Kainic acid neurotoxicity; effect of systemic injection on neurotransmitter markers in different brain regions

Brain Research, 230 (1981) 253-262

253

Elsevier/North-Holland Biomedical Press

K A I N I C ACID N E U R O T O X I C I T Y ; E F F E C T OF SYSTEMIC INJECTION ON N E U R O T R A N S M I T T E R M A R K E R S IN D I F F E R E N T BRAIN REGIONS

DAG E. HEGGLI, A. AAMODT and D. MALTHE-SORENSSEN Norwegian Defence Research Establishment, Division for Toxicology, P 0 Box 25, N-2007 K]eller, and ( A.Aa.) Laboratory of Pathology, Section of Neuropathology, Ullevdl Hospital, Oslo (Norway)

(Accepted May 21st, 1981) Key words: kainicacid lesion -- systemicinjection- - piriform cortex -- amygdala -- hippocampus --

septum neurotransmitter markers

SUMMARY Systemic injection of kainic acid (12 mg/kg) induces necrosis and neuronal degeneration in several brain regions. The most pronounced effects were observed in the piriform cortex, amygdaloid complex, hippocampus and septum. A good correlation between morphological changes and changes in some neurotransmitter markers was observed in these 4 areas. High affinity uptake of L-glutamate, as well as glutamate decarboxylase and choline acetyltransferase activities were reduced in the piriform cortex and amygdaloid complex whereas in the hippocampus and septum only the first two markers were reduced. No morphological changes or decrease in any of these neurotransmitter markers were observed in striatum or globus pallidus. A pronounced neuronal degeneration could be demonstrated in lateral thalamus and geniculate body, but this degeneration was not accompanied by any decrease in the transmitter markers tested.

INTRODUCTION Kainic acid, a rigid analogue of the putative neurotransmitter, glutamate, is a powerful neuretoxic agent 2s. It causes neuronal lesions both when injected locally in brain structures2a, 2~ and after systemic injections10, 29. The neurotoxicity of kainic acid and other 'excitotoxic' amino acids has been related to their excitatory actions since their toxicity seems to be in proportion to their excitatory potency 2s. Different mechanisms have been put forward to explain the neurotoxic action of kainic acid. These include one which is important for the direct effect of kainic acid at the injection site injuring neurons which apparently are innervated by glutamergic 0006-8993/81/0000~000/$02.75 © Elsevier/North-Holland Biomedical Press

254 fibres 4,2°,23, another which is responsible for distant effects following local, intraventricular or systemic injections which probably is dependent upon excessive stimulation of excitatory but not necessarily glutamergic neurons z42s. The latter mechanism is probably related to epileptogenic mechanisms3,25. Several investigations have been performed to study the effect of kainic acid given subcutaneously. These studies have shown that kainic acid given systemically produces profound lesions in specific areas in the blainlO, 29. Regions such like septum, amygdaloid complex, hippocampus, parts of thalamus and cortex are most frequently affected. Although extensive histological studies have been performed29, little information is available on the changes in neurotransmitter markers which occur. Systemic injection of kainic acid may reveal neurons that are particularly sensitive to kainic acid and may also indicate primary target regions for the effect of kainic acid. In the present study we have studied the neuronal damage preferentially in regions where kainic acid is known to have a neurotoxic effect and which receive glutamergic fibres. MATERIALS A N D METHODS

Male Wistar rats, 150-200 g, were given 12 mg/kg of kainic acid (KA) by a subcutaneous injection. KA was dissolved (5 mg/ml) in 10 mM sodium phosphate buffer (pH = 7.4). Only fresh solutions were used. The rats were killed by decapitation 2 or 5 days after the kainic acid injection. Slices of 600 or 800/zm were prepared by a tissue chopper and kept on ice. Dissection of the different brain structures from the slices was done on ice under microscopic guidance. The tissue samples were homogenized in ice-cold 0.32 M sucrose with a teflon-glass homogenizer, final concentration 20 mg wet weight/ml. The brain regions selected for investigation were nucleus septi lateralis, hippocampus, striatum, globus pallidus, nucleus laterialis thalami, nucleus dorsalis corporis

".2

STRIATUM A: 7600-7000/1

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/ LATERAL SEPTUM A: 8600-7800 p

GLOBUS PALLIDUS A: 6800-6000

P I R I F ~ P U S LATERAL GENICULATE CORT['X \ A . 3900-3100~ I BODY A: 5000-4200P \ LATERAL A: 3300-2500,u N AMYGDALA THALAMUS A: 5000-4200 ,u

A: 4600-3800 p

Fig. 1. Sections of rat brain slices illustrating the extension of the different structures dissected. The coordinates given (A :) represent the limits of the slices used for dissection and are according to K6nig and Klippel (1963). Abbreviations used : cp, striatum; HI, hippocampus; gl, lateral geniculate body; tv, ventral thalamus.

