0022-3042/79/0501-1473$02.00/0
Jolrrnoi of N i u r n c h e m i w ) ' Vol. 32. pp 1473 to 1477 Pergarnon Prcsa Lld 1979. Printed in Great Britain
0 International Society for Neurochemistry Ltd
GABA IN THE OLFACTORY BULB A N D OLFACTORY NUCLEUS OF THE RAT: THE DISTRIBUTION OF GAMMA-AMINOBUTYRIC ACID, GLUTAMIC ACID DECARBOXYLASE, GABA TRANSAMINASE A N D SUCCINATE SEMIALDEHYDE DEHYDROGENASE L. AUSTIN,M. RECASENS'and P. MANDEL Centre de Neurochimie du CNRS. Institut de Chimie Biologique, 11, rue Humann, 67085 Strasbourg Cedex, France (Received 27 June 1978. Reoised 19 Septemher 1978. Accepted 4 December 1978)
Abstract-The distribution of GABA and enzymes involved in its metabolism was investigated in the different regions of the olfactory bulb and olfactory nucleus. The highest levels of GABA in the olfactory bulb were found in the layers rich in nerve terminals (31 pmol/g dry wt.). A similar distribution was found in the olfactory nucleus although the overall level of GABA was only a quarter of that measured in the bulb. Glutamic acid decarboxylase (GAD) levels in the various layers of the olfactory nucleus were similar in distribution to those of GABA. However, the correlation between G A D and GABA did not hold for the olfactory bulb, particularly in the granule cell layer and the medulla. The activities of G A D and the levels of GABA are significantly higher in the bulb than in the nucleus but succinic acid scmialdehyde dehydrogenase and GABA aminotransaminase activities are almost identical in both regions.
THEOLFACTORY bulb is generally believed to play an important role in the modification of animal behaviour and it has recently been shown in this laboratory that aggressive behaviour in rats can be influenced by ablation of t h e olfactory bulb (KARLIet ul., 1969), as well as by inhibition of GABA aminotransferase (GABA-T) or by injection of y-aminobutyric acid (GABA) directly into the bulb (MACK et al., 1975; MANDELet al., 1978). GRAHAM(1973) has shown that GABA and glutamic acid decarboxylase (GAD) are unevenly distributed throughout the layers of the rat olfactory bulb, with the highest levels of each in the external plexiform layer. When compared with other brain regions, the level of GABA in the olfactory bulb of the rat is relatively high (BAXTER, 1970). Because of the possibility that GABA is an inhibitory neurotransmitter in the bulb where one of its main functions may be concerned with the modification of behavioural activity rather than regulation of olfactory input, it is important to determine not only its distribution within the bulb but also that of enzymes involved in its metabolism. This paper describes the distribution of GABA and three enzymes concerned with GABA metabolism in the olfactory bulb and the olfactory nucleus of the rat.
' To whom correspondence should be sent. Ahhreuiations used: GAD, glutamic acid decarboxylase: GABA-T, GABA transaminase; SSA-D, succinate semialdehyde dehydrogenase.
MATERIALS AND METHODS Tissue preparation. Male Wistar rats (200 g) were killed by decapitation and the heads immediately frozen in liquid nitrogen. The brains were rcmoved while still frozen and the olfactory bulb and the olfactory nucleus were sliced at -20°C with an lnternational microtome at a thickness of 16pm. The slices were freeze-dried as described by LOWRY& PASSONNEAU (1972) and, after drying, stored at - 70°C. Samplcs of cell, neuropil and fibre tract layers were micro-dissected by hand under a Nachet microscope at a magnification of x 30 t o x 40 (Fig. 1). They were weighed on 1972) and a quartz fibre balance (LOWRY& PASSONNEAU, transferred to 4 m m diameter tubes. The samples ranged from 0.3 to 3.0pg dry wt. G A B A estimation. GABA was estimated by the method of OKADA (1974) modified to suit the conditions used. This method uses a mixture of GABA-sc-aminotransaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSA-D) from Pseudomonas jfuorescens (GABAse, Sigma), followed by enzymatic cycling of the resulting NADPH formed (LOWRYet al., 1961). T o the microdissected samples werc added lop1 of 15 mM-HCl and these were heated at 80°C for 20 min. When cooled, lop1 of enzyme reagent was added. This contained 0.22 M-Tris-HC1, p H 8.6; 2.2 mM-a-ketoglutaric acid; 30 ~ M - N A D P ;' 2-mercaptoethanol, 1 pl/ml; and GABAse, 0.1 units/ml. The reaction was allowed to proceed for 30min at room temperature and then 5 p 1 of 1 M-NaOH was added. The tubes were then heated at 80°C for 20min to destroy N A D P + . The tube contents were quantitatively transferred t o 10 mm fluorometer tubes, in ice, containing 100 pl of cycling reagent. This consisted of 0.2 M-Tris-HCI, p H 7.8; 10 mM-a-ketoglutaric acid; 30 mMammonium acetate; bovine serum albumin, 0.2 mg/ml;
1473
L. A u s n N , M. RECASENSand P. MANDEL
1474
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.cORTE
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OLFACTORY TRACT PLEXIFORM ’
.OLFACTORY
’.._
--GLOMERULUS
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-ANTERIOR COMMISSURE
OLFACTORY BULB AND NUCLEUS
FIG.
