Brain Research 923 (2001) 39–44www.elsevier.com/ locate /bres

Research report

Quantitative analysis of glutamic acid decarboxylase-immunoreactiveneurons in the anterior thalamus of the human brain

a,b,c , b*Gavin Dixon , Clive G. HarperaNeuroscience Institute of Schizophrenia and Allied Disorders, 384 Victoria Street, Darlinghurst, NSW 2010, Australia

bDepartments of Pathology and Pharmacology, The University of Sydney, NSW 2006 AustraliacDepartments of Pharmacology, The University of Sydney, NSW 2006 Australia

Accepted 5 October 2001

Abstract

Local circuit neurons in the human anterior thalamus (AT) were identified on the basis of glutamic acid decarboxylase immuno-reactivity (GAD-IR). GAD-IR neurons of the AT displayed small diameter somas with thin, sparsely-branching dendrites, consistent withthe morphological characteristics of local circuit neurons found in the thalami of other mammals. Sampling techniques revealed anaverage of 42% of all neurons within the AT were GAD-IR, one of the highest reported percentages of local circuit neurons in themammalian thalamus. The presence of high proportion of local circuit neurons in the AT may indicate the extent to which the Papezcircuit has evolved within the human brain in comparison to other mammals. Crown copyright 2001 Published by Elsevier ScienceB.V. All rights reserved.

Theme: Other systems of the CNS

Topic: Limbic system and hypothalamus

Keywords: Anteroventral; GABA; Interneuron; Memory; Papez; Limbic

1. Introduction neurons than is predicted on the basis of brain size incomparison to other primate species [4]. A majority of

The anterior thalamus (AT) is an important node of the neurons in the anteroventral nucleus (AVN), the dominantPapez circuit [22] that participates in learning and memory structure within the AT of the human brain [4,12], displayacquisition [1]. Reciprocal projections exist between AT characteristics of thalamocortical relay neurons [6,8,9].and both the posterior cingulate cortex [26] and subiculum Both the axons and parent cell bodies of presumptive relay[2]. Additionally, a major projection from the hippocampal neurons are immunoreactive for the calcium-binding pro-complex reaches the AT via the mamillary bodies [25]. tein parvalbumin [8,16] but it is unclear whether theQuantitative analysis of neuronal phenotypes is important presence of this protein is an exclusive marker of relayfor a functional understanding of the Papez circuit and is neurons [9]. Quantitative estimates based on parvalbuminparticularly warranted for the human anterior thalamus, immunoreactivity [8] suggest that 41% of total neurons inwhich has a greater volume and contains significantly more AVN belong to the second class of neuron found the

mammalian thalamus, the local circuit neuron. However,sampling techniques based on lipofuscin staining deter-Abbreviations: AT, anterior thalamus; AVN, anteroventral thalamic

nucleus; GABA, gamma-amino butyric acid; GAD, glutamic acid de- mined 28% of all neurons in the AVN to be local circuitcarboxylase; GAD-IR, glutamic acid decarboxylase immunoreactivity; neurons [6]. Local circuit neurons are known to use GABACA Caucasian /Australian; CE Caucasian /Egyptian; HE Haematoxylin- as a neurotransmitter and thus can be phenotyped by theirEosin; PMD, post-mortem delay; M, male; F, female

immunoreactivity to either GABA [14], or its biosynthetic*Corresponding author. Tel.: 161-2-9351-6105; fax: 161-2-9351-enzyme, glutamic acid decarboxylase (GAD) [21]. The3429.

