1 Departments of Neuroscience, , 2 Psychiatry and , 3 Statistics, University of Pittsburgh, PA 15260, USA and , 4 Institute of Pharmacology, University of Zurich, CH-8057 Zurich, Switzerland
David A. Lewis, University of Pittsburgh, 3811 OHara Street, W1650 BST, Pittsburgh, PA 15213, USA. Email: lewisda{at}msx.upmc.edu.
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Abstract |
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Introduction |
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The status of GABA activity at the AIS of pyramidal neurons may be further informed by an analysis of postsynaptic GABAA receptors at the AIS. Indeed, compensatory changes in GABAA receptors in response to alterations in extracellular GABA levels have been previously reported (Montpied et al., 1991a; Mhatre and Ticku, 1994
). However, previous radioligand binding and mRNA expression studies of PFC GABAA receptors in schizophrenia (Hanada et al., 1987
; Akbarian et al., 1995b
; Benes et al., 1996
; Huntsman et al., 1998
; Impagnatiello et al., 1998
; Dean et al., 1999
; Ohnuma et al., 1999
) have not selectively analyzed the pyramidal neuron AIS.
GABAA receptors are composed of pentamers of subunits, most commonly including at least one of each of the , ß and
subunit classes (Sieghart et al., 1999
). In addition, the different GABAA receptor
subunits have distinctive subcellular distributions (Fritschy and Mohler, 1995
). For example, in the superficial layers of human cerebral cortex, the
2 subunit is prominently localized at pyramidal neuron AIS (Loup et al., 1998
). Indeed, although associated with only ~15% of all GABAA receptors in the cortex (Fritschy and Mohler, 1995
), the
2 subunit is found at >80% of inhibitory synapses onto pyramidal neuron AIS, at least in rat hippocampus (Nusser et al., 1996
; Nyíri et al., 2001
). Furthermore, only 0.1% of all GABA synapses are found at pyramidal AIS, whereas nearly 25% of all
2- immunoreactive synapses are found at pyramidal AIS. Finally, GABAA receptors containing the
2 subunit have a higher affinity for GABA and faster activation and slower de-activation times, compared to GABAA receptors containing the more commonly expressed
1 subunit (Levitan et al., 1988
; Lavoie et al., 1997
). Thus, GABAA receptors containing the
2 subunit appear to be anatomically positioned and functionally adapted to mediate a potent inhibitory influence on the output of pyramidal neurons and, thus, may provide critical insight into the status of inhibitory activity at pyramidal neuron AIS.
Therefore, we examined human post-mortem brain tissue containing PFC area 46 in order to determine: (i) whether the density of pyramidal neuron AIS immunoreactive for the GABAA receptor 2 subunit (a2-AIS) is altered in subjects with schizophrenia; (ii) whether changes in a2-AIS density are specific to the diagnosis of schizophrenia; and (iii) whether a2-AIS density is related to presynaptic GABA markers in chandelier axon cartridges in subjects with schizophrenia.
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Materials and Methods |
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Brain specimens were obtained during autopsies conducted at the Allegheny County Coroners Office after obtaining consent from the surviving next of kin. The 14 triads of subjects used in this study each consisted of one subject with schizophrenia matched to one control subject and one subject with major depressive disorder (MDD) for sex, and as closely as possible for age and post-mortem interval (PMI; Table 1). All subjects with schizophrenia, all control subjects and all but five subjects with MDD (613, 689, 693, 698 and 803; Table 1
) were included in previous studies of chandelier axon cartridges immunoreactive for GAT-1 GAT-1-cartridges (Woo et al., 1998
; Pierri et al., 1999
). All subjects were under the age of 70 years and all subjects with schizophrenia were previously determined to have at least a 10% decrease in the density of GAT-1-cartridges compared to matched control subjects.