Schema of the regions of the olfactory bulb and olfactory nucleus after frontal sections.
0.2 mM-EDTA; m~-glucose-6-phosphate; 0.2 mM-ADP; glutamate dehydrogenase (Boehringer) 30 pg/ml; and glucose-6-phosphate dehydrogenase (Boehringer) 6 pgiml. Cycling proceeded for 1 h at 37°C. The reaction was stopped by immersing the tubes in boiling water for 2 min. To each tube was added 1 ml of a reagent mixture con8.0; 0.1 mM-EDTA; taining 20 m~-Tris-HcI, pH 0.1 mM-NADP’. Blank fluorescence was determined at 345 nm (excitation) and 455 nm (fluorescence) in an Aminco Bowman Spectrofluorometer, modified to accept the tubes. Quinine sulphate standards were used to regulate sensitivity. Five bl (lojig) of 6-phosphogluconate dehydrogenase (Boehringer) were added to each tube and after 30 rnin the fluorescence was read again. The cycling rate under these conditions was 800-900 cycles,%. Fluorescence readings were linear with respect to GABA up to 100pmol per tube. Glutarnic ucid decurboxylase activity. The method of OKADA (1976) was used, except that the samples were placed in 5 mm tubes to which were added 10 p1 of a solution containing 100 mM-sodium phosphate buffer, pH 6.8; 5 m~-glutamicacid; 250 pM-pyridoxal; and 2-mercaptoethanol, 4 pllml. After a 60min incubation at 37°C the reaction was stopped by addition of 3 p1 of 0.25 M-HC1.The tubes were heated for 10min at 60°C to destroy enzymes and the GABA formed was determined as described above. At pH 6.8 the activity of GABA-T is very low and thus GABA formed during the incubation is not enzymatically destroyed. GABA amino transaminase activity. This was determined enzymatically as follows: Step 1, formation of glutamate: to the microdissected samples were added 10 pl of a reaction mixture containing 200 mM-Tris-HC1 buffer, pH 8.0; 10 mw-a-ketogiutarate; 50 mM-GABA; 100p~-2aminoethylisoth~ouronium bromide (AET); and 20 pM-pyridoxal phosphate. After a 1 h incubation at 3 7 T , the reaction was stopped by addition of 1 pl of 1 M-NaOH and subsequent heating at 80°C for 12 min. Step 2, formation of NADH: to each sample was added loop1 of the following mixture: 100mM-Tris-HC1, pH 8.6; 100 p ~ - A D P ;300 p ~ - N A D and , the reaction was started by addition of 5 p1 of glutamate dehydrogenase (Boehringer, 100 mg/ml solution in glycerol) diluted 1/10, After 30min incubation at room temperature, the reaction was
stopped by addition of 3 pl of 1 M-NaOH and the samples were heated for 20 min at 60°C. Step 3, NADH cycling: this cycling reaction was essentially that of KATOel al. (1973) modified as follows: 20y1 of the above mixture was transferred to a fluorescence tube containing 1OOpI of a reagent consisting of 100mM-Tris-HC1 buffer, pH 8.6; 0.01% bovine serum albumin; 3 m~-mercaptoethanol; 350 mM-ethanol; and 2.25 mM-oxaloacetic acid. The reaction was started by addition of 3 pI of an enzyme mixture containing alcohol dehydrogenase (Boehringer-suspension 30 mgiml from yeast) and malate dehydrogenase (Boehringer ; 10 mg/ml solution in glycerol) 2:l (v/v) diluted 1/10, Cycling proceeded for 1 h at 25°C. The reaction was stopped by heating at 100°C for 2 mi;. The cycling rate under these conditions was 3000-5000 cycles/h. To each sample was added 1 ml of the following mixture: 50 m~-2-amino-2-methylpropanol buffer, pH 9.9; IOmM-sodium glutamate; and 0.6 mM-NAD’. The reaction was started by addition of 15pI of a mixture of glutarnate-oxaloacetate transaminase and malate dehydrogenase (2:1, v/v diluted 1/10) (glutamate-oxaloacetate transaminase-Boehringer, suspension 10 mg/ml). The fluorescence was read at 345 m (excitation) and 455 nm (fluorescence) after 20 min. Standard solutions of glutamate were run with each determination and activities were calculated using these standards. The reaction was linear with respect to tissue weight within any one layer and for at least 75 min. Succinic semialdehyde dehydrogenase activity. The & SCOTT(1959) was modified for use method of JACOBY with small samples. To samples in 5 mm tubcs were added 50 pl of a solution containing 100mM-Tris-HC1, pH 8.6; 100 PM-EDTA; 300 ~ M - N A D +2; mercaptoethanol, 2 pl/ml; and BSA, 0.5 mg/ml. The tubes were equilibrated at 37°C and the reaction commenced by addition of 5 4 of a solution of 0.55 M-succinic semialdehyde (a gift of Dr. C. CASH).After 60min at 37°C the tubes were placed in ice and the contents quantitatively transferred to fluorometer tubes containing 1 ml of 25 rnM-carbonate-bicarbonate buffer, pH 10.0. The fluorescence was read at 345 nm (excitation) 455 nm (fluorescence). The activity was determined from suitable standards of reduced pyridine nucleotide. Preliminary experiments showed that the reaction was linear for at least 60min.