E-mail address: gavind@med.usyd.edu.au (G. Dixon). aim of the present study was to use GAD immuno-

0006-8993/01/$ – see front matter Crown copyright 2001 Published by Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 01 )03194-8

40 G. Dixon, C.G. Harper / Brain Research 923 (2001) 39 –44

histochemistry to identify neurons within the human AT weeks in 15% buffered formalin and subsequently storedthat possess a local circuit neuron phenotype and hence in 10% buffered formalin prior to being cut into 3 mmdetermine the relative proportions of local circuit neurons coronal slices. Thalamus was dissected from the slices andand relay neurons contained within this thalamic region. cryoprotected in buffered 30% sucrose before cryostat

sectioning. Coronal sections of anterior thalamus were cutat a thickness of 50 mm, collected with a moistened

2. Materials and methods paintbrush and transferred to compartmentalized storageboxes containing 0.1 M Tris buffered saline (TBS) with

2.1. Tissue collection 0.01% NaN as a preservative. Every 25th section, repre-3

senting an interval of 1250 mm, was immediately mountedHuman brain tissue was obtained from the NISAD/ onto subbed slides and stained with Cresyl violet ([M84;

NH&MRC Tissue Resource Centre. All brain collection Chroma, Stuttgart, Germany) to reveal Nissl cytoarchitec-procedures and experimental protocols were approved by ture. The remaining sections were stored at 48C untilthe ethics committees of the Central Sydney Area Health required.Service and The University of Sydney. Table 1 summa-rizes the clinical and tissue storage data for the six cases 2.2. Gad immunohistochemistryused in the study. The criteria for inclusion of casesincluded a post-mortem delay no greater than 36 h, no No specific GAD labelling could be obtained in sectionsclinical or pathological evidence of CNS disease and no of thalamus using our standard immunohistochemicalhistory of psychiatric, drug or alcohol-related illnesses. (IHC) protocol [9]. An antigen retrieval technique wasClinical histories of each patient were evaluated by exami- therefore used to uncover antigen masked by the fixationnation of clinical records. In the case of the 60 year old process [7]. Sections adjacent to the above-mentionedmale (Case I) who committed suicide by hanging, exten- Nissl-stained sections were heated to 908C for 60 min insive clinical notes demonstrated no evidence of any 0.1 M TBS prior to IHC. Sections were then IHCpsychiatric disorder that could have contributed to his processed in a free-floating environment as previouslydeath. Neuropathological examination consisted of both described [9]. Sections from each case were processedmacroscopic and microscopic reports, the latter of which together to minimize variations due to IHC. A polyclonalwas compiled following analysis of HE-stained sections GAD 65/67 anti-serum ([G5163, Sigma, St. Louis, USA),from superior frontal, motor, cingulate, inferior temporal diluted 1/100,000 in 3% normal goat serum and 0.1 Mand entorhinal regions of the cerebral cortex, hippocampus, TBS was used as the primary antibody. Label was visual-caudate, cerebellar vermis, rostral pons and caudal brain- ized with a goat anti-mouse secondary ([BA-1000, Vec-stem. torLabs, Burlingame, USA; 1/1000 dilution) and a stan-

Complete hemispheres from all cases were fixed for 2 dard ABC detection kit ([PK-6100, VectorLabs) with

Table 1Clinical data

Case Age (years) Ethnicity Cause of death Agonal PMD Laterality Time inacode gender state (h) fixative

(months)

A 55 M CE cardiac arrest 3 20 right 32thalamus

B 52 F CA ischaemic 3 9.5 right 31heart disease thalamus

C 60 M CA suicide by 1 22 left thalamus 12bhanging

D 54 M CA coronary heart 2 30 left thalamus 9disease

E 37 M CA ischaemic 1 21 right 24heart disease thalamus

F 50 M CA ischaemic 2 29 right 13heart disease thalamua

Mean 51 2.0 21.9 4 right /2 left 20SD 8 0.9 7.4 10

Abbreviations: M, male; F, female; CA, Caucasian /Australian; CG, Caucasian /German; CE, Caucasian /Egyptian; PMD, post-mortem delay; SD, standarddeviation.a No history of depressive illness.b Agonal state: 1 – best, 2 – good, 3 – average, 4 – poor, 5 – worst.

