selective deficits in prefrontal cortical gabaergic neurons in schizophrenia defined by the presence...

Selective Deficits in Prefrontal Cortical GABAergicNeurons in Schizophrenia Defined by the Presence ofCalcium-Binding Proteins

Clare L. Beasley, Zhi J. Zhang, Iain Patten, and Gavin P. Reynolds

Background: Postmortem studies have provided evidencefor abnormalities of the �-aminobutyric acid (GABA)-ergic system in schizophrenia, including deficits of GABA-containing interneurons. The calcium-binding proteinsparvalbumin, calbindin, and calretinin can be used asmarkers for specific subpopulations of cortical GABAer-gic interneurons.

Methods: Following our previous observation of a reduc-tion in the density of parvalbumin- but not calretinin-immunoreactive cells in the prefrontal cortex (Brodmannarea 10) in schizophrenia, we have quantified the laminardensity of neurons immunoreactive for the calcium-bind-ing proteins parvalbumin, calbindin, and calretinin in afurther prefrontal cortical region (Brodmann area 9) inpatients with schizophrenia, bipolar disorder, major de-pression, and in matched control subjects (each group n �15).

Results: Initial statistical analysis revealed reductions inthe total cortical density of parvalbumin- and calbindin-but not calretinin-immunoreactive neurons in schizophre-nia relative to control subjects. Further analysis compar-ing individual laminar densities between groups indicatedthat, following correction for multiple comparisons, only areduction in calbindin-immunoreactive neurons in corticallayer II in the schizophrenic group attained statisticalsignificance.

Conclusions: These findings suggest that deficits of spe-cific GABAergic neurons, defined by the presence ofcalcium-binding proteins, are present in schizophrenia.Trends toward similar reductions are observed in bipolardisorder. Biol Psychiatry 2002;52:708–715 © 2002 So-ciety of Biological Psychiatry

Key Words: Parvalbumin, calbindin, calretinin, schizo-phrenia, bipolar disorder, interneuron

Introduction

There is some intriguing evidence indicative of anom-alous �-aminobutyric acid (GABA)-ergic neurotrans-

mission in the prefrontal cortex in schizophrenia. Inparticular, postmortem analysis of brain tissue from thisregion has provided insight into cellular abnormalities ofthe GABAergic system, including a loss of presumptiveinterneurons in more superficial cortical layers (Benes etal 1991) and a reduction in the density of interneuronsexpressing mRNA for glutamic acid decarboxylase(GAD), the synthesizing enzyme for GABA (Akbarian etal 1995; Volk et al 2000). Further studies have foundincreased binding to the GABAA receptor in the prefrontalcortex in schizophrenia (Benes et al 1996; Hanada et al1987). This increase in GABAA receptors has been inter-preted as reflecting a compensatory upregulation ofpostsynaptic receptors, due to losses of GABAergic inter-neurons in this region (Benes et al 1996). Although similarstudies of bipolar disorder and major depression arelimited, there are indications that expression of GADmRNA and the density of GAD-immunoreactive terminalsare reduced in the prefrontal cortex in bipolar disorder(Benes et al 2000; Guidotti et al 2000).

To understand the functional significance of thesefindings, it is important to further define the GABAergicchanges observed in schizophrenia. A number of criteria,including morphologic appearance and biochemical prop-erties, can be utilized to discriminate distinct classes ofGABAergic interneurons. Cortical GABAergic cells canbe subdivided on the basis of co-localized neuropeptides,including somatostatin, cholecystokinin, neuropeptide Y,and vasoactive intestinal polypeptide (Somogyi et al1984). Losses of cholecystokinin, somatostatin, and vaso-active intestinal polypeptide have been described in thecortex in some schizophrenic patients (Gabriel et al 1996;Nemeroff et al 1983). Consistent with this, deficits incholecystokinin mRNA, have been reported in the frontaland temporal cortex (Virgo et al 1995) as well as in theentorhinal cortex (Bachus et al 1997). Essentially nonover-lapping subpopulations of GABAergic neurons can also be

From the Department of Biomedical Science, University of Sheffield, Sheffield,United Kindom.

Address reprint requests to Clare L. Beasley, Ph.D., Institute of Psychiatry, Sectionof Experimental Neuropathology and Psychiatry, DeCrespigny Park, LondonSE5 8AF, United Kingdom.

Received July 23, 2001; revised December 10, 2001; revised February 1, 2002;accepted February 11, 2002.