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Tissue Processing
Upon retrieval, brain specimens were blocked coronally, placed into 4% paraformaldehyde for 48 h, cryoprotected and then stored at 30°C. Subject groups did not significantly differ in mean storage time [F(2,42) = 2.36, P = 0.11; Table 1]. Blocks containing the left middle frontal gyrus were sectioned coronally at 40 µm on a calibrated cryostat. Nissl-stained sections were used to identify PFC area 46 in each subject using cytoarchitectonic criteria (Daviss and Lewis, 1995
; Rajkowska and Goldman-Rakic, 1995
). Neuropathological examination of each brain revealed no abnormalities in the region of interest and Alzheimers disease was ruled out in each subject using clinical and neuropathological criteria.
Four tissue sections containing PFC area 46, in serial order and ~400 mm apart, from each subject were processed in a randomized block design (i.e. with one section from each subject in a triad always processed together, and with different combinations of triads in each run). Using an antibody raised in guinea pig against the cDNA-derived N-terminal sequence (amino acid residues 19) of the human GABAA receptor 2 subunit (Marksitzer et al., 1993
), sections were processed using the avidinbiotin procedure, followed by diaminobenzidine and hydrogen peroxide as previously described (Woo et al., 1998
). Reaction product was intensified through serial immersions in aqueous osmium tetroxide and thiocarbohydrizide, followed by silver nitrate and gold chloride (Pucak et al., 1996
).
The specificity of the antibody for the GABAA receptor 2 subunit was previously demonstrated by Western blot experiments whereby immunoprecipitation of GABAA receptor
2 subunit by the antibody revealed a single band whose signal intensity was reduced in a dose-dependent manner following preadsorption with increasing concentrations of GABAA receptor
2 subunit peptide (Marksitzer et al., 1993
). Specificity was also confirmed by anatomical observations described in the Results section.
Quantification
All quantification was conducted by one rater (D.W.V.), who was blinded to diagnosis and subject number. Using the Stereo Investigator fractionator program (MicroBrightField Inc., Colchester, VT), a region of the middle frontal gyrus containing area 46 and of uniform cortical depth was sampled in each subject. Using a 5x objective, a contour containing layers 23a, defined as 25% of the total cortical width immediately below the layer 12 border, was drawn on each section (mean ± SD total contour area per subject: 10.7 ± 2.6 mm2). This zone was chosen because it contains ~80% of the total number of 2-AIS in the PFC and in order to maintain consistency with a previous study on the density of GAT-1-cartridges in this laminar location in the same cohort of subjects (Pierri et al., 1999
). Between 40 and 50 counting frames, 70 x 70 µm, were systematically and randomly placed within the contour for each tissue section and two sides of each counting frame were identified as exclusion boundaries. Using a 100x oil-immersion objective (NA 1.4), all a2-AIS (see Results for criteria) within each counting frame were identified on a video monitor at a final magnification of 2450x. The total number of a2-AIS counted in each subject ranged from 4 to 616 and the mean (±SD) coefficient of error for a2-AIS counts per sampling frame in each subject was 0.13 ± 0.08. Assessments of intra-rater reliability in identifying a2-AIS throughout the course of the quantification procedure revealed an intraclass correlation coefficient of 0.99 (95% CI = 0.591.0).
In a separate analysis using the Neurolucida program (MicroBright-Field Inc., Colchester, VT), mean length of a2-AIS was also determined in each schizophrenic and control subject. In one random tissue section from each subject, the length of every detectable a2-AIS was measured in a series of 100 mm wide cortical traverses. The average (±SD) number of a2-AIS measured in each subject was 54 ± 21.
Statistical Analysis
Measures of the density of a2-AIS per mm2 in each of the four tissue sections for each subject were treated as four correlated observations. A multivariate analysis of covariance (MANCOVA) model assuming a compound symmetric covariance structure (Neter et al., 1996) was employed to test for a main effect of diagnosis, with triad and immuno-histochemistry run included as blocking factors and tissue storage time as a covariate. In this model, the inclusion of triad accounted for matching of subjects for sex, age and PMI. Because regression analyses revealed a potential effect of PMI on
2-AIS density, two additional MANCOVA models were run. One model included both diagnosis and triad factors, with PMI and tissue storage time as covariates and run as a blocking factor; the other model included a diagnosis factor with sex, age, PMI and tissue storage time as covariates, and run as a blocking factor. As all three models yielded similar results, only the results of the primary model are reported. Comparisons between any two of the diagnostic groups were also conducted using the primary model. In addition, the effects of sex, psychotropic medication at time of death and/or alcoholism on a2-AIS density in subjects with schizophrenia were assessed using ANCOVAs with the difference in a2-AIS density within matched pairs of schizophrenic and control subjects as the dependent variable and PMI as a covariate. Finally, a one-tailed Pearson correlation was used to test the hypothesis that GAT-1-cartridge density was inversely related to a2-AIS density in subjects with schizophrenia. Analyses were implemented in SAS PROC Mixed (Littell et al., 1996
) and all statistical tests were conducted with
= 0.05.