255 geniculati lateralis, nucleus amygdala and cortex piriformis. The anterior-posterior coordinates and the extension in the frontal plane of the dissections are given in Fig. 1, according to K6nig and KlippeP 4. The high affinity uptake of L-glutamate (HAGlu) was measured as previously described s, using 5 × 10-7 M of [ZH]L-glutamate as substrate. Glutamate decarboxylase (GAD) and choline acetyltransferase (CHAT) activity were measured by using [14C]5-glutamate and acetyl-CoA, respectively 7. The protein was measured according to Lowry et al. 1~.

Histology Slices of 20 #m were cut from frozen brains by a cryostat. The slices were placed on glass slides, thawed and dried at room temperature. Staining of degenerating fibres was achieved by the Fink-Heimer techniquelL Autoradiography Autoradiography was performed as described by Soreide and Fonnum 32. In brief, transverse slices (300 #m) of hippocampus were used. The slices were incubated in Krebs-Ringer solution (final concentration): 27 mM NaHCO3, 136 mM NaC1, 5 mM KH2PO4, 2.4 mM KC1, 1.66 mM glucose, 1.5 mM CaCI~ and 0.5 mM MgClz (pH 7.2) containing 2.5 × 10-8 M, [2,3-ZH]L-glutamate (18.8 Ci/mmol). The buffer was equilibrated with 95 ~ O3 and 5 ~ COs. The slices were incubated for 20 min at room temperature. After incubation, the slices were rinsed twice in Krebs-Ringer buffer, fixed in 2.5~ glutaraldehyde, post-fixed in 3.5~ formalin, dehydrated and embedded in paraffin. Serial sections of 5/~m were cut from the paraffin-embedded slices. Every fifth section was mounted on microscopic slides and prepared for autoradiography using Kodak NTB2 and standard dipping procedures. The slides were kept in tight boxes at 4 °C for 12 days and were then developed in Kodak DI9. The autoradiogralns were not stained, but Ielevant sections from the same series were stained with toluidine blue for light microscopy. RESULTS

Animal response to kainic acid Usually 10-20~/o of the animals died after the kainic acid injection. The usual syndrome following kainic acid injections could be observed z,29. The first symptom was wet dog shakes, starting half-an-hour after the injection. Masticatory movements with foaming appeared 1 h after kainic acid injection. Shortly thereafter rearing and tremor of the forepaws could be observed. The first general convulsion appeared between 1 and 1.5 h after the injection. Whole body tremor was observed from 2 h onwards. Two days after the injection the rats were extremely aggressive and hyperreactive to stimuli of sound and movement. The symptoms varied among rats, possibly reflecting variability in the response to kainic acid. After 2 days there were no obvious correlations between the measured

* P < 0.01 ;

± ± ± ±

8.7 3.3 3.7 6.1

(n -- 9)* (n 9) (n 8) (n - 8)

152 89 54 55 90 93

--78 92 95 101

7.4 (n -- 8)** 5 . 4 ( n -- 8)**

68 ± 5.9** 59 ~ 4.8**

-- 13.6(n 12)* 97 ± 3.2 i 7.2 (n -- 12) 91 i 2.9 ± 5.5 (n = 11)** 78 ~ 2.1"* ± 4.3 (n 12)** 86 ± 4.2 ~ 3.1 (n -- 11) 97 _+. 2.4 5 4.2 (n -- 11) 94 + 2.7

53 ± 30 L

39 -- 10.4(n -- 8)** 26 & 6.3 (n - 8)** 96 90 76 75 89 93

± ~ ± + ± ±

2.0 3.9 3.7** 3.5** 5.8 3.1

38 ± 7.7** 36 ± 5.5**

5 days

2 days

2 days

5 days

Glutamate decarboxylase ( % 0/" control)

High affinity uptake of glutamate (% of control)