© 2002 Society of Biological Psychiatry 0006-3223/02/$22.00PII S0006-3223(02)01360-4

defined by the presence of the calcium-binding proteinsparvalbumin, calbindin, and calretinin (Celio 1990; De-meulemeester et al 1988). Studies have revealed that themost characteristic morphologic types of neurons thatexpress parvalbumin are large basket and chandelier cells(Akil and Lewis 1992; Lewis and Lund 1990); calbindin ispresent in many double bouquet cells (DeFelipe et al1989), whereas calretinin-immunoreactive neurons aregenerally bipolar, double bouquet, and Cajal-Retzius cells(Jacobowitz and Winsky 1991).

We have previously observed a reduction in the densityof parvalbumin-immunoreactive cells in the prefrontalcortex (Brodmann area [BA] 10) in schizophrenia (Beas-ley and Reynolds 1997), but we found no loss of calreti-nin-immunoreactive neurons in the same region (Reynoldsand Beasley 2001). This led us to suggest that theGABAergic deficits described in the frontal cortex inschizophrenia may be due to specific reductions in parval-bumin-immunoreactive cells. To test this, the density ofinterneurons immunoreactive for parvalbumin, calbindin,and calretinin was quantified in a further prefrontal corti-cal region (BA 9) in a series of brains from patients withschizophrenia, bipolar disorder, major depression, andfrom matched control subjects. As the morphology, distri-bution, function, and ontology of distinct subpopulationsof GABAergic neurons differs, identifying deficits ofspecific interneurons in the brain in schizophrenia couldthrow light on the putative mechanisms underlying thedisturbances of cortical function observed in this disorder.

Methods and Materials

Human SubjectsSamples were obtained from the Stanley Foundation Neuropa-thology Consortium brain collection. Brains were obtained frompatients diagnosed with schizophrenia, bipolar disorder, majordepression, and from matched control subjects (each group n �15). Demographic details are provided in Table 1.; for furtherdescriptions see Torrey et al (2000). Final diagnoses wereestablished using DSM-IV criteria and routine microscopic andtoxicological examinations carried out on all cases. Cause ofdeath and an estimate of total lifetime intake of antipsychoticmedication (in fluphenazine mg equivalents) were detailed for allcases.

Immunocytochemistry10-�m-thick paraffin sections of the dorsolateral prefrontalcortex were processed for parvalbumin-, calbindin-, or calretinin-immunoreactivity randomly and blind to diagnosis. For eachantibody two sections, at least 50 �m apart, were sampled foranalysis. Tissue sections were heated in a microwave oven onfull power (650 W) for 3 � 10 min in .05 mol/L tris buffer, pH9.0, to aid antigen retrieval. Sections were blocked with normalserum, then incubated for 36 hours at 4°C with either a mousemonoclonal antibody against parvalbumin (clone PA-235,Sigma, St. Louis, MO), a mouse monoclonal antibody againstcalbindin (Sigma) or a goat polyclonal antibody against calreti-nin (Swant, Bellinzona, Switzerland), each at a dilution of1:5000. For parvalbumin the antibody diluent also contained 3mmol/L calcium chloride. Following this the sections wereprocessed by the avidin–biotin method of Hsu et al (1981) using

Table 1. Summaries of Demographic, Clinical, and Histological Information of Schizophrenic, Bipolar Disorder, MajorDepression, and Control Groups

Demographic variable

Group

Control(n � 15)

Schizophrenia(n � 15)

Bipolar disorder(n � 15)

Major depression(n � 15)

Age (y, mean � SD) 48.1 � 10.7 44.2 � 13.1 42.3 � 11.7 46.4 � 9.3Gender (male, female) 9M, 6F 9M, 6F 9M, 6F 9M, 6FPostmortem interval (hr, mean � SD) 23.7 � 9.9 33.7 � 14.6 32.5 � 16.1 27.5 � 10.7Cause of death

CPD 13 7 3 7Accident 2 0 0 1Suicide 0 7 9 4Pneumonia 0 0 1 1Other 0 1 2 2

pH (mean � SD) 6.3 � .2 6.2 � .3 6.2 � .2 6.2 � .2Time in fixative (mo, mean � SD) 4.40 � 3.87 11.20 � 8.48 9.67 � 3.62 8.40 � 6.59Brain hemisphere used (Right : Left) 7:8 6:9 8:7 6:9Duration of illness (y, mean � SD) 0 � 0 21.3 � 11.4 20.1 � 9.7 12.7 � 11.1Lifetime antipsychotic dosea

(mg, minimum; median; maximum)0; 0; 0 0; 35,000; 200,000 0; 7,500; 60,000 0; 0; 0

Current alcohol/drug abuse 0 3 4 3

CPD, cardiopulmonary disease.aLifetime neuroleptic dose in fluphenazine milligram equivalent dose.

