Neuroscience Letters, 109 (1990) 7-12
7
Elsevier Scientific Publishers Ireland Ltd. NSL 06632
Tetanus toxin produces neuronal loss and a reduction in GABAA but not GABAB binding sites in rat hippocampus G. Bagetta 1, C. Knott l, G. Nistic6 2 and N.G. Bowery 1 iDepartment of Pharmacology, School of Pharmacy, London ( U.K. ) and 2Institute of Pharmacology, Facully of Medicine and Surgery of Catanzaro, University of Reggio Calabria, Reggio Calabria (Italy)
(Received 7 August 1989; Revisedversion received29 September 1989; Accepted 2 October 1989) Key words: Tetanus toxin; Hippocampal neurodegeneration; y-Aminobutyric acid-A binding site; 7-
Aminobutyric acid-B binding site; Autoradiography The neuropathological effects of tetanus toxin, microinjected in the rat CA1 hippocampal area, were studied by using a microscopicaland autoradiographical approach. Tetanus toxin produced a dose- and time-dependent neuronal loss in the CA 1 area accompanied by a reduction in the binding of ~,-[3H]aminobutyric acid (pH]GABA) to GABAAbut not GABABsites in the pyramidal cell layer. It is well documented that CA1 hippocampal neurons are vulnerable to ischaemic insult [19] and that the consequent neuropathological effects may be mediated in part by excitatory N-methyl-D-aspartate ( N M D A ) receptor stimulation. In fact, N M D A receptor antagonists (i.e. 2-amino-5-phosphonovaleric acid, 2-amino-7-phosphonoheptanoic acid and MK801) are able to prevent the neuronal death produced by ischaemia [9, 19] or by N M D A receptor agonists [18]. An increase in the concentration of synaptic glutamate in the hippocampus and the occurrence of spike discharges, recorded from the CA 1 pyramidal neurons, have also been observed following ischaemic insult [1, 20]. Moreover, ischaemia-induced hippocampal neurodegeneration can be prevented by lesioning the Schaffer collateral and commissural fibers [15] through which the CA1 pyramids receive excitatory inputs from the CA3 area [13]. However, pyramidal neurons in the CA1 area receive both excitatory and inhibitory (mainly GABAergic) inputs [16] and failure of the latter produces spike discharges which are sensitive to blockade by N M D A receptor antagonists [21]. Thus, we have hypothesized that, under experimental conditions in which the inhibitory mechanisms are disrupted, neuronal degeneration may occur in the hippocampus due to unopposed excitation. To test this hypothesis we have used tetanus toxin (TT), a neurobiological tool well known for producing a long-lasting impairment of inhibiCorrespondence: N.G. Bowery, Dept. of Pharmacology, School of Pharmacy, 29/39 Brunswick Square,
London, WClN lAX, U.K. 0304-3940/90/$ 03.50 © 1990 ElsevierScientific Publishers Ireland Ltd.
tory neurotransmission in the hippocampus [6, 10] and elsewhere in the central nervous system [4, 5]. Adult male Wistar rats (280-300 g) were anaesthetized with chloral hydrate (400 mg/kg, i.p.) and TT (specific toxicity o f 3.7 x 107 mouse m i n i m u m lethal doses, M L D s / m g o f protein) microinjected unilaterally into the C A I hippocampal area (coordinates: A P = - 4 . 0 from the bregma; L = 2 . 0 from the midtine; V = 2 . 4 below the dura mater [17]; 1 /tl volume o f injection; 1 /zl/min rate o f infusion) by means o f a Hamilton microsyringe m o u n t e d on a stereotaxic frame (David Kopf). Bovine serum albumin (BSA) and neutralized TT (with F(ab)I fragment o f the native lgG anti-TT [8]) were used as controls. After 1, 4, 7, 10 days, the rats were anaesthetized and perfused-fixed by intracardiac administration o f 100 ml 0.1% paraformaldehyde in phosphate buffered (pH = 7.4) saline; 10/,tm cryostat brain sections were cut. Sections from the same rat were used for both microscopical (Cresyl fast violet staining) and autoradiographical studies. A u t o r a d i o g r a m s o f ~,-[3H]aminobutyric acid ([3H]GABA) binding sites were generated as previously described by Bowery et el. [2], Briefly, each section was incubated (20 min) with 100/zl o f incubation fluid containing 50 n M [3H]GABA (91.6 Ci/mmol). In the presence o f ( - ) - b a c l o f e n (100/~M) only G A B A A sites were labelled whereas G A B A B sites were labelled in the presence o f 4 0 / t M isoguvacine. Background binding was determined by further addition o f isoguvacine ( 1 0 0 / I M ) or ( - ) - b a c l o f e n (100 llM) for G A B A A and G A B A R sites, respectively. Non-specific binding represented 5 30% o f the total binding in all cases. The slices were placed in contact with L K B Ultrofilm at 4°C for 3 4 weeks and A m e r s h a m Microscale standards were used as calibration markers. Analysis o f the a u t o r a d i o g r a m images was performed using a Quantimet 970 (Cambridge Instruments Plc) image analyser and the [3H]GABA binding sites density converted into CA ,
Dentate gyrus (granular cell layer)
(pyramidal cell layer)
35o[
100 ,--ul
~ 4°I ~
.J1~.