1475
GABA. GAD, GABA-T and SSA-D in the olfactory bulb GABA-
GABA ;AD
E i
SSA-D
i 3 1
I"
5
12..60
30 120
5
20 80
a -400
10 60 2
FIBRORUM GLOMERULOSA EXTERNAL MITRAL GRANULE PLEXIFORM CELLS
MEDULLA
CELLS
FIG.2. Mean levels of GABA, GAD, GABA-T and SSA-D in microdissected layers from thc olfactory bulb. Samples were obtained from 3 rats. GABA concentrations are expressed as pmol/g/dry wt. Enzyme activities are pmol/g dry wt./h. Numbers of samples are shown above columns. Error bars show stan-
dard deviations. RESULTS
The highest levels of GABA were found to be in the layers rich in nerve terminals, the external plexiform and the mitral cell-internal plexiform layers (Fig. 2). Because both the mitral cell layer and the internal plexiform layer are very narrow, no attempt was made to dissect them separately. The overall GABA level in the olfactory nucleus is much less than that of the bulb; the average of the values in the various layers of the nucleus is 5.7pmol/g dry wt. compared with 22.9 pmol/g dry wt. for the bulb. Again the highest levels in the nucleus are found in the nerve terminal regions (Fig. 3).
GAD activity in the bulb does not correlate particularly well with that of GABA (Fig. 2). The glomerular, external plexiform and mitral cell-internal plexiform layers are all high in activity but the granule cell layer activity is only about half that of the other layers rich in ccll bodies. Again, in the olfactory nucleus, there is much less activity than that of the bulb. The average of specific activities across layers are 66.1 pmol/g dry wt./h for the bulb and 22.2 pmol/g dry wt./h for the nucleus. In the olfactory nucleus the GAD activity follows the GABA levels reasonably closely (Fig. 3). In the olfactory bulb, the distribution of GABA-T follows that of GABA reasonably well. The highest
iABArAk
GABA-T SSA-D
30 120
EXTERNAL TRACT
12-600
PLEXIFORM
OLFACTORY NUCLEUS
a
MEDULLA
FIG.3. Mean levels of GAHA, GAD, GABA-T and SSA-D in microdissected layers from the olfactory nucleus. For concentrations and activities see Fig. 2.
L. AUSTIN,M. RECASENSand P. MANUEL
1476
levels are in the external plexiform and mitral cellinner plexiform layers. The granulc cell layer is relatively deficient in this enzyme. Unlike GABA and GAD, the GABA-T levels in the layers of the olfactory nucleus are relatively high. The mean activity across layers in the nucleus is 53 pmol/g dry wt./h, compared with 62 pmol/g dry wt./h in the bulb. The activity of SSA-D is distributed in the olfactory bulb in a similar fashion to that of GABA-T, except in that it is rather higher in the fibrorum and glomerulosa layers. High levels are again found in cell and terminal regions with lower levels in the fibre tracts (Fig. 2). The average activity across the layers of this enzyme in the nucleus is almost as high as that in the bulb: 359pmol NADH forrned/g dry wt./h for the bulb and 349pmol/g dry wt./h for the nucleus. Again, it is rather higher in activity in the cell and terminal regions than in the fibre layers but the protein content of these fibre tract layers will be lower than the cellular or neuropil layers, and consequently the values would be higher than shown if expressed on the basis of protein. DISCUSSION
The highest levels of GABA were found to be associated with the external plexiform and mitral cell layers. The mitral cells form an extremely narrow zone of cells adjacent to the equally narrow internal plexiform layer. Since no attempt was made to separate these two zones, the GABA may lie in either the cell bodies of the mitral cells or in the terminals of the inner plexiform. There was also a high level of GABA in the granule cell layer. The activity of GAD did not parallel the GABA levels. The most active regions with respect to this enzyme were the external plexiform, the mitral cell ( + inner plexiform) and the glomerular region. It is generally believed that the GABA synthesizing capacity is reflected by the level of GAD. Here, GAD is found in three zones rich in nerve terminals. Whether the greatest contribution to the GAD activity is made by the glomerular cell bodies or by axon terminals or dendrodentritic junctions is not known. There is some discrepancy between these results and those of GRAHAM(1973). Whereas we found that the mitral cell area has the highest GAD activity, he found a relatively low activity. It is not clear, however, whether he separated the mitral zone from the inner plexiform. It may be that the inner plexiform is a zone with relatively high G A D activity. Also, we found lower activities for GAD. This may be a reflection of the different methods used to measure the activity. In view of the demonstration of reciprocal synapses (RAMON-MOLINER, 1976) in the olfactory bulb, it cannot be assumed that a zone with relatively high levels of GABA and/vr GAD is composed largely of inhibitory synapses, although such layers are probably enriched in these synapses. If it is assumed that the
