Distribution and localization of sites of gamma aminobutyric acid

J. Anat. (1973), 114, 3, pp. 365-378 365With 11 figuresPr-inted in Great Britain

Distribution and localization of sites of gamma aminobutyricacid metabolism in the adult rat brain

N. ROBINSON AND F. WELLS

Department of Anatomy and Histology, The London Hospital Medical College,Turner Street, London El 2AD

(Accepted 20 January 1973)

INTRODUCTION

Previous investigations concerning the role of gamma aminobutyric acid (GABA)in brain have been mainly physiological and biochemical (Hirsch & Robins, 1962;Hall & Kravitz, 1967; Lovell, Lovell & Elliot, 1963; Fahn & Cote, 1968; Srinivasan,Neal & Mitchell, 1969; Kim, Bak, Hassler & Okada, 1971), but a histochemicaltechnique for the demonstration of GABA in the hindbrain of rodents has beenreported by Van Gelder (1965).The biochemical and physiological results of investigations on rat cerebral cortex

showed that GABA was mainly located in synaptosomes (Neal & Iversen, 1969) andthe indications were that GABA is concerned in post-synaptic chemical transmission(Krnjevic & Schwartz, 1967; Obata & Takeda, 1969). The absence of any substantialinformation on the precise distribution ofGABA in the brain prompted us to investi-gate the cellular distribution of this substance throughout the adult rat brain by thehistochemical method of Van Gelder.

MATERIALS AND METHODS

Adult Sprague-Dawley rats, 250-300 g weight, were anaesthetized with ether andthe whole brain removed. The part of the brain to be used was rapidly frozen andsections cut at 12,am in a cryostat. The sections were mounted on to slides whichwere at room temperature and immediately returned to the dessicated cryostat tofreeze-dry at -20 °C for a minimum time of 3 hours. Sections were then incubatedfor the histochemical demonstration of GABA according to Van Gelder (1965).The substrate was omitted in the incubation medium for control sections. Theincubation time was 10 minutes.

Critical factors in obtaining a reaction were pH and temperature. If the pH fellbelow 6-5 no reaction was obtained, the optimum pH being pH 7-4. The enzymeGABA-T was very sensitive to room temperature, especially when the sections werestill wet, and overexposure to incorrect pH and temperature conditions resultedin a loss in intensity of reaction. After the sections had been melted on to glass slidesthey had to be fully dried at freezing temperatures before being layered withincubation medium.Two types of agar were tried. BDH agar, which was a fine powder, gave a good

strong reaction but sometimes small aggregates were scattered over a section,


obscuring information. The second type of bacteriological grade agar, Oxoid ionagar No. 2, did not exhibit the aggregation phenomenon but its removal from thesection after incubation invariably incurred damage to the section. Because of thisthe BDH agar was used throughout.

RESULTS

When incubated in the presence of GABA, a-ketoglutarate and NAD (nicotina-mide adenine dinucleotide), the sections showed a reduction of Nitro BT to insolubleformazan throughout the rat brain, but no reaction was observed in control sectionswhen the substrate was omitted from the medium and incubated in the same way.The formazan precipitate appeared to be confined to specific nuclei and nervecells; in general, white matter showed no reaction except for occasional glial cells.The distribution of the GABA metabolizing areas is presented in Table 1. Details

of the intracellular localization follow.

TelencephalonThe cortex was stained in a graded manner. The intense reaction in layers IJ and

III gradually decreased towards the deeper layers, whilst the outer layer showed noreaction; layers II and III in the cortex of the cingulate gyrus were always heavilystained. The reaction of neuropil was moderate compared to that of nerve cells,which were identified by a stronger reaction confined to the cytoplasm.The nerve cell bodies and neuropil in the caudate nucleus and putamen (Fig. 1)

showed a strong reaction. The anterior commissure and corpus callosum, beingfibre tracts, showed no reaction except for some glial cells.The localization of the formazan in glial cells in the nucleus accumbens septi was

clearly distinguishable (Fig. 2), but in nerve cells it was difficult to determine whetherthe reaction was within the cell bodies or on nerve endings of the perikarya of thepost-synaptic nerve cells.The pyramidal cells of the hippocampus were more distinct than other cells and

exhibited a good localization but the hippocampus in general showed a weak reac-tion. The dentate gyrus appeared slightly more intense than the hippocampus butwith the pyramidal cells still distinct. The possible relevance of this will be dis-cussed. The fornix leading from the hippocampus to the mammillary body was seenas negatively stained nerve fibres interspersed with areas of strongly reactingneuroglia (Fig. 3). The internal capsule and tapetum, both being tracts, showed noreaction.