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Results |
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As shown in Figure 1A, immunoreactivity for the GABAA receptor
2 subunit in human PFC was greatest in the supragranular layers, particularly in layer 2. This pattern directly parallels previous reports of a2-immunoreactivity in the medial PFC of rat (Dunn et al., 1996
) and of
2 mRNA expression in human PFC (Akbarian et al., 1995b
). At higher magnification,
2 subunit-immunoreactivity was prominently found in pyramidal neuron AIS a2-AIS (Nusser et al., 1996
; Loup et al., 1998
; Nyíri et al., 2001
) identified as intensely immunoreactive, discrete, non-branching, vertically oriented processes located below unlabeled cell bodies, ranging in length from 8 to 40 µm and usually slightly tapered from superficial to deep (Figure 1BD
).
2-AIS Density in Schizophrenia and Major Depression
Statistical analysis using the primary MANCOVA model revealed a significant main effect of diagnosis [F(2,25) = 4.88, P = 0.016] on 2-AIS density. The mean (±SD) number of a2-AIS per mm2 was significantly [F(1,12) = 10.60, P = 0.007] increased by 113% in subjects with schizophrenia (302 ± 184) compared to control subjects (141 ± 119). Furthermore, in 12 of the 14 matched pairs of schizophrenic and control subjects,
2-AIS density was higher in the schizophrenic subject (Fig. 2
). As illustrated in Figure 3
, the mean increase in a2-AIS density in individual subjects with schizophrenia compared to their matched controls did not significantly differ when the subjects with schizophrenia were subdivided into groups based on gender [F(1,11) = 0.007, P = 0.934], treatment with psychotropic medications at time of death [F(1,11) = 0.201, P = 0.663], or history of an alcohol-related disorder [F(1,11) = 0.002, P = 0.968]. In addition, mean
2-AIS density was virtually identical in the three subjects with schizoaffective disorder (295 ± 203) and the 11 subjects with pure schizophrenia (304 ± 189; Fig. 4
).
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The mean density of a2-AIS was also increased in the subjects with schizophrenia by 37% compared to subjects with MDD (Fig. 2). Although this difference was not statistically significant [F(1,12) = 1.68, P = 0.22], 10 of the 14 subjects with schizophrenia had an increased density of
2-AIS compared to the matched subject with MDD. Furthermore, one subject with MDD (689; Table 1
) appeared to be an outlier in that the density of a2-AIS in this subject (a2-AIS/mm2 = 731) was 2.6 SD above the mean
2-AIS density for all subjects with MDD (Fig. 2
). Consequently, this outlier subject was included in the main statistical analysis, but excluded from subsequent analyses in which diagnosis groups were subdivided into smaller groups on the basis of demographic variables. Importantly, no demographic variable, such as psychotropic medication at time of death, history of alcoholism, presence of psychotic features, tissue storage time, or PMI, appeared to contribute to the increased a2-AIS density in this subject, or any other subject. When triad 9, which included subject 689, was excluded from analysis, the mean density of a2-AIS in subjects with schizophrenia was increased by 59% compared to subjects with MDD [182 ± 138, F(1,11) = 2.86, P = 0.12]. Furthermore, mean
2-AIS density did not significantly differ between control subjects and subjects with MDD [F(1,11) = 0.53, P = 0.48]. Finally, mean
2-AIS density in the four subjects with MDD with psychotic features did not differ from either the MDD subjects without psychotic features or the control subjects (Fig. 4
).