adjacent, opposing synaptic regions in a dendrodendritic, a dendrosomatic; or a dendroaxonic junction operate through different neurotransmitters, it is feasible that both excitatory and inhibitory synapses lie within a few angstroms of each other. The intracellular circuitry within the olfactory bulb, involving such et a/. (1976). synapses, has been discussed by SCHMITT Recently, immunocytochemical evidence has been presented by RIBAK et ul. (1977) which shows that GAD is located in cell bodies of granule cells and periglomerular cells as well as dendrites of these cells which make dendrodendritic connections with mitral cells. No positive reaction was seen in mitral cell soma, suggesting that the relativcly high levels of both GABA and GAD found here in the mitral cell-inner plexiform layer may be due to synapses in the inner plexiform. High SSA-D activity is probably associated with particular mitochondria (VANDEN BERG, 1970). The results presented here would suggest that these mitochondria are most conccntrated in regons enriched in GABA. The anterior olfactory nucleus has much lower levels of both GABA and GAD than the olfactory bulb. This is interesting in view of results reported earlier from this laboratory (MACK& MANDEL,1976; MANDELet a/., 1978) where it was shown that injection of n-dipropylacetate (nDPA), a GABA-T inhibitor, either i.p. or directly into the olfactory bulb of ‘killer’ rats reduces or eliminates aggression in these rats for a time. After nDPA administration, GABA levels rise in the bulb and the rise and subsequent fall follows the same time course as the reduction in aggression. At the same time there is a corresponding fall and subsequent rise in GABA-T activity. Removal of part of the olfactory bulb has no effect on the cffcctiveness of nDPA unless the bulb is sectioned close to the region of the nucleus. These results suggest that inhibitory synapses in the olfactory bulb play an important role in the regulation of aggressive behaviour. The olfactory nucleus of the normal rat has GABA levels of only about a quarter of those of the bulb. This may be due to the lower GAD activity and the relatively high GABA-T activity. Treatment with nDPA would presumably lead to an increase in the GABA content of the nucleus and this suggests that despite the lower overall levels of GABA in the nucleus region, interconnecting pathways in this region of the brain, involving GABA as a neurotransmitter, may be critical in controlling some forms of behaviour. REFERENCES BAXTER C. F. (1970) The nature of y-aminobutyric acid, in Hundbook oJNeurochemistry (LAJTHA A., ed) Vol. 3, pp. 289-353. Plenum Press, New York. GRAHAM L. T. (1973) Distribution of glutamic acid decarboxylase activity and GABA content in the olfactory bulb. Life Sci. 12, Part I, 4434t47.
GABA, GAD, GABA-T and SSA-D in the olfactory bulb JACOBYW. B. & SCOTTE. M. (1959) Aldehyde oxidation111. Succinic semialdehyde dehydrogenase J . biol. Chem. 234, 937-940. KARLI P., VERGNESM. & DIDIERGEORGES F. (1969) Ratmouse inter-specific aggressive behaviour and its manipulation by brain ablation and by brain stimulation, in Aggressive Behaviour (GARATTINI S. & SIGG E. B., eds) pp. 47-55. Excerpta Medica Foundation, Amsterdam. KATOT., BLRGERS. J., CARTERJ. A. & LOWRY0. H. (1973) An enzymatic cycling method for nicotinamide adenine dinucleotide with malic and alcohol dehydrogenase. Analyt. Biochem. 53, 86-97. LOWRY0. H. & PASSONEAU J. V. (1972) A Flexible System of Enzymatic Analysis. Academic Press, New York. J. V., SCHULTZD. W. & ROCK LOWRY0. H., PASSONNEAU M. K. (1961) The measurement of pyridine nucleotides by enzymatic cycling. J. biol. Chem. 236, 27462755. MACKG. & MANDELP. (1976) Inhibition du comportement muricide du rat par la taurine, le GABA et ses analogues. c.r. hebd. SZanc. Acad. Sci. D, Paris 283, 361-362. MACKG., SIMLERS. & M A N U ~P.L (1975) Systeme inhibiteur GABAergique dans l'agressivite interspecifique ratsouris. J. Physiol., Paris 71, A162. MANDELP., MACK G., KEMPFE., EBELA. & S~MLER S.
N.C.
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(1978) Molecular aspects of a model of aggressive behaviour : neurotransmitter interactions, in Interactions Between Putative Neurotransmitters in the Brain (GAKATTINI S., PUJOLS. F. & SAMAN" R. eds.) pp. 285-303. Raven Press, New York. OKAUAY. (1974) Distribution of y-aminobutyric acid (GABA) in the layers of superior colliculus of the rabbit. Brain Res. 75, 362-365. OKADA Y. (1976) Intrahippocampal distribution of GABA and G A D activity in the guinea-pig: microassay method for the determination of G A D activity, in G A B A in Nervous System Function (ROBERTSE., CHASET. W. TOWER D. B., eds.) pp. 223-228. Raven Press, New York. RAMON-MOLLNER E. (1976) Postsynaptic vesicles in olfactory bulb of rat. Brairt Res. 105. 551-556. RIBAKC. E., VAUGHNJ. E., SAITO K., BARBERR. & ROBERTSE. (1977) Glutamate decarboxyiase location in neurons of the olfactory bulb. Bruin Res. 126, 1-18. SCHMITTF. O., DEVP. & SMITHB. H. (1976) Electronic processing of information by brain cells, Science 193, 114-1 20. VANDEN BERGC. J. (1970) Glutamate and glutamine, in Handbook of Neurochemistry (LAJTHAA,, ed.) vol. 3, pp. 355-380. Plenum Press, New York.