DiencephalonThe thalamic nuclei showed varying intensities of reaction, the most intense being

in the anteroventral (Fig. 4) and the posterior thalamic nuclei; the paraventricularand anteromedial nuclei were less intense. The neuropil of the subthalamic nucleusexhibited a strong reaction but the mammillothalamic tract showed no reaction.

Isolated nerve cells in the hypothalamus exhibited a strong reaction; otherwisethe reaction in this area was generally weak. The neuropil of the habenular nucleuswas strong, masking any nerve cell reaction, whilst the fasciculus retroflexus, anefferent pathway, showed no reaction. Neuropil in both lateral and medial geniculatebodies was moderately intense.

366

GABA in brain 367

Table 1. Distribution of GABA in rat brain

Nerve cell NeuroglialRegion bodies cell bodies

TelencephalonCaudate nucleus and putamen + + + + + + +Globus pallidus - + +Stria terminalis - +Cortex of cingulate gyrus + + + + +Corpus callosumNeocortex, superior and lateral + +(+ +) + (+)Nucleus accumbens septi + + + + + + +Cingulum ++ +Hippocampus + + +Body of fomix - + +Columna fornicis -

Fimbria hippocampus -

Amygdaloid nucleus - +Dentate gyrus + + + + +Internal capsuleTapetum

DiencephalonAnteroventral nucleus of thalamus + + + + +Paraventricular and anteromedial - + +nuclei of thalamus

Posterior thalamic nucleus - + + +Subthalamic nucleus - + + +Mammillothalamic tract -

Lateral and medial geniculate - + + +bodiesZona incerta - +Habenular nucleus - + + +Fasciculus retroflexus -

Hypothalamus + + +Lateral preoptic area + + +Tuber cinerium -

MesencephalonColliculus anterior - + + +Colliculus posterior - + + +Commissure of colliculi -

Crus cerebri -

Reticular formation + + + +Substantia griseum - +periventricularis

Substantia nigra + + + + +Red nucleus + + + +Nucleus interpeduncularis - + +Lateral tegmental nucleus + + + + +Nucleus Darkschevitch - +Nucleus N. IV + + +Tectopontine tract - +Mesencephalic nucleus of N. V + + + +

RhombencephalonCerebellumGranular layer + + + +Purkinje cells + + + +


Table 1. (cont.)

Region

Rhombencephalon (cont.)Molecular layerDentate and emboliform nucleiFastigial nucleus

Pons and medullaOlivary nucleusVent. cochlear nucleusPrincipal vestibular nucleusTrapezoid bodyMotor nucleus of N. VSensory nucleus of N. VNuclei ofNs. VI, VII and XIIDorsal nucleus of N. XCuneate and gracile nucleiReticular formationNucleus ambiguusNucleus of tr. solitariusSubstantia gelatinosa

Nerve cellsbodies

++

++

++

++

+++++

++

++

++

Neuroglialcell bodies

++

+

++

+

+

++

++

+

+

+

++

+ + + + intense, + + + strong, + + moderate, + weak, - absent.

Fig. 1. Caudate nucleus and putamen (not clearly separated in rat). Note localized reactionfor GABA in or around the nerve cell bodies (arrows). No reaction in fibre tracts (clear circularareas). x 500.

368

GABA in brain 369

Fig. 2. Nucleus accumbens septi. Discrete granular reaction forGABA within the cytoplasm of glial cells. x 1250.

', I

B

Fig. 3. Cortex of cingulate gyrus (A), corpus callosum (B), body of fornix (C) and lateralseptal nucleus (D). Moderate GABA reaction in neuroglia of A, C and D, no reaction in B.x 190.

A NA II424


Fig. 4. Ventral thalamic nucleus and internal capsule (I.C.). The thalamic nucleus shows astrong reaction for GABA in nerve cells against a moderate reaction in nerve fibres and neuropil.The internal capsule (I.C.) shows no reaction. x 400.

MesencephalonThe two areas exhibiting the most intense reactions in the mesencephalon were the

red nucleus (Fig. 5) and substantia nigra (Fig. 6). In both these areas the reaction inthe cytoplasm of nerve cells was strong but the nerve cell processes were more easilyseen in the substantia nigra. In both nuclei the neuropil was moderately stained butglial cells were indistinct. Glial cells in the anterior and posterior colliculi showeda strong reaction which was absent in the commissures.

