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No evidence for selective GABAergic interneuron deficits in the anterior thalamic complex of patients with schizophrenia
Progress in Neuro-Psychopharmacology & Biological Psychiatry 28 (2004) 1045 – 1051 www.elsevier.com/locate/pnpbp
No evidence for selective GABAergic interneuron deficits in the anterior thalamic complex of patients with schizophrenia Gavin Dixona,b,c,*, Clive G. Harperb a
Neuroscience Institute of Schizophrenia and Allied Disorders (NISAD), 384 Victoria Street, Darlinghurst, NSW 2010, Australia b Department of Pathology, The University of Sydney, NSW 2006, Australia c Department of Pharmacology, The University of Sydney, NSW 2006, Australia Available online 30 July 2004
Abstract Fewer neurons have been reported in the anterior thalamic complex (AT) of individuals diagnosed with schizophrenia in comparison to control tissue. In addition, the density of presumptive thalamo-cortical relay neurons of the AT is reported to be significantly decreased in schizophrenia compared with controls whilst total AT neuron density appears unchanged. We have investigated whether schizophrenia alters either the density of presumptive interneurons or the ratio between the two fundamental neuron types within the AT by immunohistochemically visualizing GABAergic neurons in post-mortem brain tissue from individuals with a diagnosis of schizophrenia pair-matched to tissue from normal individuals. Qualitative observations indicated no obvious differences between the two cohorts in the morphology of neurons exhibiting a GABAergic phenotype. A cell counting analysis of AT neurons revealed: (1) a non-significant 1% increase in density of GABAergic neurons in schizophrenia compared with controls and (2), a non-significant 6% increase in the percentage of neurons with a GABAergic phenotype in the schizophrenia group compared with controls. These findings suggest that a reduction of AT neuron number in schizophrenia does not alter either the morphology of neurons with a GABAergic phenotype or the ratio of neuronal phenotypes within AT. D 2004 Elsevier Inc. All rights reserved. Keywords: GAD; Inhibitory; Limbic; Episodic; Memory; Papez
1. Introduction The brain regions that comprise the anterior thalamic complex (AT) form an important node of the Papez circuit (Papez, 1937) which is involved in the encoding and retrieval of information (Aggleton and Brown, 1999). The human AT, defined collectively as the anteroventral (AVN) and anteromedial (AMN) thalamic nuclei (Young et al., Abbreviations: AD, anterodorsal thalamic nucleus; AMN, anteromedial thalamic nucleus; AT, anterior thalamic complex; AVN, anteroventral thalamic nucleus; GABA, gamma-amino butyric acid; GAD, glutamic acid decarboxylase; IR, immunoreactivity; %GAD, percentage frequency of glutamic acid decarboxylase immunoreactive neurons; LCN, local circuit neuron; MDN, mediodorsal thalamic nucleus; N GC, numerical density of total neurons; N T, numerical density of GAD-IR neurons; PMD, postmortem delay; SD, standard deviation. * Corresponding author. Department of Pathology (D 06), The University of Sydney, Sydney 2006, Australia. Tel.: +61 2 9351 6105; fax: +61 2 9351 3429. E-mail address: [email protected] (G. Dixon). 0278-5846/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2004.06.004
2000), has significantly reduced numbers of neurons in individuals diagnosed with schizophrenia compared with healthy controls (Young et al., 2000). Furthermore, estimates based on the density of parvalbumin immunoreactivity suggest that schizophrenia-specific neuronal loss in the AVN , the main sub-nucleus of the human AT, is selective for the predominant class of thalamic neuron, the thalamocortical projection neuron (Danos et al., 1998). This in turn raises the additional question of whether fewer GABAergic interneurons occur in the human AT in schizophrenia. Based on immunoreactivity to glutamic acid decarboxylase (GAD) we have recently estimated that an average of 42% of all neurons in the AT express the GABAergic phenotype in a sample of control individuals (Dixon and Harper, 2001). The aim of the current study was to estimate (1) the density and relative frequency of GABAergic neurons and (2) the total number of neurons in the AT of two pair-matched cohorts of brain tissue, one group of which was characterised by a diagnosis of schizophrenia.