3o
to
7
t
7
i
tO
7
1
-7 7
10
Days after treatment BSA
tetanus toxin
BSA
tetanus t o x i n
Fig. 1. Neurotoxicity of tetanus toxin directly microinjected into the CA1 hippocampal area of rat. Sections (10 ,urn; n = 6 per brain) of perfused-fixed (0.1% paraformaldehyde in phosphate buffered (pH = 7.4) saline) rat brains (n = 3 per treatment) were stained with Cresyl fast violet. Cell counting (means + S.E.M.) was performed in the CA1 pyramidal layer and dentate gyrus granule cell layer under light microscopy (Leitz; 40 × ). Statistically significant differences between the mean cell values counted in the tetanus toxin (1000 MLDs) or BSA (300 ng//d) treated ([]) side vs the contralateral ( I ) sides were compared by using Student's t-test. Due to the interanimal variations in the cell number observed within the same hippocampal area, comparison between groups was not possible. *P < 0.05.
fmol/mg tissue. Optical density measurements were made in areas of the autoradiogram images which corresponded [17] to the pyramidal layers of the CA 1, CA2, CA3 areas and the dentate gyrus (DG) granular layer of the hippocampal formation. The density of GABAA and GABAB binding sites in the control (untreated) hippocampal areas (CA1, CA2 and CA3) was evaluated for all rats used and expressed as a percentage of the binding density observed in the DG. The results obtained in sections from untreated rats (e.g. D G = 13.5 + 1.7 and 7.6+ 1.7 fmol/mg tissue, for GABAA and GABAB sites, respectively) were in good agreement with those previously reported [2]. In order to quantify the effects of TT, GABAA and GABAB binding site densities observed in the treated side were compared with those of the control (untreated) side of each hippocampal area studied and the results expressed as mean ( _ S.E.M.) percentages. A single dose of TT (500 MLDs; n = 3 rats) produced no neurodegenerative effects 7 days after the injection into the CA1 area. Similarly, no changes were observed in GABAA and GABAB sites binding. On the contrary, time-dependent neuronal loss was induced by a dose of 1000 MLDs (n=3 rats per each group of treatment) of TT. A reduction of 21.5_+ i.3% and of 29.5_+3.1% in the cell number of the CA1 pyramidal layer was observed 7 and 10 days after the injection, respectively. No changes occurred 24 h after the injection of the same dose of TT (Fig. 1). A more pronounced reduction (37.5_+ 2.3%) was induced by the highest dose of TT used (2000 MLDs; n = 3 rats) after a shorter period of time (4 days). The neuronal loss observed 7 and 10 days after the injection of 1000 MLDs of TT was accompanied by a statistically significant (P < 0.05) reduction in GABAA site binding in the treated versus the contralateral CA1 area (Table I and Fig. 2). TABLE I EFFECTS OF TETANUS TOXIN M1CROINJECTED INTO THE RAT CA1 HIPPOCAMPAL AREA ON [3H]GABA BINDING SITES. Data represent mean (+ S.E.M.) percentages of [3H]GABA binding sites density calculated for the TT treated side vs the contralateral (untreated) side (n = 6 slices per rat; n = 3 rats per treatment). CAt
DG
GABAA
GABAa
GABAA
GABAB
Tetanus toxin (1000 MLDs) 1 day after 7 days after 10 days after
98.9 + 3.5 81.7"+_2.3 65.1"+4.1
96.2+0.9 103.2+8.2 105.5+2.5
98.1 +2.7 114.1+5.3 86.0+7.5
110.0+8.9 104.8+1.4 116.9___5.1
BSA (300 ng) 7 days after
99.2 + 2.4
101.7_ 2.0
100.7 ___3.0
*P < 0.05 (Student's t-test).