Prefrontal GABAergic Deficits in Schizophrenia 709BIOL PSYCHIATRY2002;52:708–715

a Vectastain ABC kit (Vector Laboratories, Burlingame, CA).Peroxidase was visualized using diaminobenzidine, intensifiedwith nickel chloride, and sections counterstained with toluidineblue. Negative control sections, in which the primary antibodywas omitted from the staining protocol, were run alongside thetest series.

Image AnalysisParvalbumin-, calbindin-, and calretinin-immunoreactive neu-rons were plotted at 100� magnification using an Olympusmicroscope (Olympus Optical Co (Europa) GmbH Hamburg,Germany) equipped with camera lucida. The density of neuronalprofiles was expressed as mean values (� SE) per mm2 percortical layer and was the result of counts from a total of 10500-�m-wide cortical traverses, each from the pial surface to thewhite matter border, selected at random from the two slides. Theplacement of the traverses was restricted to areas of the cortexthat were comparatively level, avoiding the depths of the sulci.Cortical width was also measured for each of these traverses.Counts were made in cortical region BA 9 in each case. Theboundaries of BA 9 with BA 46 were determined according tothe criteria of Rajkowska and Goldmann-Rakic (1995) andDaviss and Lewis (1995).

Statistical AnalysisTo test our initial hypothesis that the GABAergic deficitspreviously described in schizophrenia result from a reduction inparvalbumin-immunoreactive neurons but not calbindin- or cal-retinin-containing cells, we compared mean total cortical densi-ties of each of these cell populations between the schizophrenicand control groups, using univariate analysis of variance(ANOVA). The demographic and histologic variables listed inTable 1 were included as covariates in the analysis if 1) theydiffered between the schizophrenic group and the control groupat the 10% significance level (ANOVA); and 2) they could beshown empirically to predict densities at the 10% significancelevel (ANOVA or Spearman’s Rank correlation). Following onfrom this, the mean density of parvalbumin-, calbindin-, andcalretinin-immunoreactive neurons in each cortical layer for eachof the three patient groups was compared with that of the controlgroup. This allowed us to look for disease and laminar specific-ity. Mean densities were analyzed by univariate ANOVA, usingdiagnoses as contrasts. Confounding variables were determinedas above. To account for multiple layer-wise testing, p values of.010 (for calbindin and calretinin) and .013 (for parvalbumin)were determined to be statistically significant; these valuesrepresent the 5% level divided by the number of layers analyzed.In the schizophrenic and bipolar disorder groups the effects oftotal antipsychotic drug dosage on parvalbumin-, calbindin-, andcalretinin-immunoreactive neuronal densities were was alsoassessed. All statistical analysis was carried out in SPSS 10(CSPSS Inc, Chicago, IL).

Results

Parvalbumin-immunoreactive neurons in the prefrontalcortex were typically intensely stained. Immunoreactive

neurons were present predominantly in layers III, IV, andV and were also observed in layers II and VI, but not inlayer I or in the white matter (Figure 1a). Parvalbumin-positive cells appeared to be nonpyramidal and consistedof a variety of morphologies, including small ovoidperikarya, large multipolar neurons, and occasionallybitufted cells (Figure 1 d). A dense plexus of immunore-active material was also distributed throughout the neuro-pil of layers III, IV, and V and consisted of stainedprocesses and puncta, which have been shown to representaxonal terminals (DeFelipe and Jones 1991). Calbindin-immunoreactive neurons were present throughout thecortical width and were predominant in layer II andsuperficial layer III (Figure 1b). Immunoreactive neuronscomprised both densely stained nonpyramidal cells andfaintly stained pyramidal cells (Figure 1e). Calretinin-immunoreactive cells also comprised a population ofnonpyramidal neurons that were distributed throughout allsix cortical layers and the subjacent white matter, althoughagain predominantly in the more superficial laminae (Fig-ure 1c). Positive neurons were typically bipolar in mor-phology (Figure 1f); layer I contained horizontally ori-ented processes and immunoreactive puncta in addition tolabeled neurons.

Summaries of mean laminar densities for each neuronalsubpopulation are shown in Table 2. At the 10% level,mean fixation time (p � .011) and mean postmorteminterval (p � .038) were significantly higher in theschizophrenic group than in the control group; however,Spearman’s Rank correlation indicated no significant re-lationship between these variables and the density of anyneuronal population at the 10% level, and so groupcomparisons were not adjusted for these variables. Signif-icant correlations were observed between tissue pH andparvalbumin (p � .001, r � .426) and calbindin (p � .001,r � .410) neuronal density and between age and parval-bumin neuronal density (p � .023, r � .294). At the 5%level, significant reductions in the total cortical density ofparvalbumin- (p � .029) and calbindin- (p � .035)containing cells were observed in the schizophrenic group.The density of calretinin-immunoreactive cells did notdiffer between the two groups (Figure 2.)