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2. Materials and methods 2.1. Tissue collection and characterization Human brain tissue was obtained from the New South Wales Tissue Resource Centre. All brain collection procedures and experimental protocols were approved by the Central Sydney Area Health Service and the Human Ethics Committee of The University of Sydney. Criteria for case inclusion have been previously described (Sarris et al., 2002). Individuals that had a diagnosis of schizophrenia (n=6) were pair-matched for age (F10 years) and gender to six control individuals (Table 1). Where possible, pairs were also matched for post-mortem delay. An experienced clinician reviewed the medical records of each case to create a case summary. The Diagnostic Instrument for Brain Studies (Dean et al., 1999) was used to generate the DSMIV diagnosis (APA, 1994). Cases were coded to ensure blind assessment throughout the subsequent histological and analytical procedures. Extensive neuropathological characterization at macroscopic and microscopic levels was conducted to exclude brain cases with signs of confounding neurodegenerative illnesses. 2.2. Tissue preparation Initial processing of the post-mortem brain in our laboratory has been previously described (Dixon and Harper, 2001; Sarris et al., 2002). Formalin-fixed thalami were dissected from 3 mm coronal brain slices and cryoprotected in buffered 30% sucrose before sectioning at a thickness of 50 Am on a cryostat. Cryostat calibration by test sectioning caliper-verified 3 mm blocks of tissue at a thickness of 50 Am routinely produced 58–62 sections (Harding et al., 1994). Pair-matched tissue was sectioned
at the same time to avoid possible artifact associated with different post-sectioning intervals. Sections of anterior thalamus were transferred to storage boxes containing 0.1 M Tris buffered saline (TBS) with 0.01% NaN3 as a preservative. Every 25th section, representing an interval of 1250Am, was immediately mounted onto a gelatinized slide and stained with Cresyl violet (Chroma, Stuttgart, Germany) to reveal Nissl cytoarchitecture. The remaining sections were stored at 4 8C until required for immunohistochemistry. 2.3. Antigen retrieval and GAD immunohistochemistry A previously described antigen retrieval process was used to prepare tissue sections for GAD-specific immunohistochemical labelling (Dixon and Harper, 2001). Sections from matched brains (control, schizophrenia) were processed in a free-floating environment using the same antigen retrieval conditions and immunohistochemical solutions. Briefly, sections were incubated (72 h at 4 8C with gentle agitation) in a polyclonal GAD 65/67 anti-serum (Sigma #G-5163, St. Louis, MI, USA), diluted 1/105 in 3% normal horse serum (Hunter Antisera, Jessmond, NSW, Australia). A goat anti-rabbit secondary (1/1000 dilution; #BA-1000, VectorLabs, Burlingame, CA, USA ), a standard ABC detection kit (#PK-6100, VectorLabs) and a solution of 0.05% DAB/0.005% H2O2 was used to visualize label. The DAB reaction, performed over ice for approximately 30 s, was intensified with nickel ammonium sulphate and cobalt chloride (0.03% w/v each). Sections for quantification were lightly counter-stained with Cresyl violet prior to coverslipping. Some duplicate sections were cover-slipped without counter-staining to determine the range of morphologies of GAD-labelled neurons. Sections incubated without primary antibody showed no labelling.
Table 1 Clinical data Pair code
Psychiatric diagnosisa
Age/gender
Duration of illness (years)
Cause of death
PMD (h)
Tissue pH
Laterality
Time in fixative (months)
1
SZ – SZ – SZ – SZ – SZ – SZ – SZ CON
51/M 55/M 51/F 52/F 57/M 60/M 50/M 54/M 27/M 37/M 44/M 50/M 47F11 51F8
24 – 16 – 27 – 21 – 4 – 16 – 18F8 –
IHD IHD MOF IHD arrhythmia suicide by hangingb IHD IHD myocarditis IHD suicide by hanging IHD
21 20 12 9.5 36 22 36 30 30 21 36 29 28.5F10.0 21.9F7.4
6.02 6.45 5.80 5.75 6.35 6.75 6.18 6.34 6.22 6.35 6.55 6.68 6.18F0.26 6.39F0.36
R R R R R L R L R R R R 6R 4R/2L
17 32 20 31 15 12 7 9 19 24 11 13 15F5 20F10
2 3 4 5 6
IHD, ischaemic heart disease; MOF, multiple organ failure. a DSM-IV classifications. b No history of depressive illness.
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2.4. Volume analysis of AT Tissue was analyzed in section pairs (a Nissl section plus an adjacent GAD-labelled section) using a computerassisted morphometry program (Halasz and Martin, 1984) connected to a microscope by a camera lucida. The boundary of the AT, defined as the combination of the anteroventral and anteromedial thalamic nuclei (Young et al., 2000), was traced in each Nissl section to calculate cross-sectional area. The third region commonly associated with the AT, the anterodorsal thalamic nucleus was not included in the trace because of its small size relative to total AT volume (Danos et al., 1998; Young et al., 2000). Total AT volume was determined by summing the cross-sectional areas of each AT trace and multiplying the result by the inter-section distance (Cavalieri’s estimation; coefficient of error=0.07). The cross-sectional area of the AT was also measured for each GAD-labelled section to determine shrinkage resulting from the immunohistochemical procedures, which was used to correct numerical density value (via a shrinkage ratio) against the area of each corresponding Nissl-stained section.
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be considered. Thus, for the GAD-labelled sections, frame volumes were multiplied by the shrinkage fraction to give a true estimation of frame volume. Corrected numerical densities of GAD neurons (N GC) for each case were determined by dividing the summed GAD-IR counts by the total corrected frame volume. N T and N GC were averaged within each sample group to obtain mean estimates (N T coefficient of error range=0.05–0.12). 2.6. GAD-IR neuron percentages The percentage of total AT neurons which expressed a GAD-IR phenotype (%GAD) was calculated by dividing N GC by N T and then multiplying by 100%. %GAD values for each case were averaged within each sample group to obtain mean estimates. 2.7. Total neuron number Total numbers of Nissl-stained neurons were estimated for each case using the principle of multiplying total AT volume by total numerical density (N T) (Harding et al., 1994).