** P < 0.001 c o m p a r e d to c o n t r o l a n i m a l s , W i l c o x o n ' s t w o - s a m p l e test.

N. a m y g d a l a P ir ifo r m cortex D o rs al l ate ra l geniculate b od y L ater al t h a l a m u s Hippocampus Lateral septum S tr iatu m G l o b u s pallidus

Brain region

94 96 98 90 92 100

± ± ± ± ± ±

5.1 4.7 4.0 4.7 4.1 5.0

69 ~ 4.4** 76 i 5.0*

2 days

102 ± 5.4 98 -- 5.2 100 ± 2.5 87 ~ 2.8 93 ± 3.7 94 ~ 2 . 6

77 ± 7.5* 68 ± 7.0*

5 days

Choline acetyltran~ferase ( % of control)

Each val ue represents the m e a n ~ S.E.M. of n n u m b e r of brains. Ea c h s a m p l e f r o m each b r a i n region was m e a s u r e d in triplicate.

Changes in neurotransmitter markers 2 and 5 days after systemic injection of kainic acid

TABLE 1

C7~

257 toxic effect on brain structures and the physical condition of the rats; thus, rats which seemed healthy could be among the more seriously affected, and rats which had a rather sick appearance could be among th~ lesser affected. On basis of this variability we made a selection of the brains for analysis after inspecting the slices by light microscopy. In some instances the microscopic appearance were confirmed by Fink-Heimer histological staining of degenerative processes. The analyses were performed on structures from slices showing necl osis in the piriform cortex, amygdaloid complex and hippocampus. Approximately 80 ~ of the animals were used.

Effect on neurotransmitter markers Several brain structures were tested for HAGlu-uptake, G A D and ChAT activities 2 or 5 days after the systemic injection of kainic acid. Nucleus amygdala and piriform cortex Usually a severe necrosis was observed in piriform cortex, whereas a lesser necrosis developed in n. amygdala. The necrosis was usually fully developed after 2 days. All 3 neurotransmitter markers were significantly reduced in both piriform cortex and amygdala (Table I). Hippocampus and septum A pronounced necrosis was seen in the pyramidal cell layer of the hippocampus,

Fig. 2. Fink-Heimer-stained transverse slices of hippocampus. Slices (20 /~m) were cut from frozen brains by a cryostat, dried and stained for degenerating fibres: (a) kainic acid-treated animal; (b) control animal. Note the distinct layered appearance in (a) demonstrating the degeneration of fibres in CA3/CAI stratum oriens and radiatum, subiculum and the inner molecular layer of the area dentata.

258

i:!!i i:!iif!i!!! !!!!iii!i!i!!it!ii;!il ~!:iii~ !!!ii ~!~!!tt!!t!~!!!?!!!il !i!ii~!i!i!i ili ii!!iii!i!~ii i!i¸~i!~!i !i!:iiil!!!iii!iii!!!!iii!!t!i!~i!!i!iiiiii! i! i!ii !!ii!!i!!i!i!~~!iii!!!! i !?i!i Fig. 3. Autoradiography of transverse hippocampal slices with [SH]L-glutamate: (a) kainic acidtreated animal; (b) control animal. Note the parallel decrease in labelling in (a) as compared to (b) stratum oriens and radiatum of CA3 and CA1, subiculum, and the inner molecular layer of area dentata in accordance with the degeneration observed in Fig. 2a. Also note the intact mossy fibre layer which is heavily labelled and probably not affected by the lesion.

259 although the extension of the neuronal degeneration varied between the animals. Fink-Heimer staining of transverse slices of hippocampus showed degeneration of fibres in CA3/CA1 stratum oriens and radiatum, parts of subiculum and the inner molecular layer of area dentata (Fig. 2a and b). The granular layer of area dentata seemed, however, intact. Autoradiography in transverse slices of hippocampus with [2,3-3H]L-Glu showed decreased labelling in CA1-CA3 stratum oriens and radiatum. The deep part of the inner molecular layer of area dentata showed less uptake than in controls, while the mossy fibres still remained labelled (Fig. 3a and b). The degeneration of fibres was accompanied by a reduced uptake of labelled Glu (Fig. 3a and b, Table I). This is in accordance with previous suggestions that these fibres may be using glutamate as their transmitter. In the lateral septum, degeneration of fibres was observed in Fink-Heimer stained slices, and the HAGlu-uptake in lateral septum was reduced (Table I).