To look for disease and laminar specificity, the meandensity of parvalbumin-, calbindin-, and calretinin-immu-noreactive neurons in each cortical layer for each of thethree patient groups were compared with that of thecontrol group. At the 10% level, mean fixation times forschizophrenia (p � .011), bipolar disorder (p � .001), andmajor depression (p � .055) and mean postmortem inter-vals for schizophrenia (p � .038) and bipolar disorder(p � .085) were higher than for control subjects; however,Spearman’s Rank correlation indicated no significant cor-relation between these variables and the density of any

710 C.L. Beasley et alBIOL PSYCHIATRY2002;52:708–715

neuronal population at the 10% level, and so groupcomparisons were not adjusted for these variables. Signif-icant correlations were observed between tissue pH andparvalbumin (p � .002, r � .399) and calbindin (p � .011,r � .326) neuronal density and between age and parval-bumin neuronal density (p � .004, r � .365). At the 5%level, significant reductions in the density of parvalbumin-immunoreactive neurons were observed in cortical layer

III in the schizophrenic group compared with the controlgroup (p � .049). The density of calbindin-immunoreac-tive neurons was reduced in schizophrenia in layers II(p � .004), III (p � .026), and V (p � .031) and in bipolardisorder in layers II (p � .013) and III (p � .016),compared with control subjects; however, using thestricter criteria to account for multiple comparisons, onlythe reduction in calbindin density in layer II in schizophre-

Figure 1. Photomicrographs showing (a) parv-albumin-, (b) calbindin-, and (c) calretinin-immunoreactive neurons throughout the corti-cal width of a control case (bar � 200 �m); (d)multipolar neurons and processes immunore-active for parvalbumin in cortical layer IV; (e)pyramidal (asterisks) and nonpyramidal neu-rons immunoreactive for calbindin in corticallayer III; and (f) bipolar neurons immunoreac-tive for calretinin in cortical layer III (bar � 30�m).

Table 2. Density (Mean � SEM cells/mm2) of Calcium-Binding Protein-Immunoreactive Neurons in the Dorsolateral PrefrontalCortex in Schizophrenic (SCZ), Bipolar Disorder (BIP), Major Depression (DEP), and Control (CON) Groups

Calcium-bindingprotein Cortical layer

Diagnosis

SCZ(n � 15)

BIP(n � 15)

DEP(n � 15)

CON(n � 15)

Parvalbumin I 0 0 0 0II 12.98 � 1.14 12.99 � 1.78 12.63 � 1.47 15.30 � 1.78III 39.57 � 2.40 39.72 � 2.78 40.69 � 1.66 46.04 � 2.11IV 83.03 � 3.92 81.92 � 4.05 92.97 � 4.11 92.91 � 3.65V/VI 17.51 � 1.43 17.29 � 1.31 17.60 � 1.08 20.01 � 1.14

Calbindin I 1.67 � 0.50 1.84 � .43 1.68 � .35 2.20 � .49II 94.15 � 9.92 98.84 � 8.67 105.22 � 5.64 127.2 � 6.27III 20.13 � 2.92 19.37 � 1.70 23.66 � 2.54 28.33 � 2.82IV 10.93 � 2.83 7.80 � 1.03 9.21 � 2.03 10.54 � 2.64V/VI 7.95 � 1.22 9.81 � .78 9.80 � .82 10.88 � .86

Calretinin I 53.39 � 4.13 49.57 � 4.56 42.02 � 3.01 52.69 � 3.53II 129.16 � 11.54 134.61 � 9.64 122.11 � 5.63 133.72 � 5.35III 49.57 � 2.26 51.35 � 2.65 51.27 � 1.81 52.47 � 1.95IV 18.63 � 2.08 19.52 � 1.60 20.07 � 2.01 22.3 � 1.60V/VI 7.26 � 1.35 6.73 � .46 7.18 � .81 6.80 � .67


nia attained statistical significance. The density of calreti-nin-immunoreactive neurons was not altered in any layerin any disorder, compared with control subjects. Nogender � diagnosis or side � diagnosis interactions were

noted for any neuronal subpopulation. In the schizophrenicand bipolar disorder groups no significant correlationswere noted between any neuronal subpopulation andlifetime antipsychotic dose. Cortical width did not differbetween groups (mm [mean � SD]: schizophrenia: 2.74 �.29; bipolar disorder: 2.93 � .25; major depression:2.72 � .20; control subjects 2.69 � .17).