2.5. Total neuron densities and GAD-IR neuron densities 2.8. Statistical analyses For each case, section pairs from four evenly-spaced rostro-caudal positions along the length of the anterior thalamic complex were chosen, the start section of which was randomly determined. A computer-generated grid of square frames (140140 Am squares separated from each other by 1 mm) was randomly positioned over each section (Harding et al., 1994; Dixon and Harper, 2001). Frames that fell within the boundary of the AT, either entirely or in part, were scanned using a 40 objective lens (0.75 NA). The number of frames varied between cases (range 23–72). In each Nissl section, neurons with a visible nucleolus were counted if the nucleolus was contained within, or in contact with either the lower or right-hand border of each frame. Frame height was the full z-axis section thickness, since (1) the same calibrated cryostat was used for all sectioning and (2) the variable amount of z-axis shrinkage associated with GAD immunohistochemistry (Dixon and Harper, 2001). For each adjacent GAD section, counted neurons fulfilled the additional requirement of containing immuno-label within the cytoplasm. An average of 235 Nissl-stained neurons (range 172–341) and 106 GAD-IR neurons (range 68–171) were counted across the six pairs of thalami (coefficient of error range=0.06–0.11). Repeated measures of the same Nissl and GAD-labelled sections over successive weeks by the same investigator (GD) produced a 93–97% concordance of numerical density estimates. Total numerical densities (N T) for each section pair were determined by dividing total counts by the total frame volume and expressed as neurons/mm3. Since the one frame size was used throughout the analysis, the numerical density estimation of GAD-IR neurons required tissue shrinkage to
Estimates of total AT volume, total AT neuron number and percentage frequency of GAD-IR neurons (%GAD) were statistically compared between groups using a multivariate analysis of variance (MANOVA; StatView v4.01, Cary, NC, USA). All dependent variables were tested against the potential confounding effects of age, postmortem delay, tissue pH and time in fixative by regression analysis. Similarly, for the group with a diagnosis of schizophrenia, dependent variables were tested via regression analysis against the additional potential interactions: age of onset and duration of illness. The significance threshold was set at 0.05 for all analyses. A post-hoc power analysis was performed on the AT volume data (PASS 2002; NCSS Statistical Software, Kaysville, UT, USA).
3. Results 3.1. GAD immunohistochemistry in human AT The anterior thalamic complex could be clearly identified in all sections of thalamus that underwent GAD immunohistochemistry. Numerous GAD-IR neurons were seen in the AT when viewed under high magnification, the morphology of which varied from elongated fusiform to stellate shapes (Fig. 1A,B). No qualitative difference in the size, shape, labelling intensity or distribution of GAD-IR neurons were observed between sections from any of the 12 brains (Fig. 1A,B). In particular, no labelling intensity differences could be detected between sections that had
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Fig. 1. High power photomicrographs of the anterior thalamic complex following GAD immunohistochemistry and Cresyl violet counter-staining from an individual with a diagnosis of schizophrenia (A) and from a matched control individual (B). GAD-IR neurons (arrowheads) are typically small and fusiform in shape. Note the typical clustering of GAD-IR neurons in panel B. Large diameter neurons with no GAD immunoreactivity (arrows) are considered to represent thalamo-cortical relay neurons and contain visible quantities of yellow lipofuscin. Scale bar=25 Am.
undergone immunohistochemistry at varying times following cryostat sectioning (range: 1 week–4 months) and no regional differences in GAD immunoreactivity were observed in rostral sections that contained both AV and AM. Additionally, no GAD-labelled cells with a nonneuronal morphology were observed (e.g. astrocytes) and no neurons within AT were observed to have more than one nucleolus. 3.2. Anterior thalamic volumes AT volumes were reduced by an average of 27% in the schizophrenia group (154F78 mm3) compared with the control group (210F22 mm3). However, the reduction did not reach significance ( p=0.12). AT volume did not correlate with any of the confounding variables (age at death, age of onset, duration of illness, mean medication dosages, agonal status, or time in fixative).
neurons/mm3; CON: 5100F750 neurons/mm3; p=0.64). Total neuron density did not correlate with any of the confounding variables. Densities of GAD-IR neurons in AT were not significantly different between the two groups (SCH: 2144F491 neurons/mm3; CON: 2117F325 neurons/mm3; p=0.91). No evidence of correlation was found between GAD-IR neuron density and any of the confounding variables. The percentage of total AT neurons that displayed a GAD-IR phenotype (% GAD) showed a non-significant 6% increase in the schizophrenia cohort when compared with the control group (SCH: 44.8F10.1 %; CON: 42.4F6.0%; p=0.67). A graphical comparison of % GAD between the two cohorts on a pair-wise basis showed no trend (Fig. 2A). Again, no correlation was found between % GAD values and any of the confounding variables. 3.4. Total neuron numbers
3.3. Total neuron densities, GAD-IR neuron densities and phenotype percentages Total neuronal densities in AT were not significantly different between the two groups (SCH: 4820F580
Total AT neuron numbers were decreased by an average of 28% in the schizophrenia group cf. controls (SCH: 0.77F0.42107 neurons; CON: 1.07F0.15107 neurons; Fig. 2B), hence showed a similar decrease to that seen with
Fig. 2. Comparison of percentage GAD-IR neuron frequencies (A) and total AT neuron number estimates (B) between control and schizophrenia cohorts. Each graph point (numbers 1–6) corresponds to the pairing defined in Table 1. Lines join matched pairs.
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the AT volume data. However, the observed trend was not significant ( p=0.13). Total neuron number did not correlate with any of the confounding variables.
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Finally, mean pH values did not differ between groups and were comparable to that seen in other quantitative studies in which neuronal densities were obtained on the basis of immunohistochemical labelling (Cotter et al., 2002).