Thalamus No losses in the neurotransmitter markers measured were observed in lateral thalamus and geniculate body although both regions showed degeneration of fibres with Fink-Heimer staining. There was a pronounced increase in the HAGlu-uptake in lateral geniculate body 5 days after the kainic acid injection (Table I). This increase may be due to a transient increase in the HAGlu-uptake as observed by others in striatum.

Striatum and globus pallidus No changes in the 3 neurotransmitter markers could be detected in either striatum or globus pallidus and only a sparse degeneration of fibres could be seen in striatum with Fink-Heimer staining. DISCUSSION Systemic injection of kainic acid in adult rats was accompanied by neuronal damage in several brain regions. The degree of necrosis varied between different regions. Histological examination revealed that the piriform cortex was most severely affected, as previously observed 29. Other commonly affected areas were the amygdaloid complex, hippocampus, parts of thalamus and septum. Most of the affected areas are known to receive excitatory neuronal fibres which probably use glutamate or aspartate as transmitter. In several of the regions investigated a pronounced degeneration of glutamergic/aspartergic neurons occurred.

Hippocampus and septum The structures affected in hippocampus and septum were all regions which either are the terminal area of presumably glutamergic/aspartergic fibres or areas containing neurons which use glutamate/aspartate as neurotransmitter6,9,20,~t,22,34.

260 Accordingly, the HAGlu-uptake in both hippocampus and lateral septum decreased and autoradiography with tritiated L-Glu demonstrated labeling in CA1/CA3 stratum oriens and radiatum compared to controls (Fig. 3). Less labelling was also seen in the inner molecular layer of area dentata (Fig. 3). However, the granular cells and the mossy fibres, which probably also use glutamate/aspartate as transmitter 6,2v,~1, were unaffected. Some of the explanations to this differential vulnerability of glutamergic/aspartergic neurons in hippocampus may be found in the following observations. Both the granular cells and the pyramidal cells are innervated by presumably glutamergic/aspartergic fibres, the former by fibres from the perforant pathway1,27, 31, the latter by the mossy fibres ~,5. When the perforant pathway fibres activate the granular cellslV,is, the mossy fibre will activate a large number of pyramidal cells giving rise to a massive activation of pyramidal cells in CA3/CA11, 2. In this sequential activation of the neuronal loop from perforant path to the pyramidal cells, it has been shown that the granular cells require powerful tetanic stimulation of the perforant pathway to dischargelV, is. Furthermore, both histological and electrophysiological data suggest the transmission through the en passage giant synapse (mossy fibres to CA3) to be effective5,17,1s,3~. Powerful activation of the granular cells by the perforant path, which the granular cells themselves are able to sustain leads to a damaging activation of the pyiamidal cells. Kainic acid may then, by the activation of neurons in entorhinal cortex through the perforant pathway, induce a massive activation of the pyramidal cells through the granular cells which results in neuronal death in CA3/CA1 stratum oriens, stratum radiatum and subiculum (Fig. 2a and b). Although the systemic injections of kainic acid probably expose both the pyramidal and the granular cells to kainic acid, the concentration may be too low to damage the granular cells by a direct effect as seen after intrahippocampal injection of kainic acid 13. Thus depending on the route of administration of kainic acid, a differential neurotexicity can be obtained. This is supported by Nadler et al. 24 who recently could demonstrate that intraventricular and intrahippocampal injection of kainic acid affected different neuronal pathways in the hippocampus, pxobably by activating different excitatory pathways. As observed after local injectionV, 2°, the systemic injection of kainic acid was also accompanied by a loss of GABAergic neurons in the hippocampus and lateral septum. In both structures the GABAergic neurons are probably innervated by glutamergic terminals 7,2°,a°, which has been suggested to be important for kainic acid to exert a direct neurotoxic effect11,23. Piriform cortex and amygdala Neuronal structures in both piriform cortex and amygdaloid complex were highly sensitive to the neurotoxic effect of kainic acid. Approximately 30 ~o of the glutamergic/aspartergic terminals in piriform cortex probably originate from cortical structures 33, whereas the amygdaloid complex probably receives most of its glutamergic/aspartergic fibres (70 ~o) from the piriform cortex 33. Despite a common origin from glutamergic/aspartergie neurons in cortical