G. Dixon, C.G. Harper / Brain Research 923 (2001) 39 –44 41

0.05% DAB as the chromagen. The DAB reaction was either the lower or right-hand border of each disectorintensified with nickel ammonium sulphate and cobalt frame [13].chloride (0.03% w/v each). Sections were air-dried onto For GAD-labelled sections of each section pair, neuronssubbed slides, counter-stained with Cresyl Violet, dehy- were counted if (1), the nucleolus was visible (as detaileddrated and cover-slipped with Pertex (Medite, Burgdorf, above), and (2), the cytoplasm contained immunolabel.Germany). Some duplicate sections were cover-slipped The interrater reliability of this technique ranged from 92without counter-staining to allow for a description of the to 96%. For each section pair, numerical densities of allfull dendritic arbors of GAD-IR cells. neurons (N ) and of GAD-IR neurons (N ) were de-T G

Preliminary attempts at visualizing both GAD-IR neu- termined by dividing the neuron counts by the total frame24 3rons (with counter-stained nucleoli) and unlabelled neurons volume (1403140350 mm59.8310 mm ). To account

in the same section proved unreliable. In order to clearly for the observed distortion that occurred in tissue process-visualise both the nucleolus and cellular GAD-IR within ing, N was divided by the shrinkage fraction to obtain theG

individual neurons it was important to use a highly corrected numerical density of GAD-IR neurons (N ). NGC T

differentiated Nissl counterstain. This procedure would and N were averaged for the 4 sample positions in eachGC

frequently make both the nucleoli and endoplasmic re- brain and the percentage of total AT neurons expressing aticulum of large, unlabelled neurons difficult to distinguish. GAD-IR phenotype (%GAD neurons) calculated by theHence, immediately adjacent sections, collectively referred formula:to as section pairs, were used to determine numerical

%GAD-IR neurons 5 (N /N ) 3 100%GC Tdensities of GAD-IR neurons and total neurons. Onesection in each section pair underwent GAD-labellingfollowed by a Nissl counterstain, while the other section 2.5. Statistical testswas processed for Nissl staining alone. Corrections weremade for the subsequent differences in the cross-sectional Multiple linear regression analysis was performed usingarea of the AT within each section pair (see below). a statistical program (StatView4) with %GAD-IR neurons

and total AT neurons as dependent variables and age,agonal status, post-mortem delay and time in fixative as2.3. Estimates of AT cross-sectional area and correctionindependent variables (P50.05 for significance).for section shrinkage

Section pairs were analysed using a computer-assistedmorphometry program [11,13] connected to a microscope

3. Resultsby a camera lucida. Outlines of the AT were traced and theprogram calculated the enclosed area (Cavalieri principle).

3.1. GAD-immunoreactivity in human anterior thalamusFor each section pair, the change in cross-sectional area ofthe AT in the GAD-labelled section was expressed as a

GAD-labelled sections at low power showed the im-fraction of the AT area measured in the corresponding

munoreactive profile of the AT to be clearly discernibleNissl section. The resulting ‘shrinkage fraction’ was then

from surrounding fibre tracts (Fig. 1A). The anterior poleused to correct numerical density estimates of GAD-IR

of the nearby thalamic reticular nucleus, the mediodorsalneurons (see below).

nucleus and the tail of the caudate nucleus were also foundto be GAD-IR (Fig. 1A). Sections processed without the

2.4. Estimates of numerical densities and phenotype inclusion of GAD antiserum showed no labelling (notratios shown).

At higher magnification, GAD-IR was found within theFor each case, section pairs from four evenly-spaced somata of cells exhibiting neuronal morphologies (Fig. 1B,

rostro-caudal positions along the length of the AT were C). Labelled cell bodies were either fusiform or round andchosen, the start point of which was randomly determined. typically displayed 2–3 primary dendrites. SecondaryA computer-generated grid of disector frames (1403140 dendritic processes containing GAD-IR could often bemm squares separated from each other by 1 mm) was followed for up to 50 mm. In sections that had undergonerandomly positioned over each section [13]. Frames that GAD-labelling and Nissl counter-staining, small GAD-IRfell within the boundary of the AT, either entirely or in somata contrasted with large diameter neurons that werepart, were used for analysis. A 403 objective lens with a devoid of immunoreactivity (Fig. 1D). Small diameternumerical aperture of 0.75 was used for the counting neurons that did not contain GAD-IR were not observed.procedure, focussing from top to bottom along the entire In addition to labelled neurons, GAD-IR was foundz-axis of the section. In the Nissl-stained section of each throughout the AT neuropil within thin, axon-like pro-section pair, neurons with a visible nucleolus were counted cesses and as distinct punctate profiles (not shown). Bothif the nucleolus was contained within, or in contact with cellular and neuropil GAD-IR was present throughout the

42 G. Dixon, C.G. Harper / Brain Research 923 (2001) 39 –44

estimations of GAD-IR and total neuron densities wereperformed in adjacent GAD-IR and Nissl sections (section-pairs).