4. Discussion 4.1. Are GABAergic neurons of the AT selectively affected in schizophrenia? Firstly, as assessed by GAD immunohistochemistry (Dixon and Harper, 2001), our results indicate no obvious qualitative differences in morphology between GABAergic neurons of the AT between the two diagnostic groups. This contrasts with evidence of interneuron dysmorphology found in some cortical regions in schizophrenia (Iritani et al., 1999). Secondly, our results suggest that GABAergic neuron density within AT is not altered between schizophrenia and control groups. Further, our results indicate the percentage frequency of GABAergic neurons within AT is not significantly altered in schizophrenia. Danos et al. (1998) found a significantly decreased density of parvalbumin immunoreactive AVN projection neurons in schizophrenia without a corresponding change in overall neuronal density (Danos et al., 1998). Our findings are supported by a recent study which reported no significant differences in optical densities of GAD mRNA transcripts in AT between schizophrenics and matched controls (Popken et al., 2002). Literature concerning cerebral cortex changes associated with schizophrenia indicate altered densities of immunohostochemically defined GABAergic neuron subtypes in the disease state (Daviss and Lewis, 1995; Beasley and Reynolds, 1997; Kalus et al., 1997; Cotter et al., 2002) without a corresponding change in overall neuron density (Selemon et al., 1998; Benes et al., 2001). Each individual within our schizophrenia cohort had a history of neuroleptic medication. Studies in rodents have demonstrated that both classical and atypical neuroleptics lead to increased expression of GAD mRNA isoforms in the basal ganglia (Yu et al., 1999). It is not known whether similar changes take place in the thalamus, although we did not observe any between-group differences in the intensity of GAD immunoreactivity. It is worth noting that the large inter-individual range in %GAD frequency in this and previous studies (Dixon and Harper, 2001) closely matches, in a complimentary fashion, the variation seen in percentages of presumptive relay neurons of the human AT (Danos et al., 1998). It must be noted that a combination of antigen retrieval and immunohistochemical techniques can result in anomalies in subsequent cell quantification unless appropriate precautions are taken. We believe that the matching of case pairs throughout the staining procedure and the calculation of shrinkage corrections for the individual section pairs minimised this complication.
4.2. The extent of reduced total AT neuron numbers in schizophrenia The present study found a non-significant reduction of total AT neuron numbers in schizophrenia compared with controls ( 28%, p=0.13). Young et al. (2000) reported significantly fewer neurons in the AT of individuals with schizophrenia compared with controls ( 16%, p=0.012, n=8). In both studies, total AT volume was the predominant factor contributing to reduced total neuron numbers whilst the total neuronal density was relatively preserved (Young et al., 2000). Byne et al. (2002) also found that a modest sample size was insufficient to find a significant reduction in the AT volume of a schizophrenia cohort when compared with a control cohort. Total neuron number changes between the two cohorts of the present study remained non-significant even when the outlier point (case #2 of the schizophrenia cohort) was excluded from the calculations. A post-hoc power analysis of our total AT volume data indicated that a sample size of at least 19 would be needed to achieve significance. Although the large variability within total AT neuron number (14%) may be due to the limited sample size of our study, evidence of similar variability from other studies (Harding et al., 2000; Young et al., 2000) suggests this extent of variation represents a normal inter-individual phenomenon. Similar quantification techniques (nucleoli counted in thick cryostat sections using an objective lens of similar NA) minimised bias towards an overestimation of large diameter cell types (Harding et al., 1994; Guillery, 2002). It should be mentioned that the sample groups of our study had an average age (and duration of illness) 13–20 years younger than most other studies (Pakkenberg, 1990; Popken et al., 2000; Young et al., 2000; Byne et al., 2002). In view of the current literature, one would have to conclude that the anterior thalamus is less affected in schizophrenia than adjacent structures such as the mediodorsal thalamic complex (Pakkenberg, 1990; Popken et al., 2000; Young et al., 2000; but see DorphPetersen et al., 2004) and the pulvinar complex (Byne et al., 2002). 4.3. Implications for AT function in schizophrenia A modest reduction in total AT neuron numbers in schizophrenia (Young et al., 2000) in the absence of ratio changes between constituent neuron phenotypes (current study) could be interpreted as a reduced AT bcapacityQ without significant change to activity modulation (Pare,
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1991). What are the implications for brain regions to which the AT interconnects? Data from other primate species indicate that AT relay neurons project axons to both the anterior (Xiao and Barbas, 2003) and posterior cingulate cortex (Morris et al., 1999). Studies have documented schizophrenia-specific alterations in the densities of calbindin-IR interneuron subtypes in anterior cingulate cortex (Cotter et al., 2002) without a corresponding change in overall neuron density (Cotter et al., 2001). It is not known whether the posterior cingulate cortex exhibits similar changes in schizophrenia. Studies in a variety of mammals have implicated the mammalian AT in the encoding and retrieval of information (Aggleton and Brown, 1999; Harding et al., 2000). Future cross-discipline investigations will be needed to determine if altered neuronal indices within the AT in schizophrenia are associated with memory deficits, one of the core features of the illness (Saykin et al., 1991).
5. Conclusions The results of this study suggest that neither the basic morphology nor the percentage frequency of GABAergic neurons is altered in schizophrenia. As suggested by previous studies (Popken et al., 2000), further investigation is warranted to determine whether this pattern of neuron alteration in schizophrenia applies to other thalamic nuclei. Acknowledgements This work was supported by NISAD, utilising infrastructure funding from NSW Health. The authors would like to thank the New South Wales Tissue Resource Centre (funded by NIH and NISAD) for access to the human brain tissue. Particular thanks go to Dr Maria Sarris, Andrew Fortis and Therese Garrick for providing advice on human brain tissue processing and the interpretation of the associated clinical data, Clive Jeffery for photography, Associate Professor Sing Kai Lo for advice on statistical analysis and Associate Professor Phillip Ward for helpful advice on the manuscript. References Aggleton, J.P., Brown, M.W., 1999. Episodic memory, amnesia, and the hippocampal-anterior thalamic axis. Behav. Brain Sci. 22, 425 – 489. APA, 1994. Diagnostic and Statistical Manual of Mental Disorders (4th ed.). Washington, DC7 American Psychiatric Association. Beasley, C.L., Reynolds, G.P., 1997. Parvalbumin-immunoreactive neurons are reduced in the prefrontal cortex of schizophrenics. Schizophr. Res. 24, 349 – 355. Benes, F.M., Vincent, S.L., Todtenkopf, M., 2001. The density of pyramidal and nonpyramidal neurons in anterior cingulate cortex of schizophrenic and bipolar subjects. Biol. Psychiatry 50, 395 – 406.