94.8 + 11.6
10
T
A
C
)
C
C
)
B
)
)
D
)
T
Fig. 2. Receptor autoradiographs of GABAA binding to rat brain sections. Transverse cryostat sections (10 ,urn) were incubated with 50 nM [3H]GABAfor 20 rain at 21 23°C. Sections C and D were additionally incubated in 100/~M isoguvacine to obtain background levels. Note in A that a single injection of tetanus toxin (1000 MLDs) produced, 7 days after the treatment, a decrease in GABAA binding in the treated (T) CA! pyramidal layer in comparison to the contralateral (C) side. In B is shown an example of the lack of effect 7 days after treatment with BSA (300 ng//~l; T, treated side; C, control). A time-dependency for the effects on G A B A A binding was also observed. In fact, no changes were induced by T T (1000 M L D s ) 24h after the injection whereas approximately 35 % reduction was observed 10 days after. N o changes were observed in G A B A B site binding (Table I). Treatment with BSA (300 ng//,tl; n = 3 rats) or with neutralized T T (n = 3 rats) did not affect the density (analyses performed 7 days after the injection) o f G A B A A or G A B A B sites. The present experiments indicate that T T is able to produce dose- and time-dependent neuropathological effects when microinjected into the rat CA1 hippocampal area. The neuronal loss was confined to a region very close to the injection site even after the administration o f 2000 M L D s . This confirms its inability to diffuse within the hippocampus, as already demonstrated in experiments in which 125I-labelled T T was used [12]. N o direct evidence has been shown in the present study to indicate that the neuropathological effects induced by T T were accompanied by a reduced inhibitory tone in the hippocampus; however, the results are most likely to be explained on the basis o f an unopposed excitation occurring in the neurons at the site o f injection. In fact, it has been widely demonstrated that TT is able to produce an excitatory focus when applied onto the hippocampus [6, 10], an effect linked to its ability to block the release o f G A B A in this area [3]. This effect has also been demonstrated in other brain regions [4, 5] and in no instance has a direct enhancement o f excitatory transmission been reported for TT (see ref. 11). As a consequence o f the neuronal loss occurring in the C A ! pyramidal layer, the concomitant decrease in the density o f G A B A A binding sites was not unexpected since these are present on the neuronal cell bodies. But quite surprisingly, no significant changes were
11
observed in the binding to GABA~ sites. This might be explained on the basis of a different rate of disappearance, a longer period of time being necessary for the number of GABAB sites to diminish. However, a more attractive hypothesis is that GABAB sites are present on nerve terminals within the pyramidal cell layer rather than on the pyramidal cells. Whilst they are present on pyramidal cell dendrites, they appear to be absent from the cell soma [14]. Their presence on the terminals of Schaffer collaterals and commissural fibres has also been described [7]. Thus, even if the pyramidal cells disappear the GABAB sites would still remain. Unfortunately, the high mortality rate of rats treated with TT did not allow the effects on the GABAB site to be followed for longer periods of time. However, the existence of a very small number of GABAB sites located on the dendrites of pyramidal neurons within the CA1 pyramidal cell layer cannot be excluded and the reduction eventually produced by TT could be masked by sprouting of terminals where the largest number of GABAB sites is present. In conclusion, the present results clearly demonstrate that TT produces neuronal loss and a reduction in GABAA but not GABAB binding sites into the rat hippocampus. Further studies are in progress in our laboratory to clarify the mechanism through which these effects are mediated. Tetanus toxin was a kind gift from Prof. B. Bizzini (Pasteur Institute, Paris). Partial financial support from the Italian Council of Research (CNR, Rome) is gratefully acknowledged. 