3.2. AT volumes and shrinkage corrections

Despite the antigen retrieval process being standardizedfor time and temperature, the areas of AT outlines inindividual sections processed for GAD-IR varied between67 and 102% of the AT area measured in adjacent Nissl-stained sections. Therefore, shrinkage corrections werecalculated and used to adjust the numerical density esti-mates of GAD-IR neurons for each section-pair prior to thedetermination of phenotype ratios (Table 2).

3.3. Neuronal densities and phenotype ratios

Table 2 summarizes the estimated total volumes, nu-merical densities, shrinkage corrections, total neuron num-ber estimates and the percentage of GAD-IR neurons forthe six brains. An average of 239 Nissl-stained neurons(range 188–337) and 103 GAD-IR neurons (range 158–68) were counted per case. Both total numbers of Nissl-stained and GAD-IR neurons, along with the percentagefrequency of GAD-IR neurons were not significantlyrelated to age, agonal status, post-mortem delay or time infixative. Although the sample group contained 5 malecases and 1 female case, the %GAD-IR value of 41.1% forthe one female was closest to the group average (42.4%)of any of the six cases.

4. Discussion

4.1. Local circuit neuron frequency in the AT of humanthalamus

Previous studies have shown GAD-IR neurons of theFig. 1. Brightfield photomicrographs of GAD-IR in coronal sections of mammalian thalamus (with the exception of the thalamichuman anterior thalamus. (A) GAD immunoreactivity within the an- reticular nucleus) to possess the morphological characteris-teroventral nucleus (denoted by asterisk) is clearly delineated from the tics of local circuit neurons [20]. On the basis of GAD-IR,surrounding fibre tracts. Scale bar54 mm. (B–C) GAD-IR somata,

our results indicate that 42% of all neurons in the humandendrites and neuropil within AVN at higher magnification. LabelledAT are local circuit neurons. The remaining 58% ofneurons have either fusiform (B) or stellate (C) morphologies. (D) Dark

GAD-IR neurons (arrows) contrast with the pale, counter-stained profiles neurons are assumed to represent the only other type ofof presumptive projection neurons (arrowheads). Scale bar for B–D530 neuron described in the mammalian thalamus, themm. thalamocortical projection neuron [15]. Parvalbumin im-

munoreactivity is found in thalamocortical projectionentire z-axis of each GAD-labelled section, which varied in neurons in the AT of both the monkey [16] and human [8],thickness between 18 and 23 mm. although in the human AT the same antiserum labels a

Attempts at visualizing both GAD-IR neurons (with small population of neurons with the morphologicalcounter-stained nucleoli) and unlabelled neurons in the characteristics of local circuit neurons [9]. Our estimatesame section proved unreliable. Typically, the highly compares favourably with that of Danos et al. [8] whosedifferentiated Nissl counterstain that was needed to visual- results indicate that an average of 41% of neurons in theize the nucleolus of a GAD-IR neuron frequently made human AVN are immuno-negative for parvalbumin. How-both the nucleolus and endoplasmic reticulum of large, ever, percentage estimates of neuron subtype on the basisunlabelled neurons impossible to distinguish. Hence, the of lipofuscin pigmentation found local circuit neurons to

G. Dixon, C.G. Harper / Brain Research 923 (2001) 39 –44 43

Table 2Determination of neuron ratios in hyuman anterior thalamus

Case Numerical densities Shrinkage Numerical densities %GAD-IRacode of total (Nissl) fractions of GAD-IR neurons

6 6neurons (310 neurons (310 (mean6SD)3 3 bneurons /mm 6SD) neurons /mm 6SD)

