontal, parietal, temporal, occipital, and cerebellum), but appeared most robust in the frontal and parietal regions. Stereological analysis of the parietal region using Cavalieris principle revealed similar volume reductions in both gray and white matter. In addition, we assessed the subsequent tissue shrinkage due to standard histological processing and found no evidence of differential shrinkage due to drug exposure. However, we observed a pronounced general shrinkage effect of ∼20% and a highly significant variation in shrinkage across brain regions. In conclusion, chronic exposure of non-human primates to antipsychotics was associated with reduced brain volume. Antipsychotic medication may confound post-mortem studies and longitudinal imaging studies of subjects with schizophrenia that depend upon volumetric measures.

Altered GABA neurotransmission and prefrontal cortical dysfunction in schizophrenia

Dysfunction of the dorsolateral prefrontal cortex appears to be a central feature of the pathophysiology of schizophrenia, and this dysfunction may be related to alterations in gamma aminobutyric acid (GABA) neurotransmission. Determining the causes and consequences of altered GABA neurotransmission in schizophrenia, and the relationship of these changes to other abnormalities in prefrontal cortical circuitry, requires an understanding of which of the multiple subpopulations of cortical GABA neurons are affected. The chandelier class of GABA neurons, especially those located in the middle layers of the prefrontal cortex (PFC), have been hypothesized to be preferentially involved in schizophrenia because they 1) receive direct synaptic input from dopamine axons, 2) exert powerful inhibitory control over the excitatory output of layer 3 pyramidal neurons, and 3) undergo substantial developmental changes during late adolescence, the typical age of onset of schizophrenia. Consistent with this hypothesis, the axon terminals of chandelier neurons, as revealed by immunoreactivity for the GABA membrane transporter, are reduced substantially in the middle layers of the PFC in schizophrenic subjects. This alteration appears to be selective for the chandelier class of GABA neurons and for the disease process of schizophrenia. These findings provide insight into the pathophysiologic mechanisms underlying prefrontal cortical dysfunction in schizophrenia, and they reveal new targets for therapeutic intervention in this illness.

Effect of chronic antipsychotic exposure on astrocyte and oligodendrocyte numbers in macaque monkeys

BACKGROUND Both in vivo and postmortem studies suggest that oligodendrocyte and myelination alterations are present in individuals with schizophrenia. However, it is unclear whether prolonged treatment with antipsychotic medications contributes to these disturbances. We recently reported that chronic exposure of macaque monkeys to haloperidol or olanzapine was associated with a 10%-18% lower glial cell number in the parietal grey matter. Consequently, in this study we sought to determine whether the lower glial cell number was due to fewer oligodendrocytes as opposed to lower numbers of astrocytes. METHODS With fluorescent immunocytochemical techniques, we optimized the visualization of each cell type throughout the entire thickness of tissue sections, while minimizing final tissue shrinkage. As a result, we were able to obtain robust stereological estimates of total oligodendrocyte and astrocyte numbers in the parietal grey matter with the optical fractionator method. RESULTS We found a significant 20.5% lower astrocyte number with a non-significant 12.9% lower oligodendrocyte number in the antipsychotic-exposed monkeys. Similar effects were seen in both the haloperidol and olanzapine groups. CONCLUSIONS These findings suggest that studies investigating glial cell alterations in schizophrenia must take into account the effect of antipsychotic treatment.

Magnetic resonance imaging and spectroscopy in offspring at risk for schizophrenia : Preliminary studies

1. Studies of first-degree relatives of persons with schizophrenia provide an opportunity to characterize risk factors for the development of this illness. In this report the authors will provide preliminary data from an ongoing study of neurobiological alterations in the offspring of schizophrenia patients. 2. A series of offspring of schizophrenic patients (OS) were compared with age and sex matched healthy controls (HC) without psychiatric history in first degree relatives on psychiatric, volumetric Magnetic Resonance Imaging (MRI) of whole brain and proton Magnetic Resonance Spectroscopy (1H MRS) evaluations of the ventral prefrontal cortex. 3. Compared with HC group, high risk subjects had reduced left amygdala volume, enlarged third ventricular volume, and smaller overall brain volume. 4. 1H MRS studies showed a trend for decreased NAA/choline ratios in the anterior cingulate region in the OS group as compared to HC subjects. 5. Follow-up studies of these subjects are needed to confirm the predictive value of these measures for future emergence of schizophrenia in subjects at risk for this illness.

Effect of chronic exposure to antipsychotic medication on cell numbers in the parietal cortex of macaque monkeys.