Jolrrnoi of N i u r n c h e m i w ) ' Vol. 32. pp 1473 to 1477 Pergarnon Prcsa Lld 1979. Printed in Great Britain
0 International Society for Neurochemistry Ltd
GABA IN THE OLFACTORY BULB A N D OLFACTORY NUCLEUS OF THE RAT: THE DISTRIBUTION OF GAMMA-AMINOBUTYRIC ACID, GLUTAMIC ACID DECARBOXYLASE, GABA TRANSAMINASE A N D SUCCINATE SEMIALDEHYDE DEHYDROGENASE L. AUSTIN,M. RECASENS'and P. MANDEL Centre de Neurochimie du CNRS. Institut de Chimie Biologique, 11, rue Humann, 67085 Strasbourg Cedex, France (Received 27 June 1978. Reoised 19 Septemher 1978. Accepted 4 December 1978)
Abstract-The distribution of GABA and enzymes involved in its metabolism was investigated in the different regions of the olfactory bulb and olfactory nucleus. The highest levels of GABA in the olfactory bulb were found in the layers rich in nerve terminals (31 pmol/g dry wt.). A similar distribution was found in the olfactory nucleus although the overall level of GABA was only a quarter of that measured in the bulb. Glutamic acid decarboxylase (GAD) levels in the various layers of the olfactory nucleus were similar in distribution to those of GABA. However, the correlation between G A D and GABA did not hold for the olfactory bulb, particularly in the granule cell layer and the medulla. The activities of G A D and the levels of GABA are significantly higher in the bulb than in the nucleus but succinic acid scmialdehyde dehydrogenase and GABA aminotransaminase activities are almost identical in both regions.
THEOLFACTORY bulb is generally believed to play an important role in the modification of animal behaviour and it has recently been shown in this laboratory that aggressive behaviour in rats can be influenced by ablation of t h e olfactory bulb (KARLIet ul., 1969), as well as by inhibition of GABA aminotransferase (GABA-T) or by injection of y-aminobutyric acid (GABA) directly into the bulb (MACK et al., 1975; MANDELet al., 1978). GRAHAM(1973) has shown that GABA and glutamic acid decarboxylase (GAD) are unevenly distributed throughout the layers of the rat olfactory bulb, with the highest levels of each in the external plexiform layer. When compared with other brain regions, the level of GABA in the olfactory bulb of the rat is relatively high (BAXTER, 1970). Because of the possibility that GABA is an inhibitory neurotransmitter in the bulb where one of its main functions may be concerned with the modification of behavioural activity rather than regulation of olfactory input, it is important to determine not only its distribution within the bulb but also that of enzymes involved in its metabolism. This paper describes the distribution of GABA and three enzymes concerned with GABA metabolism in the olfactory bulb and the olfactory nucleus of the rat.
' To whom correspondence should be sent. Ahhreuiations used: GAD, glutamic acid decarboxylase: GABA-T, GABA transaminase; SSA-D, succinate semialdehyde dehydrogenase.
MATERIALS AND METHODS Tissue preparation. Male Wistar rats (200 g) were killed by decapitation and the heads immediately frozen in liquid nitrogen. The brains were rcmoved while still frozen and the olfactory bulb and the olfactory nucleus were sliced at -20°C with an lnternational microtome at a thickness of 16pm. The slices were freeze-dried as described by LOWRY& PASSONNEAU (1972) and, after drying, stored at - 70°C. Samplcs of cell, neuropil and fibre tract layers were micro-dissected by hand under a Nachet microscope at a magnification of x 30 t o x 40 (Fig. 1). They were weighed on 1972) and a quartz fibre balance (LOWRY& PASSONNEAU, transferred to 4 m m diameter tubes. The samples ranged from 0.3 to 3.0pg dry wt. G A B A estimation. GABA was estimated by the method of OKADA (1974) modified to suit the conditions used. This method uses a mixture of GABA-sc-aminotransaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSA-D) from Pseudomonas jfuorescens (GABAse, Sigma), followed by enzymatic cycling of the resulting NADPH formed (LOWRYet al., 1961). T o the microdissected samples werc added lop1 of 15 mM-HCl and these were heated at 80°C for 20 min. When cooled, lop1 of enzyme reagent was added. This contained 0.22 M-Tris-HC1, p H 8.6; 2.2 mM-a-ketoglutaric acid; 30 ~ M - N A D P ;' 2-mercaptoethanol, 1 pl/ml; and GABAse, 0.1 units/ml. The reaction was allowed to proceed for 30min at room temperature and then 5 p 1 of 1 M-NaOH was added. The tubes were then heated at 80°C for 20min to destroy N A D P + . The tube contents were quantitatively transferred t o 10 mm fluorometer tubes, in ice, containing 100 pl of cycling reagent. This consisted of 0.2 M-Tris-HCI, p H 7.8; 10 mM-a-ketoglutaric acid; 30 mMammonium acetate; bovine serum albumin, 0.2 mg/ml;
1473
L. A u s n N , M. RECASENSand P. MANDEL
1474
,
.cORTE
X
MITRAL CELLS
OLFACTORY TRACT PLEXIFORM ’
.OLFACTORY
’.._
--GLOMERULUS
?k!$kW”
-ANTERIOR COMMISSURE
OLFACTORY BULB AND NUCLEUS
FIG.