1 Beneviste, H., Prejer, J., Schousboe, A. and Diemer, N.H., Elevation of the extracellular concentration of glutamate and aspartate in rat hippocampus during transient cerebral ischaemia monitored by intracerebral microdialysis, J. Neurochem., 43 (I 984) 1369-1374. 2 Bowery, N.G., Hudson, A.L. and Price, W., GABAA and GABAB receptor site distribution in the rat central nervous system, Neuroscience, 20 (1987) 365 383. 3 Collingridge, G.L., Thompson, P.A., Davies, J. and Mellanby J., In vitro effect of tetanus toxin on GABA release from rat hippocampal slices, J. Neurochem., 37 (1981 ) 1039-1041. 4 Curtis, D.R., Felix, D., Game, C.J.A. and McCullogh, R.M., Tetanus toxin and the synaptic release of GABA, Brain Res., 51 (1973) 358-362. 5 Davies, J. and Tongroach, P., Tetanus toxin and synaptic inhibition in the substantia nigra and striatum of the rat, J. Physiol. (Lond.), 290 (1979) 23-26. 6 De Sarro, G.B., Bagetta, G. and Nistic6, G., Antagonism by drugs enhancing GABAergic transmission of central effects of tetanus toxin. In G. Nistic6, P. Mastroeni and M. Pitzurra (Eds.), Seventh International Conference on Tetanus, Gangemi, Rome, 1985, pp. 142-156. 7 Dutar, P. and Nicoll, R.A., Pre- and postsynaptic GABAB receptors in the hippocampus have different pharmacological properties, Neuron, 1 (1988) 585-591. 8 Gawade, S., Bon, C. and Bizzini, B., The use of antibody Fab fragments specifically directed to two different complementary parts of the tetanus toxin molecule for studying the mode of action of the toxin, Brain Res., 334 (1985) 139-146. 9 Gill, R., Foster, A.C. and Woodruff, G.N., Systemic administration of MK-801 protects against ischemia-induced hippocampal neurodegeneration in the gerbil, J. Neurosci., 7 (1987) 3343-3349. 10 Mellanby, J., George, G., Robinson, A. and Thompson, P.A., Epileptiform syndrome in rats produced by injecting tetanus toxin into the hippocampus, J. Neurol. Neurosurg. Psychiatry, 40 (1977) 404414. 11 Mellanby, J. and Green, J., How does tetanus toxin act?, Neuroscience, 6 (1981) 281-300. 12 Mellanby, J. and Thompson, P.A., Tetaus toxin in the rat hippocampus, J. Physiol. (Lond.), 269 (1977) 44-45 P.
12 13 Nadler, J.V., 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.), 260 (1976) 538 540. 14 Newberry, N.R. and Nicoll, R.A., Baclofen directly hyperpolarizes hippocampal cells, Nature (Lond.), 308 (1984) 45ff452. 15 Onodera, H., Sato, G. and Kogure, K., Lesions to Schaffer collaterals prevents ischemic death of CAI pyramidal cells, Neurosci. Lett., 68 (I 986) 169- 174. 16 Ottersen, O.P. and Storm-Mathisen, J. In A. Bj6rklund, T. H6kfelt and M.J. Kuhar (Eds.), Handbook of Chemical Neuroanatomy, Vol. 3, Elsevier, Amsterdam, 1984, pp. 141 246. 17 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1981. 18 Rothman, S.M. and Olney, J.W., Glutamate and the pathophysiology of hypoxic-ischaemic brain damage, Ann. Neurol., 19 (1986) 105- 11 I. 19 Simon, R.P., Swan, J.H., Griftith. T. and Meldrum, B.S. Blockade of N-methyl-o-aspartatc receptors may protect against ischemic damage in the brain, Science, 226 (1984) 850 852. 20 Suzuki, R., Yamaguchi, T., Li, C.L. and Klatzo, I., The effects of 5-minute ischemia in mongolian gerbils: I1. Changes of the spontaneous neuronal activity in cerebral cortex and CA~ sector of hippocampus, Acta Neuropathol., 60 (1983) 217 222. 21 Taylor, C.P., How do seizurcs begin'? Clues from hippocampal slices, Trends Neurosci . 11 (1988) 375 378.