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No evidence for selective GABAergic interneuron deficits in the anterior thalamic complex of patients with schizophrenia Gavin Dixona,b,c,*, Clive G. Harperb a
Neuroscience Institute of Schizophrenia and Allied Disorders (NISAD), 384 Victoria Street, Darlinghurst, NSW 2010, Australia b Department of Pathology, The University of Sydney, NSW 2006, Australia c Department of Pharmacology, The University of Sydney, NSW 2006, Australia Available online 30 July 2004
Abstract Fewer neurons have been reported in the anterior thalamic complex (AT) of individuals diagnosed with schizophrenia in comparison to control tissue. In addition, the density of presumptive thalamo-cortical relay neurons of the AT is reported to be significantly decreased in schizophrenia compared with controls whilst total AT neuron density appears unchanged. We have investigated whether schizophrenia alters either the density of presumptive interneurons or the ratio between the two fundamental neuron types within the AT by immunohistochemically visualizing GABAergic neurons in post-mortem brain tissue from individuals with a diagnosis of schizophrenia pair-matched to tissue from normal individuals. Qualitative observations indicated no obvious differences between the two cohorts in the morphology of neurons exhibiting a GABAergic phenotype. A cell counting analysis of AT neurons revealed: (1) a non-significant 1% increase in density of GABAergic neurons in schizophrenia compared with controls and (2), a non-significant 6% increase in the percentage of neurons with a GABAergic phenotype in the schizophrenia group compared with controls. These findings suggest that a reduction of AT neuron number in schizophrenia does not alter either the morphology of neurons with a GABAergic phenotype or the ratio of neuronal phenotypes within AT. D 2004 Elsevier Inc. All rights reserved. Keywords: GAD; Inhibitory; Limbic; Episodic; Memory; Papez
1. Introduction The brain regions that comprise the anterior thalamic complex (AT) form an important node of the Papez circuit (Papez, 1937) which is involved in the encoding and retrieval of information (Aggleton and Brown, 1999). The human AT, defined collectively as the anteroventral (AVN) and anteromedial (AMN) thalamic nuclei (Young et al., Abbreviations: AD, anterodorsal thalamic nucleus; AMN, anteromedial thalamic nucleus; AT, anterior thalamic complex; AVN, anteroventral thalamic nucleus; GABA, gamma-amino butyric acid; GAD, glutamic acid decarboxylase; IR, immunoreactivity; %GAD, percentage frequency of glutamic acid decarboxylase immunoreactive neurons; LCN, local circuit neuron; MDN, mediodorsal thalamic nucleus; N GC, numerical density of total neurons; N T, numerical density of GAD-IR neurons; PMD, postmortem delay; SD, standard deviation. * Corresponding author. Department of Pathology (D 06), The University of Sydney, Sydney 2006, Australia. Tel.: +61 2 9351 6105; fax: +61 2 9351 3429. E-mail address: [email protected] (G. Dixon). 0278-5846/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2004.06.004
2000), has significantly reduced numbers of neurons in individuals diagnosed with schizophrenia compared with healthy controls (Young et al., 2000). Furthermore, estimates based on the density of parvalbumin immunoreactivity suggest that schizophrenia-specific neuronal loss in the AVN , the main sub-nucleus of the human AT, is selective for the predominant class of thalamic neuron, the thalamocortical projection neuron (Danos et al., 1998). This in turn raises the additional question of whether fewer GABAergic interneurons occur in the human AT in schizophrenia. Based on immunoreactivity to glutamic acid decarboxylase (GAD) we have recently estimated that an average of 42% of all neurons in the AT express the GABAergic phenotype in a sample of control individuals (Dixon and Harper, 2001). The aim of the current study was to estimate (1) the density and relative frequency of GABAergic neurons and (2) the total number of neurons in the AT of two pair-matched cohorts of brain tissue, one group of which was characterised by a diagnosis of schizophrenia.
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2. Materials and methods 2.1. Tissue collection and characterization Human brain tissue was obtained from the New South Wales Tissue Resource Centre. All brain collection procedures and experimental protocols were approved by the Central Sydney Area Health Service and the Human Ethics Committee of The University of Sydney. Criteria for case inclusion have been previously described (Sarris et al., 2002). Individuals that had a diagnosis of schizophrenia (n=6) were pair-matched for age (F10 years) and gender to six control individuals (Table 1). Where possible, pairs were also matched for post-mortem delay. An experienced clinician reviewed the medical records of each case to create a case summary. The Diagnostic Instrument for Brain Studies (Dean et al., 1999) was used to generate the DSMIV diagnosis (APA, 1994). Cases were coded to ensure blind assessment throughout the subsequent histological and analytical procedures. Extensive neuropathological characterization at macroscopic and microscopic levels was conducted to exclude brain cases with signs of confounding neurodegenerative illnesses. 2.2. Tissue preparation Initial processing of the post-mortem brain in our laboratory has been previously described (Dixon and Harper, 2001; Sarris et al., 2002). Formalin-fixed thalami were dissected from 3 mm coronal brain slices and cryoprotected in buffered 30% sucrose before sectioning at a thickness of 50 Am on a cryostat. Cryostat calibration by test sectioning caliper-verified 3 mm blocks of tissue at a thickness of 50 Am routinely produced 58–62 sections (Harding et al., 1994). Pair-matched tissue was sectioned
at the same time to avoid possible artifact associated with different post-sectioning intervals. Sections of anterior thalamus were transferred to storage boxes containing 0.1 M Tris buffered saline (TBS) with 0.01% NaN3 as a preservative. Every 25th section, representing an interval of 1250Am, was immediately mounted onto a gelatinized slide and stained with Cresyl violet (Chroma, Stuttgart, Germany) to reveal Nissl cytoarchitecture. The remaining sections were stored at 4 8C until required for immunohistochemistry. 2.3. Antigen retrieval and GAD immunohistochemistry A previously described antigen retrieval process was used to prepare tissue sections for GAD-specific immunohistochemical labelling (Dixon and Harper, 2001). Sections from matched brains (control, schizophrenia) were processed in a free-floating environment using the same antigen retrieval conditions and immunohistochemical solutions. Briefly, sections were incubated (72 h at 4 8C with gentle agitation) in a polyclonal GAD 65/67 anti-serum (Sigma #G-5163, St. Louis, MI, USA), diluted 1/105 in 3% normal horse serum (Hunter Antisera, Jessmond, NSW, Australia). A goat anti-rabbit secondary (1/1000 dilution; #BA-1000, VectorLabs, Burlingame, CA, USA ), a standard ABC detection kit (#PK-6100, VectorLabs) and a solution of 0.05% DAB/0.005% H2O2 was used to visualize label. The DAB reaction, performed over ice for approximately 30 s, was intensified with nickel ammonium sulphate and cobalt chloride (0.03% w/v each). Sections for quantification were lightly counter-stained with Cresyl violet prior to coverslipping. Some duplicate sections were cover-slipped without counter-staining to determine the range of morphologies of GAD-labelled neurons. Sections incubated without primary antibody showed no labelling.