Schema of the regions of the olfactory bulb and olfactory nucleus after frontal sections.
0.2 mM-EDTA; m~-glucose-6-phosphate; 0.2 mM-ADP; glutamate dehydrogenase (Boehringer) 30 pg/ml; and glucose-6-phosphate dehydrogenase (Boehringer) 6 pgiml. Cycling proceeded for 1 h at 37°C. The reaction was stopped by immersing the tubes in boiling water for 2 min. To each tube was added 1 ml of a reagent mixture con8.0; 0.1 mM-EDTA; taining 20 m~-Tris-HcI, pH 0.1 mM-NADP’. Blank fluorescence was determined at 345 nm (excitation) and 455 nm (fluorescence) in an Aminco Bowman Spectrofluorometer, modified to accept the tubes. Quinine sulphate standards were used to regulate sensitivity. Five bl (lojig) of 6-phosphogluconate dehydrogenase (Boehringer) were added to each tube and after 30 rnin the fluorescence was read again. The cycling rate under these conditions was 800-900 cycles,%. Fluorescence readings were linear with respect to GABA up to 100pmol per tube. Glutarnic ucid decurboxylase activity. The method of OKADA (1976) was used, except that the samples were placed in 5 mm tubes to which were added 10 p1 of a solution containing 100 mM-sodium phosphate buffer, pH 6.8; 5 m~-glutamicacid; 250 pM-pyridoxal; and 2-mercaptoethanol, 4 pllml. After a 60min incubation at 37°C the reaction was stopped by addition of 3 p1 of 0.25 M-HC1.The tubes were heated for 10min at 60°C to destroy enzymes and the GABA formed was determined as described above. At pH 6.8 the activity of GABA-T is very low and thus GABA formed during the incubation is not enzymatically destroyed. GABA amino transaminase activity. This was determined enzymatically as follows: Step 1, formation of glutamate: to the microdissected samples were added 10 pl of a reaction mixture containing 200 mM-Tris-HC1 buffer, pH 8.0; 10 mw-a-ketogiutarate; 50 mM-GABA; 100p~-2aminoethylisoth~ouronium bromide (AET); and 20 pM-pyridoxal phosphate. After a 1 h incubation at 3 7 T , the reaction was stopped by addition of 1 pl of 1 M-NaOH and subsequent heating at 80°C for 12 min. Step 2, formation of NADH: to each sample was added loop1 of the following mixture: 100mM-Tris-HC1, pH 8.6; 100 p ~ - A D P ;300 p ~ - N A D and , the reaction was started by addition of 5 p1 of glutamate dehydrogenase (Boehringer, 100 mg/ml solution in glycerol) diluted 1/10, After 30min incubation at room temperature, the reaction was
stopped by addition of 3 pl of 1 M-NaOH and the samples were heated for 20 min at 60°C. Step 3, NADH cycling: this cycling reaction was essentially that of KATOel al. (1973) modified as follows: 20y1 of the above mixture was transferred to a fluorescence tube containing 1OOpI of a reagent consisting of 100mM-Tris-HC1 buffer, pH 8.6; 0.01% bovine serum albumin; 3 m~-mercaptoethanol; 350 mM-ethanol; and 2.25 mM-oxaloacetic acid. The reaction was started by addition of 3 pI of an enzyme mixture containing alcohol dehydrogenase (Boehringer-suspension 30 mgiml from yeast) and malate dehydrogenase (Boehringer ; 10 mg/ml solution in glycerol) 2:l (v/v) diluted 1/10, Cycling proceeded for 1 h at 25°C. The reaction was stopped by heating at 100°C for 2 mi;. The cycling rate under these conditions was 3000-5000 cycles/h. To each sample was added 1 ml of the following mixture: 50 m~-2-amino-2-methylpropanol buffer, pH 9.9; IOmM-sodium glutamate; and 0.6 mM-NAD’. The reaction was started by addition of 15pI of a mixture of glutarnate-oxaloacetate transaminase and malate dehydrogenase (2:1, v/v diluted 1/10) (glutamate-oxaloacetate transaminase-Boehringer, suspension 10 mg/ml). The fluorescence was read at 345 m (excitation) and 455 nm (fluorescence) after 20 min. Standard solutions of glutamate were run with each determination and activities were calculated using these standards. The reaction was linear with respect to tissue weight within any one layer and for at least 75 min. Succinic semialdehyde dehydrogenase activity. The & SCOTT(1959) was modified for use method of JACOBY with small samples. To samples in 5 mm tubcs were added 50 pl of a solution containing 100mM-Tris-HC1, pH 8.6; 100 PM-EDTA; 300 ~ M - N A D +2; mercaptoethanol, 2 pl/ml; and BSA, 0.5 mg/ml. The tubes were equilibrated at 37°C and the reaction commenced by addition of 5 4 of a solution of 0.55 M-succinic semialdehyde (a gift of Dr. C. CASH).After 60min at 37°C the tubes were placed in ice and the contents quantitatively transferred to fluorometer tubes containing 1 ml of 25 rnM-carbonate-bicarbonate buffer, pH 10.0. The fluorescence was read at 345 nm (excitation) 455 nm (fluorescence). The activity was determined from suitable standards of reduced pyridine nucleotide. Preliminary experiments showed that the reaction was linear for at least 60min.