7
Elsevier Scientific Publishers Ireland Ltd. NSL 06632
Tetanus toxin produces neuronal loss and a reduction in GABAA but not GABAB binding sites in rat hippocampus G. Bagetta 1, C. Knott l, G. Nistic6 2 and N.G. Bowery 1 iDepartment of Pharmacology, School of Pharmacy, London ( U.K. ) and 2Institute of Pharmacology, Facully of Medicine and Surgery of Catanzaro, University of Reggio Calabria, Reggio Calabria (Italy)
(Received 7 August 1989; Revisedversion received29 September 1989; Accepted 2 October 1989) Key words: Tetanus toxin; Hippocampal neurodegeneration; y-Aminobutyric acid-A binding site; 7-
Aminobutyric acid-B binding site; Autoradiography The neuropathological effects of tetanus toxin, microinjected in the rat CA1 hippocampal area, were studied by using a microscopicaland autoradiographical approach. Tetanus toxin produced a dose- and time-dependent neuronal loss in the CA 1 area accompanied by a reduction in the binding of ~,-[3H]aminobutyric acid (pH]GABA) to GABAAbut not GABABsites in the pyramidal cell layer. It is well documented that CA1 hippocampal neurons are vulnerable to ischaemic insult [19] and that the consequent neuropathological effects may be mediated in part by excitatory N-methyl-D-aspartate ( N M D A ) receptor stimulation. In fact, N M D A receptor antagonists (i.e. 2-amino-5-phosphonovaleric acid, 2-amino-7-phosphonoheptanoic acid and MK801) are able to prevent the neuronal death produced by ischaemia [9, 19] or by N M D A receptor agonists [18]. An increase in the concentration of synaptic glutamate in the hippocampus and the occurrence of spike discharges, recorded from the CA 1 pyramidal neurons, have also been observed following ischaemic insult [1, 20]. Moreover, ischaemia-induced hippocampal neurodegeneration can be prevented by lesioning the Schaffer collateral and commissural fibers [15] through which the CA1 pyramids receive excitatory inputs from the CA3 area [13]. However, pyramidal neurons in the CA1 area receive both excitatory and inhibitory (mainly GABAergic) inputs [16] and failure of the latter produces spike discharges which are sensitive to blockade by N M D A receptor antagonists [21]. Thus, we have hypothesized that, under experimental conditions in which the inhibitory mechanisms are disrupted, neuronal degeneration may occur in the hippocampus due to unopposed excitation. To test this hypothesis we have used tetanus toxin (TT), a neurobiological tool well known for producing a long-lasting impairment of inhibiCorrespondence: N.G. Bowery, Dept. of Pharmacology, School of Pharmacy, 29/39 Brunswick Square,
London, WClN lAX, U.K. 0304-3940/90/$ 03.50 © 1990 ElsevierScientific Publishers Ireland Ltd.
tory neurotransmission in the hippocampus [6, 10] and elsewhere in the central nervous system [4, 5]. Adult male Wistar rats (280-300 g) were anaesthetized with chloral hydrate (400 mg/kg, i.p.) and TT (specific toxicity o f 3.7 x 107 mouse m i n i m u m lethal doses, M L D s / m g o f protein) microinjected unilaterally into the C A I hippocampal area (coordinates: A P = - 4 . 0 from the bregma; L = 2 . 0 from the midtine; V = 2 . 4 below the dura mater [17]; 1 /tl volume o f injection; 1 /zl/min rate o f infusion) by means o f a Hamilton microsyringe m o u n t e d on a stereotaxic frame (David Kopf). Bovine serum albumin (BSA) and neutralized TT (with F(ab)I fragment o f the native lgG anti-TT [8]) were used as controls. After 1, 4, 7, 10 days, the rats were anaesthetized and perfused-fixed by intracardiac administration o f 100 ml 0.1% paraformaldehyde in phosphate buffered (pH = 7.4) saline; 10/,tm cryostat brain sections were cut. Sections from the same rat were used for both microscopical (Cresyl fast violet staining) and autoradiographical studies. A u t o r a d i o g r a m s o f ~,-[3H]aminobutyric acid ([3H]GABA) binding sites were generated as previously described by Bowery et el. [2], Briefly, each section was incubated (20 min) with 100/zl o f incubation fluid containing 50 n M [3H]GABA (91.6 Ci/mmol). In the presence o f ( - ) - b a c l o f e n (100/~M) only G A B A A sites were labelled whereas G A B A B sites were labelled in the presence o f 4 0 / t M isoguvacine. Background binding was determined by further addition o f isoguvacine ( 1 0 0 / I M ) or ( - ) - b a c l o f e n (100 llM) for G A B A A and G A B A R sites, respectively. Non-specific binding represented 5 30% o f the total binding in all cases. The slices were placed in contact with L K B Ultrofilm at 4°C for 3 4 weeks and A m e r s h a m Microscale standards were used as calibration markers. Analysis o f the a u t o r a d i o g r a m images was performed using a Quantimet 970 (Cambridge Instruments Plc) image analyser and the [3H]GABA binding sites density converted into CA ,
Dentate gyrus (granular cell layer)
(pyramidal cell layer)
35o[
100 ,--ul
~ 4°I ~
.J1~.