Reciprocal Changes in a2-AIS and GAT-1-Cartridge Density in Schizophrenia
The density of GAT-1-cartridges in area 46 was previously determined for all subjects with schizophrenia, all control subjects and nine subjects with MDD examined in the present study (Woo et al., 1998; Pierri et al., 1999
). Whereas the mean
2-AIS density in the subjects with schizophrenia was approximately twice that in the matched control subjects, the mean GAT-1-cartridge density in the same schizophrenic subjects was less than half that in the matched control subjects (Fig. 5
). Furthermore, a significant inverse relationship (r = 0.49, P = 0.038) between the densities of
2-AIS and GAT-1-cartridges was present in the subjects with schizophrenia. In contrast, in the nine subjects with MDD included in the previous GAT-1-cartridge study, neither the mean density of a2-AIS nor of GAT-1-cartridges differed significantly from the nine matched control subjects (Fig. 5
).
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Discussion |
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Specificity of Findings for Schizophrenia
The increase in a2-AIS density appears to be relatively specific to the diagnosis of schizophrenia, or at least not a common feature of MDD, even when that disorder is accompanied by psychosis. Consistent with these findings, previous studies of the frontal cortex in subjects with MDD have failed to find differences in the concentration, synthesis, or re-uptake of GABA (Cheetham et al., 1988; Korpi et al., 1988
; Sundman et al., 1997
). Thus, alterations in PFC GABA neurotransmission at the chandelier cellpyramidal neuron synapse appear to be a characteristic of schizophrenia that is not associated with depression or psychosis, per se.
Several lines of evidence suggest that treatment with antipsychotic medications and benzodiazepines or a history of alcoholism do not account for the changes in PFC GABA markers in subjects with schizophrenia. First, a2-AIS density was increased in subjects with schizophrenia, regardless of the use of psychotropic medications at time of death or history of an alcohol-related disorder. Consistent with these observations, studies of haloperidol-treated monkeys revealed no changes in the expression of GAD67 or GAT-1 mRNAs or the density of GAT-1-cartridges in the PFC (Pierri et al., 1999; Volk et al., 2000
, 2001
). In addition, pharmacological manipulations in rats have found that: (i) antipsychotic medications result in decreased muscimol binding to GABAA receptors (Johnson et al., 1994
; Farnbach-Pralong et al., 1998
); (ii) benzodiazepine treatment causes no changes in cortical GABAA receptor
2 subunit mRNA or protein expression levels (ODonovan et al., 1992
; Holt et al., 1996
, 1997
; Impagnatiello et al., 1996
; Chen et al., 1999
; Tietz et al., 1999
); and (iii) ethanol results in either decreases or no change in cortical GABAA receptor
2 mRNA expression (Montpied et al., 1991b
; Mhatre et al., 1993
; Chen et al., 1998
)
Methodological Considerations
The stereological principle of systematic, random sampling was employed in this study to reduce sampling bias; however, the lack of clear boundaries of PFC area 46 precluded the determination of the total number of a2-AIS in this region. Importantly, measures of a2-AIS density were not confounded by differences in a2-AIS length between the schizophrenic and control groups. Furthermore, given that a2-AIS and GAT-1-cartridge densities were changed in opposite directions in nearby tissue sections from the same subjects with schizophrenia, neither the method of quantification nor potential differences in cortical volume across subject groups appear to have introduced a systematic confound.
We interpret the greater density of a2-AIS in schizophrenia as reflecting an increase in the amount of a2 subunit and the total density of GABAA receptors, per AIS, resulting in a greater number of AIS with detectable levels of immunoreactivity. Unfortunately, direct quantification of GABAA receptor subunits in pyramidal neuron AIS would require the use of procedures for example immunogold-labeling and electron microscopy or immunofluorescence at the light microscopic level (Sutoo et al., 1998) that are technically challenging in post-mortem human brain. However, it seems unlikely that the increased
2-AIS density reflects an increase in pyramidal neuron density. First, in previous studies, PFC neuron density was reported to be either unchanged (Akbarian et al., 1995a
), or, at ~20%, not increased enough (Selemon et al., 1995
, 1998
) in schizophrenia to account for the present findings. Second, in the same subjects examined in the present study, the density of GAT-1-cartridges was actually reduced by >50%, which further argues against an increase in pyramidal neuron density. Finally, re-analysis of data from a previous study (Pierri et al., 2001
) revealed no difference in deep layer 3 pyramidal neuron density between the same cohort of subjects with schizophrenia (48.4 ± 7.7 cells/0.001 mm3) and control subjects (49.1 ± 8.7 cells/0.001 mm3) examined in the present study.