Discussion

In the prefrontal cortex the total cortical density ofcalbindin- and parvalbumin- but not calretinin-immunore-active neurons was reduced in the schizophrenic group,compared with control subjects. This confirmed our pre-vious findings of deficits in parvalbumin- but not calreti-nin-containing cells in schizophrenia (Beasley and Reyn-olds 1997; Reynolds and Beasley 2001) but indicates thatthese reductions are not specific to parvalbumin-contain-ing cells, as we had previously suggested. More detailedanalyses, which examined disease and laminar specificity,revealed that significant reductions were only present incalbindin-containing cells in layer II in schizophrenia;however, the density of both parvalbumin- and calbindin-immunoreactive neurons was reduced in each corticallayer in schizophrenia and bipolar disorder, suggestingthat deficits in GABAergic interneurons are not specific todisease, cortical layer, or neuronal subtype.

Our finding of an absence of any changes in the densityof calretinin-immunoreactive neurons in schizophreniccompared with control subjects is consistent with theresults of previous studies in the dorsolateral prefrontalcortex, anterior cingulate cortex, and hippocampus (Cotteret al, 2002; Daviss and Lewis 1995; Reynolds and Beasley2001; Zhang and Reynolds, 2002); however, this is nottrue of our findings of reductions in the density ofparvalbumin- and calbindin-immunoreactive neurons. Inthe only previous study of calbindin neuronal density inthe prefrontal cortex, Daviss and Lewis (1995) describedan increase in density in a small group of five schizo-phrenic subjects compared with control subjects. Wesuggest that the discrepancy between these studies couldbe due to differences in the samples or in the methodologyused. More recently, Cotter et al 2002, who used the samebrain collection as studied here, found that the density ofcalbindin-positive interneurons was reduced in corticallayer II in the anterior cingulate cortex in schizophrenia.The deficit in total cortical parvalbumin-immunoreactivecells we observed in the schizophrenic group is consistentwith our earlier findings in BA 10 in a separate series ofschizophrenic and control cases (Beasley and Reynolds1997). Although we found no significant difference in anyindividual cortical layer, an 11%–15% reduction in thedensity of parvalbumin-immunoreactive neurons was ob-

Figure 2. Scatterplots showing total cortical density of parval-bumin-, calbindin-, and calretinin-immunoreactive neurons in thedorsolateral prefrontal cortex in schizophrenic (SCZ), bipolardisorder (BPD), major depressive disorder (MDD), and control(CON) subjects. Horizontal lines indicate mean values.


served in each layer in both the schizophrenic and bipolardisorder groups. This is consistent with the study by Cotteret al (2002), who found a nonsignificant reduction ofapproximately 20% in parvalbumin-immunoreactive cellsin the anterior cingulate cortex in both schizophrenia andbipolar disorder; however, a further study by Woo et al(1997) found no significant change in the density ofparvalbumin-immunoreactive neurons in the dorsolateralprefrontal cortex. Again, we propose that the discrepancybetween these studies could be due to differences in thesamples or the methodology used. A further possibility isthat the antigen retrieval method used in our study may notbe able to identify cells that contain only very low levelsof parvalbumin. Reductions in the density of parvalbumin-immunoreactive neurons have also been described in otherbrain regions, including the hippocampus and dorsomedialthalamus, in schizophrenia (Danos et al 1998; Zhang andReynolds, 2002). Although we were not able to determineif the reduction in parvalbumin-containing neurons wasattributable to one specific subclass, we suggest that itmight reflect a deficit in chandelier cells. A loss ofchandelier cells could result in the large reduction in thedensity of chandelier cell axonal terminals, identifiedusing an antibody directed against the GABA membranetransporter GAT-1, which has been observed in the pre-frontal cortex in schizophrenia (Woo et al 1998). Chande-lier cells are of particular interest, as they are thought toregulate the excitatory output of pyramidal neurons, thusmodulating patterns of activity in the prefrontal cortex.