3o
to
7
t
7
i
tO
7
1
-7 7
10
Days after treatment BSA
tetanus toxin
BSA
tetanus t o x i n
Fig. 1. Neurotoxicity of tetanus toxin directly microinjected into the CA1 hippocampal area of rat. Sections (10 ,urn; n = 6 per brain) of perfused-fixed (0.1% paraformaldehyde in phosphate buffered (pH = 7.4) saline) rat brains (n = 3 per treatment) were stained with Cresyl fast violet. Cell counting (means + S.E.M.) was performed in the CA1 pyramidal layer and dentate gyrus granule cell layer under light microscopy (Leitz; 40 × ). Statistically significant differences between the mean cell values counted in the tetanus toxin (1000 MLDs) or BSA (300 ng//d) treated ([]) side vs the contralateral ( I ) sides were compared by using Student's t-test. Due to the interanimal variations in the cell number observed within the same hippocampal area, comparison between groups was not possible. *P < 0.05.
fmol/mg tissue. Optical density measurements were made in areas of the autoradiogram images which corresponded [17] to the pyramidal layers of the CA 1, CA2, CA3 areas and the dentate gyrus (DG) granular layer of the hippocampal formation. The density of GABAA and GABAB binding sites in the control (untreated) hippocampal areas (CA1, CA2 and CA3) was evaluated for all rats used and expressed as a percentage of the binding density observed in the DG. The results obtained in sections from untreated rats (e.g. D G = 13.5 + 1.7 and 7.6+ 1.7 fmol/mg tissue, for GABAA and GABAB sites, respectively) were in good agreement with those previously reported [2]. In order to quantify the effects of TT, GABAA and GABAB binding site densities observed in the treated side were compared with those of the control (untreated) side of each hippocampal area studied and the results expressed as mean ( _ S.E.M.) percentages. A single dose of TT (500 MLDs; n = 3 rats) produced no neurodegenerative effects 7 days after the injection into the CA1 area. Similarly, no changes were observed in GABAA and GABAB sites binding. On the contrary, time-dependent neuronal loss was induced by a dose of 1000 MLDs (n=3 rats per each group of treatment) of TT. A reduction of 21.5_+ i.3% and of 29.5_+3.1% in the cell number of the CA1 pyramidal layer was observed 7 and 10 days after the injection, respectively. No changes occurred 24 h after the injection of the same dose of TT (Fig. 1). A more pronounced reduction (37.5_+ 2.3%) was induced by the highest dose of TT used (2000 MLDs; n = 3 rats) after a shorter period of time (4 days). The neuronal loss observed 7 and 10 days after the injection of 1000 MLDs of TT was accompanied by a statistically significant (P < 0.05) reduction in GABAA site binding in the treated versus the contralateral CA1 area (Table I and Fig. 2). TABLE I EFFECTS OF TETANUS TOXIN M1CROINJECTED INTO THE RAT CA1 HIPPOCAMPAL AREA ON [3H]GABA BINDING SITES. Data represent mean (+ S.E.M.) percentages of [3H]GABA binding sites density calculated for the TT treated side vs the contralateral (untreated) side (n = 6 slices per rat; n = 3 rats per treatment). CAt
DG
GABAA
GABAa
GABAA
GABAB
Tetanus toxin (1000 MLDs) 1 day after 7 days after 10 days after
98.9 + 3.5 81.7"+_2.3 65.1"+4.1
96.2+0.9 103.2+8.2 105.5+2.5
98.1 +2.7 114.1+5.3 86.0+7.5
110.0+8.9 104.8+1.4 116.9___5.1
BSA (300 ng) 7 days after
99.2 + 2.4
101.7_ 2.0
100.7 ___3.0
*P < 0.05 (Student's t-test).