Alternatively, an increased amount of a2 subunit at the AIS could be coupled with reductions in other subunits reported to be located at pyramidal neuron AIS, such as the a1 and a3 subunits (Nusser et al., 1996; Loup et al., 1998
), without changes in the total number of GABAA receptors. Such subunit switching between the
2 and a1 subunits occurs in rat hypothalamic oxytocin neurons during pregnancy (Brussaard and Herbison, 2000
). Unfortunately, quantification of a1-labeled AIS in schizophrenia is not possible, since the much greater density of a1 subunit at other locations (Nusser et al., 1996
) prohibits the resolution of
1-labeled AIS by light microscopy. In addition, we were not able to detect a3-immunoreactivity in pyramidal neuron AIS in our human tissue samples. Yet, even if subunit switching, without a change in total receptor number, occurs in schizophrenia, an increased proportion of GABAA receptors containing the
2 subunit would still likely confer a greater inhibitory response to GABA at pyramidal neuron AIS (Levitan et al., 1988
; Lavoie et al., 1997
). Thus, the increase in
2-AIS density, whether reflecting an increased total density of GABAA receptors or an increased proportion of GABAA receptors containing the
2 subunit, suggests that postsynaptic GABAA receptors at pyramidal neuron AIS are functionally up-regulated in schizophrenia.
Pathophysiological Significance
The inverse relationship between the densities of a2-AIS and GAT-1-cartridges in the same subjects with schizophrenia suggests that GABAA receptors are up-regulated at pyramidal neuron AIS in response to deficient GABA activity in chandelier axon terminals in schizophrenia (Fig. 6). Although experimental models of a selective decrease in GABA release at chandelier axon terminals are not available, previous studies (Mhatre and Ticku, 1994
) have demonstrated that GABAA receptor antagonists produce increased
2 subunit mRNA expression in embryonic chick neurons. Furthermore, in subjects with temporal lobe epilepsy, a loss of GABA neurons is associated with a substantial increase in a2 subunit immunoreactivity in pyramidal cells (Loup et al., 2000
).
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The pathophysiological mechanism that selectively initiates disturbances in a subset of GABA neurons that appears to include chandelier neurons, but not in the majority of PFC GABA neurons (Woo et al., 1998; Volk et al., 2000
, 2001
), remains unclear. One possibility is that reduced inhibitory neuro-transmission in chandelier neurons reflects an abnormality intrinsic to this cell class. Alternatively, inhibitory neurotransmission may be reduced in chandelier neurons in response to an alteration in their excitatory inputs. For example, morphological alterations in layer 3 pyramidal neurons in PFC area 46 (Garey et al., 1998
; Rajkowska et al., 1998
; Glantz and Lewis, 2000
; Pierri et al., 2001
) and reduced neuronal number in the mediodorsal thalamic nucleus (Pakkenberg, 1990
; Popken et al., 2000
; Young et al., 2000
; Byne et al., 2002
) have been reported in subjects with schizophrenia. Interestingly, in monkey PFC area 46, the dendrites of parvalbumin-containing cells, which include chandelier neurons, receive synaptic inputs from local pyramidal neurons (Melchitzky et al., 2001
) and from the mediodorsal thalamic nucleus (Melchitzky et al., 1999
), whereas other classes of GABA cells do not (Melchitzky and Lewis, 2000
). Further studies of the mechanism(s) responsible for altered chandelier-cellpyramidal-neuron inhibitory neurotransmission may lead to novel therapeutic strategies for remediating prefrontal cortical dysfunction in schizophrenia.
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Acknowledgments |
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