Several prominent nerve cells were seen against a weak neuropil reaction in thetrochlear nucleus, and the lateral nucleus of the tegmentum showed a similar re-sponse (Fig. 7).An intense column of nerve cells identified as part of the mesencephalic nucleus of

the trigeminal nerve was observed ventral to the locus coeruleus; fibres adjacent tothese cells showed a weak reaction (Fig. 8). Cells in the reticular substance in thisarea showed no reaction.

RhombencephalonCerebellum

In the molecular layer (Fig. 9) the dense network of dendrites and axons showeda moderate reaction. The strong reaction in many Purkinje cells was precisely local-ized within the cytoplasm (Figs. 9, 10); these cells appeared within a clear bandadjacent to the granular layer, and the absence of a glial cell reaction enhanced theclarity within Purkinje cells. The granular layer itself showed an intense reactionwithin the perikarya of granular layer cells (Fig. 10).

370

GABA in brain

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Fig. 5. Red nucleus. Nerve cell bodies showing a well localized GABA reaction. Isolatednerve cell processes can be seen against a weak neuropil reaction. x 1250.

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Fig. 6. Substantia nigra. Nerve cell processes exhibit a strong reaction for GABA. Intensity ofnerve cell cytoplasmic reaction appears increased in parts by pigment (arrows). A moderateneuropil reaction is also apparent. x 1170.

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Fig. 7. Lateral tegmental nucleus. Strong well localized cytoplasmic reaction for GABA innerve cells. A moderate glial cell cytoplasmic reaction is also apparent. x 1730.

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Fig. 8. Mesencephalic nucleus of N. V. The large round nerve cells show a strong reactionfor GABA. Longitudinal fibres adjacent to these cells exhibit a weak reaction. x 500.

372

GABA in brain 373

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Hence one is indirectly establishing the distribution and localization of this substance.


10

Fig. 10. Cerebellar cortex. Higher magnification to show clearly definable Purkinje cells (P),nerve fibres (arrows) and the molecular layer (M). x 1000.

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Fig. 11. Ventral nucleus of the reticular formation. Nerve cells exhibit a well localized cytoplas-mic reaction for GABA. A large latticework of lightly stained fibres and more heavily stainedtransected fibres can be seen. x 1250.

374

Whereas previous biological and physiological investigations have given indica-tions of the quantitative distribution of GABA throughout the central nervoussystems of various vertebrates and crustaceans (Hirsch & Robins, 1962; Lovell et al.1963; Fahn & Cote, 1968) there have been no previous comprehensive reports inwhich direct visualization of the distribution and localization of GABA by a histo-chemical technique has been demonstrated.

In carrying out the technique the two most critical factors were those of pH andtemperature, facts which emphasize that the histochemical reaction is dependent onenzymic activity. It was important to avoid overexposure of the tissue to room tem-perature during section mounting, and also any drift of pH during preparation of themedium because of too low a molarity of the buffer. If the experimental conditionsset out by Van Gelder were followed closely the reaction was rapid and the controlsalways negative.The precise localization of GABA, whether in the cytoplasm of the nerve cell or

in the nerve terminals in juxtaposition with it was sometimes difficult to discern.However, in some areas this question was found to be resolvable and is discussed.

In general, the nuclei of the cerebrum and brain stem showed a reaction forGABA, but the fibre tracts, including the corpus callosum, anterior commissure,internal capsule, mammillothalamic tract, fasciculus retroflexus and the com-missures of anterior and posterior colliculi, showed no reaction whatsoever.The intensity of reaction of a particular layer within the neocortex was consistent

from one area to another, but there were distinct differences from layer to layer.The outermost layer was invariably negative. This corresponds to molecular layer I,which is composed mainly of a superficial band of tangential myelinated fibres andneuroglia. Layers II (the external granular layer) and III (the pyramidal cell layer)were stained intensely. The cortex of the cingulate gyrus consistently showed a moremarked reaction than others, the heaviest reaction being ascribed to the layers ILand III. GABA has been shown to be a normal constituent of brain (Awapara,Landua, Fuerst & Scale, 1950; Roberts & Frankel, 1950) with a strong inhibitoryaction on cortical neurons (Krnjevic & Phillis, 1963) although this has been questionedrepeatedly (Curtis, Phillis & Watkins, 1959; Crawford & Curtis, 1964; Curtis, 1965).The intense reaction assumed to be GABA would indicate a strong inhibitory rolefor either the neurons or some of the nerve fibres terminating in this region. Thedominant cell in this region is the pyramidal cell, and the preponderance of GABAin this area suggests that it would probably take part in the control of theseneurons in the role of an inhibitory neurotransmitter.