1475
GABA. GAD, GABA-T and SSA-D in the olfactory bulb GABA-
GABA ;AD
E i
SSA-D
i 3 1
I"
5
12..60
30 120
5
20 80
a -400
10 60 2
FIBRORUM GLOMERULOSA EXTERNAL MITRAL GRANULE PLEXIFORM CELLS
MEDULLA
CELLS
FIG.2. Mean levels of GABA, GAD, GABA-T and SSA-D in microdissected layers from thc olfactory bulb. Samples were obtained from 3 rats. GABA concentrations are expressed as pmol/g/dry wt. Enzyme activities are pmol/g dry wt./h. Numbers of samples are shown above columns. Error bars show stan-
dard deviations. RESULTS
The highest levels of GABA were found to be in the layers rich in nerve terminals, the external plexiform and the mitral cell-internal plexiform layers (Fig. 2). Because both the mitral cell layer and the internal plexiform layer are very narrow, no attempt was made to dissect them separately. The overall GABA level in the olfactory nucleus is much less than that of the bulb; the average of the values in the various layers of the nucleus is 5.7pmol/g dry wt. compared with 22.9 pmol/g dry wt. for the bulb. Again the highest levels in the nucleus are found in the nerve terminal regions (Fig. 3).
GAD activity in the bulb does not correlate particularly well with that of GABA (Fig. 2). The glomerular, external plexiform and mitral cell-internal plexiform layers are all high in activity but the granule cell layer activity is only about half that of the other layers rich in ccll bodies. Again, in the olfactory nucleus, there is much less activity than that of the bulb. The average of specific activities across layers are 66.1 pmol/g dry wt./h for the bulb and 22.2 pmol/g dry wt./h for the nucleus. In the olfactory nucleus the GAD activity follows the GABA levels reasonably closely (Fig. 3). In the olfactory bulb, the distribution of GABA-T follows that of GABA reasonably well. The highest
iABArAk
GABA-T SSA-D
30 120
EXTERNAL TRACT
12-600
PLEXIFORM
OLFACTORY NUCLEUS
a
MEDULLA
FIG.3. Mean levels of GAHA, GAD, GABA-T and SSA-D in microdissected layers from the olfactory nucleus. For concentrations and activities see Fig. 2.
L. AUSTIN,M. RECASENSand P. MANUEL
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levels are in the external plexiform and mitral cellinner plexiform layers. The granulc cell layer is relatively deficient in this enzyme. Unlike GABA and GAD, the GABA-T levels in the layers of the olfactory nucleus are relatively high. The mean activity across layers in the nucleus is 53 pmol/g dry wt./h, compared with 62 pmol/g dry wt./h in the bulb. The activity of SSA-D is distributed in the olfactory bulb in a similar fashion to that of GABA-T, except in that it is rather higher in the fibrorum and glomerulosa layers. High levels are again found in cell and terminal regions with lower levels in the fibre tracts (Fig. 2). The average activity across the layers of this enzyme in the nucleus is almost as high as that in the bulb: 359pmol NADH forrned/g dry wt./h for the bulb and 349pmol/g dry wt./h for the nucleus. Again, it is rather higher in activity in the cell and terminal regions than in the fibre layers but the protein content of these fibre tract layers will be lower than the cellular or neuropil layers, and consequently the values would be higher than shown if expressed on the basis of protein. DISCUSSION
The highest levels of GABA were found to be associated with the external plexiform and mitral cell layers. The mitral cells form an extremely narrow zone of cells adjacent to the equally narrow internal plexiform layer. Since no attempt was made to separate these two zones, the GABA may lie in either the cell bodies of the mitral cells or in the terminals of the inner plexiform. There was also a high level of GABA in the granule cell layer. The activity of GAD did not parallel the GABA levels. The most active regions with respect to this enzyme were the external plexiform, the mitral cell ( + inner plexiform) and the glomerular region. It is generally believed that the GABA synthesizing capacity is reflected by the level of GAD. Here, GAD is found in three zones rich in nerve terminals. Whether the greatest contribution to the GAD activity is made by the glomerular cell bodies or by axon terminals or dendrodentritic junctions is not known. There is some discrepancy between these results and those of GRAHAM(1973). Whereas we found that the mitral cell area has the highest GAD activity, he found a relatively low activity. It is not clear, however, whether he separated the mitral zone from the inner plexiform. It may be that the inner plexiform is a zone with relatively high G A D activity. Also, we found lower activities for GAD. This may be a reflection of the different methods used to measure the activity. In view of the demonstration of reciprocal synapses (RAMON-MOLINER, 1976) in the olfactory bulb, it cannot be assumed that a zone with relatively high levels of GABA and/vr GAD is composed largely of inhibitory synapses, although such layers are probably enriched in these synapses. If it is assumed that the