We cannot conclude that the observed reduction in thedensity of calbindin- and parvalbumin-immunoreactiveneurons in this study reflects a reduction in cell numberssolely on the basis of immunohistochemical data. Al-though this is one explanation, consistent with earlyindications of interneuron deficits, the reduction in thedensity of immunoreactive cells could also be influencedby a number of other factors. As mentioned above, it maybe that the immunocytochemical method used was notable to pick up neurons that only express very low levelsof calbindin or parvalbumin. This may suggest that in theschizophrenic group the expression of these proteins isgreatly reduced in a subpopulation of neurons. An appar-ent decrease in cell density could therefore result from areduction in cellular calbindin or parvalbumin expressionin the absence of any cell loss. This would be consistentwith recent reports of a reduction in the density ofinterneurons that express mRNA for glutamic acid decar-boxylase in the prefrontal cortex in schizophrenia, whichhave been interpreted as reflecting a downregulation ofGAD expression (Akbarian et al 1995; Volk et al 2000). Itis conceivable that a potentially reversible downregulationof calcium-binding proteins may reflect a change in acell’s functional activity, possibly brought about by

changes in the activity of other neurons innervating thecell. Indeed a number of experimental conditions havebeen described in which the expression of calcium-bindingproteins or GABA is altered, including monocular depri-vation (Carder et al 1996) and kindling (Kamphuis et al1989).

Confounding Factors

The Stanley Foundation Neuropathology Consortiumbrain collection has been extensively characterized, anddetails regarding possible confounding factors have beenmade available. In this study, the influence of potentialconfounders (including postmortem interval, tissue pH,age, gender, time in fixative, antipsychotic exposure, andco-morbid alcohol/substance abuse) on measures of parv-albumin-, calbindin-, and calretinin-immunoreactive neu-rons was examined. Fixation time and postmortem intervalwere significantly different between the groups; however,we found no significant relationship between these vari-ables and measures of parvalbumin-, calbindin-, or calreti-nin-immunoreactive neurons. Although the detectability ofparvalbumin and calbindin is greatly reduced after fixationfor only 2 weeks, previous studies have indicated that fortissue preserved in fixative for longer periods, microwaveantigen-retrieval, as used in the present study, allows thevisualization of parvalbumin- and calbindin-containingcell bodies (Evers and Uylings 1994).

There is little evidence with regard to the potentialeffects of antipsychotics on the expression of these calci-um-binding proteins. In this study, we noted that the oneschizophrenic subject and three bipolar disorder subjectswho had never been medicated with neuroleptics hadrelative densities similar to those patients who weremedicated. Also, in the schizophrenic and bipolar disordergroups no correlation was observed between lifetimeneuroleptic load and the relative density of immunoreac-tive neurons in the prefrontal cortex. Furthermore, therewas no significant correlation between the relative densityof any subset of interneurons and duration of illness, acrude measure of antipsychotic exposure. This suggeststhat the observed reduction in parvalbumin- and calbindin-expressing neurons is not attributable to an effect ofantipsychotics.

The presence of co-morbid alcohol and substance abuseis a further potential confounding factor. In this braincollection three of the schizophrenic patients and four ofthe bipolar disorder patients were abusing alcohol and/orillicit drugs at the time of death. In light of this smallsample size and the fact that many of these patients wereabusing multiple drugs, we were not able to determine ifalcohol or any specific drugs alone could have suppressedthe expression of these calcium-binding proteins. Al-


though we suggest that the observed reduction in parval-bumin- and calbindin-immunoreactive neurons in theschizophrenic group was not due to alcohol abuse, thisfactor cannot be completely excluded.

In this study, the use of stereological methods was notpossible within the confines of the tissue available to us.The major source of error in any two-dimensional study islikely to be differences in cell size. Although we were notable to determine cell size in our study, Cotter et al (2002),using the same brain collection as used in this study, foundno significant reductions in the somal size of calbindin- orparvalbumin-immunoreactive neurons in the anterior cin-gulate cortex in schizophrenia, bipolar disorder, or majordepression. Two other studies have also shown that thesize of parvalbumin-containing neurons does not differbetween schizophrenic and control subjects (Kalus et al1997; Woo et al 1997). Although the concept that stereo-logical techniques represent the most accurate approachfor determining neuronal density has recently been chal-lenged (Benes and Lange 2001), the inconsistencies ob-served between the two-dimensional studies in this areaindicate the need for a stereological assessment of cellvolumes and densities of GABAergic neuronal subtypes inpsychiatric disorders. A further possible source of error istissue shrinkage during the processing, sectioning, andstaining of tissue sections. Although ideally a correctionfor shrinkage should be employed when calculating neu-ronal densities in both two-dimensional and three-dimen-sional studies, this is often not feasible and was notpossible within the confines of this investigation.