261 structures to striatum 4, lateral geniculate b o d y 19, piriform cortex and amygdaloid complex 3~, their responsiveness towards kainic acid differs. The glutamergic/aspartergic neurons in piriform cortex and amygdala were particularly sensitivie to kainic acid and the degeneration t o o k place in only two days with no further effect after 5 days. Both in piriform cortex and amygdala GABAergic and cholinergic neurons were lesioned by kainic acid. A more pronounced decrease in G A D activity than in C h A T activity was observed in both structures especially after 5 days. The decrease in C h A T activity in the amygdala is consistent with the decrease observed after local injection of kainic acid in amygdala. This could reflect that the effect on G A D and C h A T in piriform cortex and amygdala is a direct action of kainic acid on neul ons innervated by glutamergic/aspartergic fibres. Since there was no effect in striatum on G A D or C h A T wheie kainic acid is k n o w n to exert a toxic effect on GABAergic and cholinergic structures after local injections, these neurons must be less sensitive to systemic kainic acid than neurons in the other structures. Interestingly there is no decrease in C h A T in hippocampus either, although the cholinergic cell bodies located in diagonal band2,15, are sensitive to locally injected kainic acid and are probably innervated by glutamergic fibres 20. ACKNOWLEDGEMENT A. Aa. was a recipient o f G r a n t (C23.38-8) from the Norwegian Research Council for Science and Humanities.

REFERENCES 1 Andersen, P., Organization of hippocampal neurons and their interconnections. In R. L. Isaacson and K. H. Pribram (Eds.), The Hippocamp~s, Vol. 1, Plenum Press, New York, 1975 pp. 155-175. 2 Andersen, P., Bliss, T. V. P. and Skrede, K. K., Unit analysis of hippocampal population spikes, Exp. Brain Res., 13 (1971) 208-221. 3 Ben-Ari, Y., Tremblay, E., Ottersen, O. P. and Naquet, R., Evidence suggesting secondary epileptogenic lesions after kainic acid: Pretreatment with diazepam reduces distant but not local brain damage, Brain Research, 165 (1979) 362-365. 4 Biziere, K. and Coyle, J. T., Influence of corticostriatal afferents on striatal kainic acid neurotoxicity, Neurosci. Lett., (1978) 303-310. 5 Blackstad, T. W., Brink, K., Ham, J. and Jeune, B., Distribution of hippocampal mossy fibres in the rat. An experimental study with silver impregnations methods, J. comp. Neurol., 138 (1970) 433450. 6 Crawford, I. L. and Connor, J. D., Localization and release of glutamic acid in relation to the hippocampal mossy fibre pathway, Nature (Lond.), 244 (1973) 442~,43. 7 Fonnum, F. and Walaas, I., The effect of intrahippocampal kainic acid injections and surgical lesions on neurotransmitters in hippocampus and septum, J. Neurochem., 31 (1978) 1173-1181. 8 Fonnum, F., Walaas, I. and Iversen, E., Localization of GABAergic, cholinergic and aminergic structures in the mesolimbic system, J. Neurochem., 29 (1977) 221-230. 9 Fonnum, F., Iversen, E., Malthe-Sorenssen, D., Odden, E. and Skrede, K. K., Evidence for glutamate as transmitter of the commissural fibres to stratum oriens in hippocampus, Neuroscience, in press. 10 Fuller, A. T. and Olney, J. W., Effects of morphine or naloxon on kainic acid neurotoxicity, Life Sci., 24 (1979) 1793-1798.