adjacent, opposing synaptic regions in a dendrodendritic, a dendrosomatic; or a dendroaxonic junction operate through different neurotransmitters, it is feasible that both excitatory and inhibitory synapses lie within a few angstroms of each other. The intracellular circuitry within the olfactory bulb, involving such et a/. (1976). synapses, has been discussed by SCHMITT Recently, immunocytochemical evidence has been presented by RIBAK et ul. (1977) which shows that GAD is located in cell bodies of granule cells and periglomerular cells as well as dendrites of these cells which make dendrodendritic connections with mitral cells. No positive reaction was seen in mitral cell soma, suggesting that the relativcly high levels of both GABA and GAD found here in the mitral cell-inner plexiform layer may be due to synapses in the inner plexiform. High SSA-D activity is probably associated with particular mitochondria (VANDEN BERG, 1970). The results presented here would suggest that these mitochondria are most conccntrated in regons enriched in GABA. The anterior olfactory nucleus has much lower levels of both GABA and GAD than the olfactory bulb. This is interesting in view of results reported earlier from this laboratory (MACK& MANDEL,1976; MANDELet a/., 1978) where it was shown that injection of n-dipropylacetate (nDPA), a GABA-T inhibitor, either i.p. or directly into the olfactory bulb of ‘killer’ rats reduces or eliminates aggression in these rats for a time. After nDPA administration, GABA levels rise in the bulb and the rise and subsequent fall follows the same time course as the reduction in aggression. At the same time there is a corresponding fall and subsequent rise in GABA-T activity. Removal of part of the olfactory bulb has no effect on the cffcctiveness of nDPA unless the bulb is sectioned close to the region of the nucleus. These results suggest that inhibitory synapses in the olfactory bulb play an important role in the regulation of aggressive behaviour. The olfactory nucleus of the normal rat has GABA levels of only about a quarter of those of the bulb. This may be due to the lower GAD activity and the relatively high GABA-T activity. Treatment with nDPA would presumably lead to an increase in the GABA content of the nucleus and this suggests that despite the lower overall levels of GABA in the nucleus region, interconnecting pathways in this region of the brain, involving GABA as a neurotransmitter, may be critical in controlling some forms of behaviour. REFERENCES BAXTER C. F. (1970) The nature of y-aminobutyric acid, in Hundbook oJNeurochemistry (LAJTHA A., ed) Vol. 3, pp. 289-353. Plenum Press, New York. GRAHAM L. T. (1973) Distribution of glutamic acid decarboxylase activity and GABA content in the olfactory bulb. Life Sci. 12, Part I, 4434t47.
GABA, GAD, GABA-T and SSA-D in the olfactory bulb JACOBYW. B. & SCOTTE. M. (1959) Aldehyde oxidation111. Succinic semialdehyde dehydrogenase J . biol. Chem. 234, 937-940. KARLI P., VERGNESM. & DIDIERGEORGES F. (1969) Ratmouse inter-specific aggressive behaviour and its manipulation by brain ablation and by brain stimulation, in Aggressive Behaviour (GARATTINI S. & SIGG E. B., eds) pp. 47-55. Excerpta Medica Foundation, Amsterdam. KATOT., BLRGERS. J., CARTERJ. A. & LOWRY0. H. (1973) An enzymatic cycling method for nicotinamide adenine dinucleotide with malic and alcohol dehydrogenase. Analyt. Biochem. 53, 86-97. LOWRY0. H. & PASSONEAU J. V. (1972) A Flexible System of Enzymatic Analysis. Academic Press, New York. J. V., SCHULTZD. W. & ROCK LOWRY0. H., PASSONNEAU M. K. (1961) The measurement of pyridine nucleotides by enzymatic cycling. J. biol. Chem. 236, 27462755. MACKG. & MANDELP. (1976) Inhibition du comportement muricide du rat par la taurine, le GABA et ses analogues. c.r. hebd. SZanc. Acad. Sci. D, Paris 283, 361-362. MACKG., SIMLERS. & M A N U ~P.L (1975) Systeme inhibiteur GABAergique dans l'agressivite interspecifique ratsouris. J. Physiol., Paris 71, A162. MANDELP., MACK G., KEMPFE., EBELA. & S~MLER S.
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(1978) Molecular aspects of a model of aggressive behaviour : neurotransmitter interactions, in Interactions Between Putative Neurotransmitters in the Brain (GAKATTINI S., PUJOLS. F. & SAMAN" R. eds.) pp. 285-303. Raven Press, New York. OKAUAY. (1974) Distribution of y-aminobutyric acid (GABA) in the layers of superior colliculus of the rabbit. Brain Res. 75, 362-365. OKADA Y. (1976) Intrahippocampal distribution of GABA and G A D activity in the guinea-pig: microassay method for the determination of G A D activity, in G A B A in Nervous System Function (ROBERTSE., CHASET. W. TOWER D. B., eds.) pp. 223-228. Raven Press, New York. RAMON-MOLLNER E. (1976) Postsynaptic vesicles in olfactory bulb of rat. Brairt Res. 105. 551-556. RIBAKC. E., VAUGHNJ. E., SAITO K., BARBERR. & ROBERTSE. (1977) Glutamate decarboxyiase location in neurons of the olfactory bulb. Bruin Res. 126, 1-18. SCHMITTF. O., DEVP. & SMITHB. H. (1976) Electronic processing of information by brain cells, Science 193, 114-1 20. VANDEN BERGC. J. (1970) Glutamate and glutamine, in Handbook of Neurochemistry (LAJTHAA,, ed.) vol. 3, pp. 355-380. Plenum Press, New York.