These data provide further evidence for deficits inspecific populations of GABAergic interneurons in theprefrontal cortex in schizophrenia. Similar reductions wereobserved in bipolar disorder. Although specific interneu-ron populations have not previously been quantified in theprefrontal cortex in this disorder, these reductions areconsistent with a reduction in the expression of GADmRNA and density of GAD-immunoreactive terminals(Benes et al 2000; Guidotti et al 2000). GABAergicdeficits are likely to have wide-ranging effects on theneuronal circuitry of the prefrontal cortex and its outputconnections to other brain regions. Any change in theactivity of GABAergic neurons, which play a vital role inmodulating the activity of the cortex, would lead toimbalances in other systems, for example via their inter-actions with dopaminergic and glutamatergic neurons.This would inevitably have effects on many functions,such as cognitive processing. Studies of the GABAergicsystem may be valuable in the search to define theneuropathological changes in psychiatric disorders andelucidate possible putative mechanisms underlying thedisturbances of cortical function observed.

This study was funded by a project award from the Theodore and VadaStanley Foundation. Postmortem brains were donated by the StanleyFoundation Brain Bank Consortium, courtesy of Drs. Llewellyn B.Bigelow, Juraj Cervenak, Mary M. Herman, Thomas M. Hyde, JoelKleinman, Jose D. Paltan, Robert M. Post, E. Fuller Torrey, Maree J.Webster, and Robert Yolken.

ReferencesAkbarian S, Kim JJ, Potkin SG, Hagman JO, Tafazzoli A,

Bunney WE Jr, et al (1995): Gene expression for glutamicacid decarboxylase is reduced without loss of neurons inprefrontal cortex of schizophrenics. Arch Gen Psychiatry52:258–266.

Akil M, Lewis DA (1992): Postnatal development of parvalbu-min immunoreactivity in axon terminals of basket and chan-delier neurons in monkey neocortex. Prog Neuropsychophar-macol Biol Psychiatry 16:329–337.

Bachus SE, Hyde TM, Herman MM, Eyan MF, Kleinman JE(1997): Abnormal cholecystokinin mRNA levels in entorhi-nal cortex of schizophrenics. J Psychiatr Res 31:233–256.

Beasley CL, Reynolds GP (1997): Parvalbumin-immunoreactiveneurons are reduced in the prefrontal cortex of schizophren-ics. Schizophr Res 24:349–355.

Benes FM, Lange N (2001): Two-dimensional versus three-dimensional cell counting: A practical perspective. TrendsNeurosci 24:11–17.

Benes FM, McSparren J, Bird ED, SanGiovanni JP, Vincent SL(1991): Deficits in small interneurons in prefrontal andcingulate cortices of schizophrenic and schizoaffective pa-tients. Arch Gen Psychiatry 48:996–1001.

Benes FM, Todtenkopf MS, Logiotatos P, Williams M (2000):Glutamate decarboxylase(65)-immunoreactive terminals incingulate and prefrontal cortices of schizophrenic and bipolarbrain. J Chem Neuroanat 20:259–269.

Benes FM, Vincent SL, Marie A, Khan Y (1996): Up-regulationof GABAA receptor binding on neurons of the prefrontalcortex in schizophrenic subjects. Neuroscience 74:1021–1031.

Carder R, LeClerc SL, Hendry SHC (1996): Regulation ofcalcium-binding protein immunoreactivity in GABA neuronsof macaque primary visual cortex. Cereb Cortex 6:271–287.

Celio MR (1990): Calbindin D-28K and parvalbumin in the ratnervous system. Neuroscience 35:375–475.

Cotter D, Landau S, Beasley C, Stevenson R, Chana G, Mac-Millan L, Everall I (2002): The density and spatial distribu-tion of GABAergic neurons in the anterior cingulate cortex inmajor depressive disorder, bipolar disorder and schizophre-nia. Biol Psychiatry 51:377–386.

Danos P, Baumann B, Bernstein H-G, Franz M, Stauch R,Northoff G-M, et al (1998): Schizophrenia and anteroventralthalamic nucleus: Selective decrease of parvalbumin-immu-noreactive thalamocortical projection neurons. PsychiatryRes 82:1–10.

Daviss SR, Lewis DA (1995): Local circuit neurons of theprefrontal cortex in schizophrenia: Selective increase in thedensity of calbindin-immunoreactive neurons. Psychiatry Res59:81–96.


DeFelipe J, Hendry SHC, Jones EG (1989): Visualisation ofchandelier cell axons by parvalbumin-immunoreactivity inmonkey cerebral cortex. Proc Natl Acad Sci USA 86:2093–2097.

DeFelipe J, Jones EG (1991): Parvalbumin immunoreactivityreveals layer IV of monkey cerebral cortex as a mosaic ofmicrozones of thalamic afferent terminations. Brain Res562:39–47.