262 11 Herndon, R. M., Coyle, J. T. and Addicks, E., Ultrastructural analysis of kainic acid lesion t~ cerebellar cortex, Neuroscience, 5 (1980) 1015-1026. 12 Hjort-Simonson, A., Fink Heimer silver impregnation of degenerating axons and terminals in mounted cryostat sections of fresh and fixed brains, Stain. Technol., 45 (1978) 199-204. 13 K6hler, C., Schwarcz, R. and Fuxe, K., Perforant path transections protect hippocampal granule cells from kainate lesion, Neurosci. Lett., 10 (1978) 241-246. 14 K6nig, J. F. R. and Klippel, R. A., The Rat Brain. A Stereotaxic Atlas o f the Forebrain and Lower Parts o f the Brain Stem, Williams and Wilkins, Baltimore, 1963. 15 Lewis, P. R., Shute, C. C. D. and Silver, A., Confirmation from choline acetylase analysis of a massive cholinergic innervation to the rat hippocampus, J. Physiol. (Lond.), 191 (1967) 215-244. 16 Lowry, O. H., Rosebrough, N. J., Farr, H. L. and Randall, R. J., Protein measurement with the folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 17 Lomo, T., Patterns of activation in a monosynaptic cortical pathway: the perforant path input to the dentate area of the hippocampal formation, Brain Research, 12 (1971) 18-45. 18 Lomo, T., Potentiation of monosynaptic EPSP in the perforant path - dentate granule cell synapse, Brain Research, 12 (1971) 46-63. 19 Lurid Karlsen, R. and Fonnum, F., Evidence for glutamate as a neurotransmitter in the corticofugal fibres to the dorsal lateral geniculate body and the superior colliculus in rats, Brain Research, 151 (1978) 457-467. 20 Malthe-Sorenssen, D., Odden, E. and Walaas, I., Selective destruction by kainic acid of neurons innervated by putative glutamergic afferents in septum and nucleus of the diagonal band, Brain Research, (1980) 461-465. 21 Malthe-Sorenssen, D., Skrede, K. K. and Fonnum, F., Calcium dependent release of o-[ZH] aspartate evoked by selective stimulation of excitatory afferent fibres to hippocampal pyramidal cells in vitro, Neuroscience, 4 (1979) 1255-1263. 22 Malthe-Sorenssen, D., Skrede, K. K. and Fonnum, E., Release ofo-[3H]aspartate from the dorsolateral septum after electrical stimulation of the fimbria in vitro, Neuroscience, 5 (1980) 127-133. 23 McGeer, P. L., McGeer, E. G. and Hattori, T., Kainic acid as a tool in neurobiology. In E. G. McGeer, J. W. Olney and P. L. McGeer (Eds.), Kainic Acidas a Tool in Neurobiology, Raven Press, New York, 1978, pp. 123-138. 24 Nadler, J. V. and Cuthbertson, G. J., Kainic acid neurotoxicity towards hippocampal formation : dependence on specific excitatory pathways, Brain Research, 195 (1980) 47-56. 25 Nadler, J. V., Evenson, D. A., and Smith, E. M., Evidence from lesion studies for epileptogenic and non-epileptogenic-neurotoxic interactions between kainic acid and excitatory innervation, Brain Research, 205 (1981 ) 405-410. 26 Nadler, J. V., Perry, B. W. and Cotman, P. W., Preferential vulnerability of hippocampus to intraventricular kainic acid. In E. G. McGeer, .1. W. Olney and P. L. McGeer (Eds.), Kainic Acidas a Tool in Neurobiology, Raven Press, New York, 1978, pp. 219-237. 27 Nadler, J. N., Vaca, K. W., White, W. F., Lynch, G. S. and Cotman, C. W., Aspartate and glutamate as possible transmitters of excitatory hippocampal afferents, Nature (Lond.), (1976) 538-540. 28 Olney, J. W., Neurotoxicity of excitatory amino acids. In E. G. McGeer, J. W. Olney and P. L. McGeer (Eds.), Kainic Acid as a Tool in Neurobiology, Raven Press, New York, 1978. 29 Schwob, J. E., Fuller, T., Price, J. L. and Olney, J. W., Widespread patterns of neuronal damage following systemic or intracerebral injections of kainic acid: a histological study, Neuroscience, 5 (1980) 991-1014. 30 Skrede, K. K. and Malthe-Sorenssen, D., Differential release of D-[SH]aspartate and [14C]7aminobutyric acid following activation of commissural fibres in a longitudinal slice preparation of guinea pig hippocampus, Neurosci. Lett., 21 (1981) 71-76. 31 Storm-Mathisen, J. and lversen, L. L., Uptake of [ZH]L-glutamic acid in excitatory nerve endings: light and electron microscopic observations in the hippocampal formation of the rat, Neuroscience, 4 (1979) 1237-1253. 32 Soreide, A. J. and Fonnum, F., High affinity uptake of D-aspartate in the barrel subfield of the mouse somatic sensory cortex, Brain Research, 201 (1980) 427-430. 33 Walker, J. and Fonnum, F., Glutamate as a possible neurotransmitter in piriform cortex and nucleus amygdala, Brain Research, in press. 34 Wieraszko, A. and Lynch, G., Stimulation-dependent release of possible transmitter substances from hippocampal slices studied with localized perfusion, Brain Research, 160 (1979) 372-376. 35 Yamamoto, C., Activation of hippocampal neurons by mossy fibre stimulation in thin brain sections in vitro, Exp. Brain Res., 14 (1972) 423-435.