Both in vivo and post-mortem investigations have demonstrated smaller volumes of the whole brain and of certain brain regions in individuals with schizophrenia. It is unclear to what degree such smaller volumes are due to the illness or to the effects of antipsychotic medication treatment. Indeed, we recently reported that chronic exposure of macaque monkeys to haloperidol or olanzapine, at doses producing plasma levels in the therapeutic range in schizophrenia subjects, was associated with significantly smaller total brain weight and volume, including an 11.8–15.2% smaller gray matter volume in the left parietal lobe. Consequently, in this study we sought to determine whether these smaller volumes were associated with lower numbers of the gray matters constituent cellular elements. The use of point counting and Cavalieris principle on Nissl-stained sections confirmed a 14.6% smaller gray matter volume in the left parietal lobe from antipsychotic-exposed monkeys. Use of the optical fractionator method to estimate the number of each cell type in the gray matter revealed a significant 14.2% lower glial cell number with a concomitant 10.2% higher neuron density. The numbers of neurons and endothelial cells did not differ between groups. Together, the findings of smaller gray matter volume, lower glial cell number, and higher neuron density without a difference in total neuron number in antipsychotic-exposed monkeys parallel the results of post-mortem schizophrenia studies, and raise the possibility that such observations in schizophrenia subjects might be due, at least in part, to antipsychotic medication effects.

Altered Cortical Glutamate Neurotransmission in Schizophrenia

Abstract: Multiple lines of evidence from pharmacological, neuroimaging, and postmortem studies implicate disturbances in cortical glutamate neurotransmission in the pathophysiology of schizophrenia. Given that pyramidal neurons are the principal source of cortical glutamate neurotransmission, as well as the targets of the majority of cortical glutamate‐containing axon terminals, understanding the nature of altered glutamate neurotransmission in schizophrenia requires an appreciation of both the types of pyramidal cell abnormalities and the specific class(es) of pyramidal cells that are affected in the illness. In this chapter, we review evidence indicating that a subpopulation of pyramidal neurons in the dorsolateral prefrontal cortex exhibits reductions in dendritic spine density, a marker of the number of excitatory inputs, and in somal volume, a measure correlated with a neurons dendritic and axonal architecture. Specifically, pyramidal neurons located in deep layer 3 of the dorsolateral prefrontal cortex and that lack immunoreactivity for nonphosphorylated neurofilament protein may be particularly involved in the pathophysiology of schizophrenia. The presence of similar changes in pyramidal neurons located in deep layer 3 of auditory association cortex suggests that a shared property, which remains to be determined, confers cell type‐specific vulnerability to a subpopulation of cortical glutamatergic neurons in schizophrenia.

Stereological analysis of the mediodorsal thalamic nucleus in schizophrenia: volume, neuron number, and cell types.

The mediodorsal thalamic nucleus (MD) is the principal relay nucleus for the prefrontal cortex, a brain region thought to be dysfunctional in schizophrenia. Several, but not all, postmortem studies of the MD in schizophrenia have reported decreased volume and total neuronal number. However, it is not clear whether the findings are specific for schizophrenia nor is it known which subtypes of thalamic neurons are affected. We studied the left MD in 11 subjects with schizophrenia, 9 control subjects, and 12 subjects with mood disorders. Based on morphological criteria, we divided the neurons into two subclasses, presumably corresponding to projection neurons and local circuit neurons. We estimated MD volume and the neuron number of each subclass using methods based on modern unbiased stereological principles. We also estimated the somal volumes of each subclass using a robust, but biased, approach. In addition, we investigated the left MD in four cynomolgus monkeys chronically exposed to haloperidol and in four control monkeys in order to assess the possible effects of antipsychotic medications. The three human subject groups did not differ in any of the measures. In addition, no differences were observed between the two groups of monkeys. Thus, these findings do not support the hypothesis that the MD is a locus of pathology in schizophrenia, although they cannot rule out important functional or structural changes in parameters not measured. Like other studies, this investigation is subject to the limitations involved in sampling from a heterogeneous population emphasizing the need to continue to improve the application of robust, unbiased techniques to quantitative studies of this complex brain disorder. J. Comp. Neurol. 472:449–462, 2004.

Reduced pyramidal cell somal volume in auditory association cortex of subjects with schizophrenia.

Subjects with schizophrenia have decreased gray matter volume of auditory association cortex in structural imaging studies, and exhibit deficits in auditory sensory memory processes subserved by this region. In dorsal prefrontal cortex (dPFC), similar in vivo observations of reduced regional volume and working memory deficits in subjects with schizophrenia have been related to reduced somal volume of deep layer 3 pyramidal cells. We hypothesized that deep layer 3 pyramidal cell somal volume would also be reduced in auditory association cortex (BA42) in schizophrenia. We used the nucleator to estimate the somal volume of pyramidal neurons in deep layer 3 of BA42 in 18 subjects with schizophrenia, each of whom was matched to one normal comparison subject for gender, age, and post-mortem interval. For all subject pairs, somal volume of pyramidal neurons in deep layer 3 of dPFC (BA9) had previously been determined. In BA42, somal volume was reduced by 13.1% in schizophrenic subjects (p=0.03). Reductions in somal volume were not associated with the history of antipsychotic use, alcohol dependence, schizoaffective disorder, or death by suicide. The percent change in somal volume within-subject pairs was highly correlated between BA42 and BA9 (r=0.67, p=0.002). Deep layer 3 pyramidal cell somal volume is reduced in BA42 of subjects with schizophrenia. This reduction may contribute to impairment in auditory function. The correlated reductions of somal volume in BA42 and BA9 suggest that a common factor may affect deep layer 3 pyramidal cells in both regions.