Demeulemeester H, Vandesande F, Orban GA, Brando C (1988):Heterogeneity of GABAergic cells in cat visual cortex.J Neurosci 8:988–1000.

Evers P, Uylings HB (1994): Effects of microwave pretreatmenton immunocytochemical staining of vibratome sections andtissue blocks of human cerebral cortex stored in formalde-hyde fixative for long periods. J Neurosci Methods 55:163–72.

Gabriel SM, Davidson M, Haroutunian V, Powchik P, BiererLM, Purohit DP, et al (1996): Neuropeptide deficits inschizophrenia vs. Alzheimer’s disease cerebral cortex. BiolPsychiatry 39:82–91.

Guidotti A, Auta J, Davis JM, Gerevini VD, Dwivedi Y, GraysonDR, et al (2000): Decrease in reelin and glutamic aciddecarboxylase 67 (GAD67) expression in schizophrenia andbipolar disorder: A postmortem brain study. Arch Gen Psy-chiatry 57:1061–1069.

Hanada S, Mita T, Nishinok N, Tanaka C (1987): 3H-Muscimolbinding sites are increased in autopsied brains of chronicschizophrenics. Life Sci 40:259–266.

Hsu SM, Raine L, Fanger H (1981): Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques:A comparison between ABC and unlabelled antibody (PAP)procedures. J Histochem Cytochem 29:577–580.

Jacobowitz DM, Winsky L (1991): Immunocytochemical local-isation of calretinin in the forebrain of the rat. J Comp Neurol304:198–218.

Kalus P, Senitz D, Beckmann H (1997): Altered distribution ofparvalbumin-immunoreactive local circuit neurons in theanterior cingulate cortex of schizophrenic patients. PsychiatryRes 75:49–59.

Kamphuis W, Huisman E, Wadman WJ, Lopez Da Silva FH(1989): Decrease in GABA immunoreactivity and alterationof GABA metabolism after kindling in the rat hippocampus.Exp Brain Res 74:375–386.

Lewis DA, Lund JS (1990): Heterogeneity of chandelier neuronsin monkey neocortex: Corticotropin-releasing factor and par-valbumin-immunoreactive populations. J Comp Neurol 293:599–615.

Nemeroff CB, Youngblood WW, Manberg PJ, Prange AJ Jr,Kizer JS (1983): Regional brain concentrations of neuropep-tides in Huntington’s chorea and schizophrenia. Science221:972–975.

Rajkowska G, Goldman-Rakic PS (1995): Cytoarchitectonicdefinition of prefrontal areas in the normal human cortex: I.Remapping of areas 9 and 46 using quantitative criteria.Cereb Cortex 5:307–322.

Reynolds GP, Beasley CL (2001): GABAergic neuronal sub-types in the human frontal cortex—development and deficitsin schizophrenia. J Chem Neuroanat 22:95–100.

Somogyi P, Hodgson AJ, Smith AD, Nunzi MG, Gorio A, WuJY (1984): Different populations of GABAergic neurons inthe visual cortex and hippocampus of cat contain somatosta-tin- or cholecystokinin-immunoreactive material. J Neurosci4:2590–2603.

Torrey EF, Webster M, Knable M, Johnston N, Yolken RH(2000): The Stanley foundation brain collection and neuro-pathology consortium. Schizophr Res 44:151–155.

Virgo L, Humphries C, Mortimer A, Barnes T, Hirsch S,DeBelleroche J (1995): Cholecystokinin messenger RNAdeficit in frontal and temporal cerebral cortex in schizophre-nia. Biol Psychiatry 37:694–701.

Volk DW, Austin MC, Pierri JN, Sampson AR, Lewis DA(2000): Decreased glutamic acid decarboxylase67 messengerRNA expression in a subset of prefrontal cortical �–aminobu-tyric acid neurons in subjects with schizophrenia. Arch GenPsychiatry 57:237–245.

Woo TU, Miller JL, Lewis DA (1997): Schizophrenia and theparvalbumin-containing class of cortical local circuit neurons.Am J Psychiatry 154:1013–1015.

Woo TU, Whitehead RE, Melchitzky DS, Lewis DA (1998): Asubclass of prefrontal gamma-aminobutyric acid axon termi-nals are selectively altered in schizophrenia. Proc Natl AcadSci USA 95:5341–5346.

Zhang ZJ, Reynolds GP (2002): A selective decrease in therelative density of parvalbumin-immunoreactive neurons inthe hippocampus in schizophrenia. Schizophr Res 55:1–10.


selective deficits in prefrontal cortical gabaergic neurons in schizophrenia defined by the presence...

Documents