Table 1 Clinical data Pair code
Psychiatric diagnosisa
Age/gender
Duration of illness (years)
Cause of death
PMD (h)
Tissue pH
Laterality
Time in fixative (months)
1
SZ – SZ – SZ – SZ – SZ – SZ – SZ CON
51/M 55/M 51/F 52/F 57/M 60/M 50/M 54/M 27/M 37/M 44/M 50/M 47F11 51F8
24 – 16 – 27 – 21 – 4 – 16 – 18F8 –
IHD IHD MOF IHD arrhythmia suicide by hangingb IHD IHD myocarditis IHD suicide by hanging IHD
21 20 12 9.5 36 22 36 30 30 21 36 29 28.5F10.0 21.9F7.4
6.02 6.45 5.80 5.75 6.35 6.75 6.18 6.34 6.22 6.35 6.55 6.68 6.18F0.26 6.39F0.36
R R R R R L R L R R R R 6R 4R/2L
17 32 20 31 15 12 7 9 19 24 11 13 15F5 20F10
2 3 4 5 6
IHD, ischaemic heart disease; MOF, multiple organ failure. a DSM-IV classifications. b No history of depressive illness.
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2.4. Volume analysis of AT Tissue was analyzed in section pairs (a Nissl section plus an adjacent GAD-labelled section) using a computerassisted morphometry program (Halasz and Martin, 1984) connected to a microscope by a camera lucida. The boundary of the AT, defined as the combination of the anteroventral and anteromedial thalamic nuclei (Young et al., 2000), was traced in each Nissl section to calculate cross-sectional area. The third region commonly associated with the AT, the anterodorsal thalamic nucleus was not included in the trace because of its small size relative to total AT volume (Danos et al., 1998; Young et al., 2000). Total AT volume was determined by summing the cross-sectional areas of each AT trace and multiplying the result by the inter-section distance (Cavalieri’s estimation; coefficient of error=0.07). The cross-sectional area of the AT was also measured for each GAD-labelled section to determine shrinkage resulting from the immunohistochemical procedures, which was used to correct numerical density value (via a shrinkage ratio) against the area of each corresponding Nissl-stained section.
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be considered. Thus, for the GAD-labelled sections, frame volumes were multiplied by the shrinkage fraction to give a true estimation of frame volume. Corrected numerical densities of GAD neurons (N GC) for each case were determined by dividing the summed GAD-IR counts by the total corrected frame volume. N T and N GC were averaged within each sample group to obtain mean estimates (N T coefficient of error range=0.05–0.12). 2.6. GAD-IR neuron percentages The percentage of total AT neurons which expressed a GAD-IR phenotype (%GAD) was calculated by dividing N GC by N T and then multiplying by 100%. %GAD values for each case were averaged within each sample group to obtain mean estimates. 2.7. Total neuron number Total numbers of Nissl-stained neurons were estimated for each case using the principle of multiplying total AT volume by total numerical density (N T) (Harding et al., 1994).
2.5. Total neuron densities and GAD-IR neuron densities 2.8. Statistical analyses For each case, section pairs from four evenly-spaced rostro-caudal positions along the length of the anterior thalamic complex were chosen, the start section of which was randomly determined. A computer-generated grid of square frames (140140 Am squares separated from each other by 1 mm) was randomly positioned over each section (Harding et al., 1994; Dixon and Harper, 2001). Frames that fell within the boundary of the AT, either entirely or in part, were scanned using a 40 objective lens (0.75 NA). The number of frames varied between cases (range 23–72). In each Nissl section, neurons with a visible nucleolus were counted if the nucleolus was contained within, or in contact with either the lower or right-hand border of each frame. Frame height was the full z-axis section thickness, since (1) the same calibrated cryostat was used for all sectioning and (2) the variable amount of z-axis shrinkage associated with GAD immunohistochemistry (Dixon and Harper, 2001). For each adjacent GAD section, counted neurons fulfilled the additional requirement of containing immuno-label within the cytoplasm. An average of 235 Nissl-stained neurons (range 172–341) and 106 GAD-IR neurons (range 68–171) were counted across the six pairs of thalami (coefficient of error range=0.06–0.11). Repeated measures of the same Nissl and GAD-labelled sections over successive weeks by the same investigator (GD) produced a 93–97% concordance of numerical density estimates. Total numerical densities (N T) for each section pair were determined by dividing total counts by the total frame volume and expressed as neurons/mm3. Since the one frame size was used throughout the analysis, the numerical density estimation of GAD-IR neurons required tissue shrinkage to
Estimates of total AT volume, total AT neuron number and percentage frequency of GAD-IR neurons (%GAD) were statistically compared between groups using a multivariate analysis of variance (MANOVA; StatView v4.01, Cary, NC, USA). All dependent variables were tested against the potential confounding effects of age, postmortem delay, tissue pH and time in fixative by regression analysis. Similarly, for the group with a diagnosis of schizophrenia, dependent variables were tested via regression analysis against the additional potential interactions: age of onset and duration of illness. The significance threshold was set at 0.05 for all analyses. A post-hoc power analysis was performed on the AT volume data (PASS 2002; NCSS Statistical Software, Kaysville, UT, USA).