A 5.34 0.92 2.50 46.7B 4.97 0.82 1.77 41.1C 4.05 0.96 1.64 40.3D 5.75 0.72 1.97 34.7E 4.48 0.72 2.32 52.4F 6.02 0.87 2.45 40.7

Mean6SD 5.1060.75 0.8260.10 2.1560.33 42.466.0a Area of AT outlilne in GAD-IR stection /area of AT outline in adjacent Nissl section.b Values after correctin for shrinkage.

comprise only 28% of the total AVN population [6]. between brains. We interpret these findings to indicate thatAlthough specific patterns of pigmentation have been multiple antigens on the GAD molecule are relativelyshown to correlate well with the morphology of Golgi- insensitive to extended post mortem delay, formalin fixa-impregnated cells [5], classification on the basis of a tion and time in fixative. By contrast, autolysis-relatedneurotransmitter-related features is arguably a more reli- changes in small molecules such as GABA can occurable method of determining neuronal phenotype. Identifi- within min of death [23]. We found no correlation betweencation of the local circuit neuron phenotype in the AT of estimates of AT volume and time in fixative. The currentother primate species has been reliably demonstrated by study used a random systematic sampling technique on 50immunoreactivity to the neurotransmitter GABA [14]. In mm cryostat sections, similar in design to methods ofparticular, the anteromedial thalamic nucleus of the stereology used to estimate total neuron numbers in themacaque, a component region of the AT in the human human AT ([13], Young, 2000 [40). Importantly, it shouldthalamus, revealed 47% of neurons to be GABAergic [14]. be noted that the full thickness counting protocol used in

this and other studies does not lead to a significant ‘lost4.2. Methodological considerations caps’ error at a section thickness of 50 mm [13,19]. Our

analytical method assumed that each neuron possessed oneOur clinical data indicated that Case I committed nucleolus at most. No evidence of AT neurons possessing

suicide. A significant proportion of adult suicides can be more than one nucleolus was seen, in agreement withattributed to diagnoses of either major depression or previous studies [6,13]. We took into consideration themelancholia, conditions that may involve abnormal func- observed shrinkage of sections during the immunohistoch-tioning within the thalamus [10]. However, extensive emical process by adjusting the GAR-IR neuron countsclinical notes demonstrated no evidence of any psychiatric prior to the determination numerical densities of neurons.disorder that could have contributed to his death.

The observed pattern of immunoreactivity in thethalamic reticular nucleus and caudate nucleus of the 4.3. Functional significancecurrent study suggest that the label observed in the AT wasGAD specific. However, it is possible that high numbers of A relationship between numbers of local circuit neuronsGAD-IR neurons were a result of either ante-mortem or and brain complexity has recently been hypothesized forpost-mortem changes within the thalamus, or a combina- the mammalian thalamus [3]. A comparative analysistion thereof. Several observations suggest that this is not amongst primate species revealed the human anteriorthe case. Labelled profiles with ‘astrocytic’ morphologies thalamus to contain a greater volume and significantlywere no observed in the AT of the current study, unlike more neurons than was predicted on the basis of brain sizethat seen in the rat hippocampus following transient [4], a finding which is suggestive of an increased process-ischaemia [18]. No evidence of anoxia, as determined by ing capacity. Our results demonstrate that close to half ofthe loss of CA1 hippocampal neurons, was found in any of all AT neurons display the neurochemical phenotype ofneuropathology reports. Similarly, although post-mortem local circuit neurons. Discriminative avoidance learningdelay (PMD) times prior to fixation in the current study tasks in rabbits suggest that the cingulo-thalamic com-ranged from 9.5 to 30 h, no relationship between PMD and ponent of the Papez circuit is involved in the production of%GAD neurons was found. Additionally, the intensity of attentional responses to discrete, associatively significantGAD label within the caudate nucleus, the reticular cues [17]. Further investigation is needed to determine thethalamic nucleus or the AT did not noticeably vary role of local circuit neurons in AT function.

44 G. Dixon, C.G. Harper / Brain Research 923 (2001) 39 –44

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