Striking features in the telencephalon were the intensity of reaction seen in thecaudate nucleus and the putamen, and the absence of a reaction in the fibre tractspassing through these nuclei. Several neurons and their processes can be observedagainst the background neuropil. However, it is possible that GABA is not localizedin the cytoplasm of these neurons but in the nerve terminals in juxtaposition withthem (Kuhar, Green, Snyder & Gfeller, 1970).The reaction in the pyramidal cell layer of the hippocampus was strong in contrast

to the remainder of this region. This distribution in the pyramidal cell layer correlateswell with other reports on the functional significance of the pyramidal cells. Some ofthe first observations on post-synaptic inhibition were made in the hippocampus.

GABA in brain 375


Physiological studies by Anderson, Eccles & L0yning (1964) gave evidence that thesynapses which exerted an inhibitory action on the hippocampal pyramidal cellswere located on the cell soma; this also fits the histochemical observations in thepresent study. Kandel, Spencer & Brinley (1961) and Anderson et al. (1964) have alsoreported that all pyramidal cells in the hippocampal formation display very powerfuland long-lasting inhibitory potentials, and that the IPSPs are more easily recordedthan excitatory potentials, suggesting that there is a powerful recurrent inhibitionin hippocampal pyramidal cells. The high GABA content seen histochemically inthis region supports the possibility of its role as an inhibitory transmitter. In contrast,the fornix, the main fibre pathway from the hippocampus, showed no reaction forGABA.

In the diencephalon the strongest reaction was localized mainly within neuropilin the anteroventral and posterior thalamic nuclei and the habenular nucleus, witha moderate reaction in the ventral thalamic nucleus. In all these nuclei the neuropilexhibited a strong reaction which masked most nerve cells. In contrast, neuropil ofthe hypothalamic nuclei stained very lightly, but isolated nerve cells were distinct incertain nuclei, particularly in the lateral preoptic area.The substantia nigra and red nucleus were conspicuous features of the mesen-

cephalon. In the substantia nigra the reaction was localized in the nerve cell bodiesand their processes, with some response in the neuropil and glial cells. The nervecells of the red nucleus also showed up very clearly but the reaction was of quitea different pattern from that in the substantia nigra. In the red nucleus the formazanprecipitate was localized in the soma of the nerve cells but sparse in their processes:furthermore, the neuropil in this area was not so heavily stained as in the substantianigra, and so glial cells were identified. Recent morphological studies of the sub-stantia nigra in the rat showed evidence of strionigral fibre terminals containingelongated vesicles (Type I) or small synaptic vesicles (30 nm) (Bak, Hassler, Parisek& Wagner, 1970) and that the GABA level in the substantia nigra decreased markedlyafter destruction of the strionigral pathway (Kim et al. 1971). According toUchizono's hypothesis (1965) these fibres may be inhibitory to the nigral cells. Insupport of this, electrophysiological studies in the cat showed monosynaptic IPSPsin pallidal and nigral cells after stimulation of the caudate nucleus (Yoshida &Precht, 1971). Okada, Nitsch-Hassler, Kim, Bak & Hassler (1971) demonstratedby a sensitive enzymic and fluoreometric assay that a high concentration of GABAwas present in the substantia nigra.There was a noticeable contrast in the intensity of reaction and localization

between the molecular and granular layers of the cerebellum. In the molecular layera preponderance of fibres could be made out, whereas in the granular layer thereaction was localized mainly within cells, with some reaction in nerve fibres travers-ing the granular layer. Between these two layers the perikarya of the Purkinje cellsshowed one of the most intense reactions of the whole CNS, a finding which supportsthe supposed inhibitory nature of the Purkinje cells (Obata & Takeda, 1969).The localization of GABA in the cranial nerve nuclei in the brain stem was

generally well defined in the nerve cells, but although variations in distributionbetween different nuclei were apparent, there were no particular distinctive featuresfor comment.

376

GABA in brain 377

SUMMARY

The distribution and localization of GABA within the brain of the adult rat,seen histochemically, is reported.Nerve cells in different regions showed distinct differences in the level of the GABA

reaction, which was intense in the caudate nucleus and putamen, nucleus accum-bens septi, and the Purkinje and granular layer cells of the cerebellum, and weak orabsent in most diencephalic and mesencephalic nuclei. Reactions in the neurogliavaried from strong to absent and seldom exceeded reactions in nerve cells. Thesehistochemical observations on GABA are related to earlier biochemical and physio-logical reports on its distribution and function.

We gratefully acknowledge financial assistance to one of us (F.W.) from theFriedreich's Ataxia Group and Action for the Crippled Child Fund.

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378 N. ROBINSON AND F. WELLS

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Distribution and localization of sites of gamma aminobutyric acid