3. Results 3.1. GAD immunohistochemistry in human AT The anterior thalamic complex could be clearly identified in all sections of thalamus that underwent GAD immunohistochemistry. Numerous GAD-IR neurons were seen in the AT when viewed under high magnification, the morphology of which varied from elongated fusiform to stellate shapes (Fig. 1A,B). No qualitative difference in the size, shape, labelling intensity or distribution of GAD-IR neurons were observed between sections from any of the 12 brains (Fig. 1A,B). In particular, no labelling intensity differences could be detected between sections that had
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Fig. 1. High power photomicrographs of the anterior thalamic complex following GAD immunohistochemistry and Cresyl violet counter-staining from an individual with a diagnosis of schizophrenia (A) and from a matched control individual (B). GAD-IR neurons (arrowheads) are typically small and fusiform in shape. Note the typical clustering of GAD-IR neurons in panel B. Large diameter neurons with no GAD immunoreactivity (arrows) are considered to represent thalamo-cortical relay neurons and contain visible quantities of yellow lipofuscin. Scale bar=25 Am.
undergone immunohistochemistry at varying times following cryostat sectioning (range: 1 week–4 months) and no regional differences in GAD immunoreactivity were observed in rostral sections that contained both AV and AM. Additionally, no GAD-labelled cells with a nonneuronal morphology were observed (e.g. astrocytes) and no neurons within AT were observed to have more than one nucleolus. 3.2. Anterior thalamic volumes AT volumes were reduced by an average of 27% in the schizophrenia group (154F78 mm3) compared with the control group (210F22 mm3). However, the reduction did not reach significance ( p=0.12). AT volume did not correlate with any of the confounding variables (age at death, age of onset, duration of illness, mean medication dosages, agonal status, or time in fixative).
neurons/mm3; CON: 5100F750 neurons/mm3; p=0.64). Total neuron density did not correlate with any of the confounding variables. Densities of GAD-IR neurons in AT were not significantly different between the two groups (SCH: 2144F491 neurons/mm3; CON: 2117F325 neurons/mm3; p=0.91). No evidence of correlation was found between GAD-IR neuron density and any of the confounding variables. The percentage of total AT neurons that displayed a GAD-IR phenotype (% GAD) showed a non-significant 6% increase in the schizophrenia cohort when compared with the control group (SCH: 44.8F10.1 %; CON: 42.4F6.0%; p=0.67). A graphical comparison of % GAD between the two cohorts on a pair-wise basis showed no trend (Fig. 2A). Again, no correlation was found between % GAD values and any of the confounding variables. 3.4. Total neuron numbers
3.3. Total neuron densities, GAD-IR neuron densities and phenotype percentages Total neuronal densities in AT were not significantly different between the two groups (SCH: 4820F580
Total AT neuron numbers were decreased by an average of 28% in the schizophrenia group cf. controls (SCH: 0.77F0.42107 neurons; CON: 1.07F0.15107 neurons; Fig. 2B), hence showed a similar decrease to that seen with
Fig. 2. Comparison of percentage GAD-IR neuron frequencies (A) and total AT neuron number estimates (B) between control and schizophrenia cohorts. Each graph point (numbers 1–6) corresponds to the pairing defined in Table 1. Lines join matched pairs.
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the AT volume data. However, the observed trend was not significant ( p=0.13). Total neuron number did not correlate with any of the confounding variables.
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Finally, mean pH values did not differ between groups and were comparable to that seen in other quantitative studies in which neuronal densities were obtained on the basis of immunohistochemical labelling (Cotter et al., 2002).
4. Discussion 4.1. Are GABAergic neurons of the AT selectively affected in schizophrenia? Firstly, as assessed by GAD immunohistochemistry (Dixon and Harper, 2001), our results indicate no obvious qualitative differences in morphology between GABAergic neurons of the AT between the two diagnostic groups. This contrasts with evidence of interneuron dysmorphology found in some cortical regions in schizophrenia (Iritani et al., 1999). Secondly, our results suggest that GABAergic neuron density within AT is not altered between schizophrenia and control groups. Further, our results indicate the percentage frequency of GABAergic neurons within AT is not significantly altered in schizophrenia. Danos et al. (1998) found a significantly decreased density of parvalbumin immunoreactive AVN projection neurons in schizophrenia without a corresponding change in overall neuronal density (Danos et al., 1998). Our findings are supported by a recent study which reported no significant differences in optical densities of GAD mRNA transcripts in AT between schizophrenics and matched controls (Popken et al., 2002). Literature concerning cerebral cortex changes associated with schizophrenia indicate altered densities of immunohostochemically defined GABAergic neuron subtypes in the disease state (Daviss and Lewis, 1995; Beasley and Reynolds, 1997; Kalus et al., 1997; Cotter et al., 2002) without a corresponding change in overall neuron density (Selemon et al., 1998; Benes et al., 2001). Each individual within our schizophrenia cohort had a history of neuroleptic medication. Studies in rodents have demonstrated that both classical and atypical neuroleptics lead to increased expression of GAD mRNA isoforms in the basal ganglia (Yu et al., 1999). It is not known whether similar changes take place in the thalamus, although we did not observe any between-group differences in the intensity of GAD immunoreactivity. It is worth noting that the large inter-individual range in %GAD frequency in this and previous studies (Dixon and Harper, 2001) closely matches, in a complimentary fashion, the variation seen in percentages of presumptive relay neurons of the human AT (Danos et al., 1998). It must be noted that a combination of antigen retrieval and immunohistochemical techniques can result in anomalies in subsequent cell quantification unless appropriate precautions are taken. We believe that the matching of case pairs throughout the staining procedure and the calculation of shrinkage corrections for the individual section pairs minimised this complication.
4.2. The extent of reduced total AT neuron numbers in schizophrenia The present study found a non-significant reduction of total AT neuron numbers in schizophrenia compared with controls ( 28%, p=0.13). Young et al. (2000) reported significantly fewer neurons in the AT of individuals with schizophrenia compared with controls ( 16%, p=0.012, n=8). In both studies, total AT volume was the predominant factor contributing to reduced total neuron numbers whilst the total neuronal density was relatively preserved (Young et al., 2000). Byne et al. (2002) also found that a modest sample size was insufficient to find a significant reduction in the AT volume of a schizophrenia cohort when compared with a control cohort. Total neuron number changes between the two cohorts of the present study remained non-significant even when the outlier point (case #2 of the schizophrenia cohort) was excluded from the calculations. A post-hoc power analysis of our total AT volume data indicated that a sample size of at least 19 would be needed to achieve significance. Although the large variability within total AT neuron number (14%) may be due to the limited sample size of our study, evidence of similar variability from other studies (Harding et al., 2000; Young et al., 2000) suggests this extent of variation represents a normal inter-individual phenomenon. Similar quantification techniques (nucleoli counted in thick cryostat sections using an objective lens of similar NA) minimised bias towards an overestimation of large diameter cell types (Harding et al., 1994; Guillery, 2002). It should be mentioned that the sample groups of our study had an average age (and duration of illness) 13–20 years younger than most other studies (Pakkenberg, 1990; Popken et al., 2000; Young et al., 2000; Byne et al., 2002). In view of the current literature, one would have to conclude that the anterior thalamus is less affected in schizophrenia than adjacent structures such as the mediodorsal thalamic complex (Pakkenberg, 1990; Popken et al., 2000; Young et al., 2000; but see DorphPetersen et al., 2004) and the pulvinar complex (Byne et al., 2002). 4.3. Implications for AT function in schizophrenia A modest reduction in total AT neuron numbers in schizophrenia (Young et al., 2000) in the absence of ratio changes between constituent neuron phenotypes (current study) could be interpreted as a reduced AT bcapacityQ without significant change to activity modulation (Pare,
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1991). What are the implications for brain regions to which the AT interconnects? Data from other primate species indicate that AT relay neurons project axons to both the anterior (Xiao and Barbas, 2003) and posterior cingulate cortex (Morris et al., 1999). Studies have documented schizophrenia-specific alterations in the densities of calbindin-IR interneuron subtypes in anterior cingulate cortex (Cotter et al., 2002) without a corresponding change in overall neuron density (Cotter et al., 2001). It is not known whether the posterior cingulate cortex exhibits similar changes in schizophrenia. Studies in a variety of mammals have implicated the mammalian AT in the encoding and retrieval of information (Aggleton and Brown, 1999; Harding et al., 2000). Future cross-discipline investigations will be needed to determine if altered neuronal indices within the AT in schizophrenia are associated with memory deficits, one of the core features of the illness (Saykin et al., 1991).
5. Conclusions The results of this study suggest that neither the basic morphology nor the percentage frequency of GABAergic neurons is altered in schizophrenia. As suggested by previous studies (Popken et al., 2000), further investigation is warranted to determine whether this pattern of neuron alteration in schizophrenia applies to other thalamic nuclei. Acknowledgements This work was supported by NISAD, utilising infrastructure funding from NSW Health. The authors would like to thank the New South Wales Tissue Resource Centre (funded by NIH and NISAD) for access to the human brain tissue. Particular thanks go to Dr Maria Sarris, Andrew Fortis and Therese Garrick for providing advice on human brain tissue processing and the interpretation of the associated clinical data, Clive Jeffery for photography, Associate Professor Sing Kai Lo for advice on statistical analysis and Associate Professor Phillip Ward for helpful advice on the manuscript. References Aggleton, J.P., Brown, M.W., 1999. Episodic memory, amnesia, and the hippocampal-anterior thalamic axis. Behav. Brain Sci. 22, 425 – 489. APA, 1994. Diagnostic and Statistical Manual of Mental Disorders (4th ed.). Washington, DC7 American Psychiatric Association. Beasley, C.L., Reynolds, G.P., 1997. Parvalbumin-immunoreactive neurons are reduced in the prefrontal cortex of schizophrenics. Schizophr. Res. 24, 349 – 355. Benes, F.M., Vincent, S.L., Todtenkopf, M., 2001. The density of pyramidal and nonpyramidal neurons in anterior cingulate cortex of schizophrenic and bipolar subjects. Biol. Psychiatry 50, 395 – 406.
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