94.8 + 11.6
10
T
A
C
)
C
C
)
B
)
)
D
)
T
Fig. 2. Receptor autoradiographs of GABAA binding to rat brain sections. Transverse cryostat sections (10 ,urn) were incubated with 50 nM [3H]GABAfor 20 rain at 21 23°C. Sections C and D were additionally incubated in 100/~M isoguvacine to obtain background levels. Note in A that a single injection of tetanus toxin (1000 MLDs) produced, 7 days after the treatment, a decrease in GABAA binding in the treated (T) CA! pyramidal layer in comparison to the contralateral (C) side. In B is shown an example of the lack of effect 7 days after treatment with BSA (300 ng//~l; T, treated side; C, control). A time-dependency for the effects on G A B A A binding was also observed. In fact, no changes were induced by T T (1000 M L D s ) 24h after the injection whereas approximately 35 % reduction was observed 10 days after. N o changes were observed in G A B A B site binding (Table I). Treatment with BSA (300 ng//,tl; n = 3 rats) or with neutralized T T (n = 3 rats) did not affect the density (analyses performed 7 days after the injection) o f G A B A A or G A B A B sites. The present experiments indicate that T T is able to produce dose- and time-dependent neuropathological effects when microinjected into the rat CA1 hippocampal area. The neuronal loss was confined to a region very close to the injection site even after the administration o f 2000 M L D s . This confirms its inability to diffuse within the hippocampus, as already demonstrated in experiments in which 125I-labelled T T was used [12]. N o direct evidence has been shown in the present study to indicate that the neuropathological effects induced by T T were accompanied by a reduced inhibitory tone in the hippocampus; however, the results are most likely to be explained on the basis o f an unopposed excitation occurring in the neurons at the site o f injection. In fact, it has been widely demonstrated that TT is able to produce an excitatory focus when applied onto the hippocampus [6, 10], an effect linked to its ability to block the release o f G A B A in this area [3]. This effect has also been demonstrated in other brain regions [4, 5] and in no instance has a direct enhancement o f excitatory transmission been reported for TT (see ref. 11). As a consequence o f the neuronal loss occurring in the C A ! pyramidal layer, the concomitant decrease in the density o f G A B A A binding sites was not unexpected since these are present on the neuronal cell bodies. But quite surprisingly, no significant changes were
11
observed in the binding to GABA~ sites. This might be explained on the basis of a different rate of disappearance, a longer period of time being necessary for the number of GABAB sites to diminish. However, a more attractive hypothesis is that GABAB sites are present on nerve terminals within the pyramidal cell layer rather than on the pyramidal cells. Whilst they are present on pyramidal cell dendrites, they appear to be absent from the cell soma [14]. Their presence on the terminals of Schaffer collaterals and commissural fibres has also been described [7]. Thus, even if the pyramidal cells disappear the GABAB sites would still remain. Unfortunately, the high mortality rate of rats treated with TT did not allow the effects on the GABAB site to be followed for longer periods of time. However, the existence of a very small number of GABAB sites located on the dendrites of pyramidal neurons within the CA1 pyramidal cell layer cannot be excluded and the reduction eventually produced by TT could be masked by sprouting of terminals where the largest number of GABAB sites is present. In conclusion, the present results clearly demonstrate that TT produces neuronal loss and a reduction in GABAA but not GABAB binding sites into the rat hippocampus. Further studies are in progress in our laboratory to clarify the mechanism through which these effects are mediated. Tetanus toxin was a kind gift from Prof. B. Bizzini (Pasteur Institute, Paris). Partial financial support from the Italian Council of Research (CNR, Rome) is gratefully acknowledged. 1 Beneviste, H., Prejer, J., Schousboe, A. and Diemer, N.H., Elevation of the extracellular concentration of glutamate and aspartate in rat hippocampus during transient cerebral ischaemia monitored by intracerebral microdialysis, J. Neurochem., 43 (I 984) 1369-1374. 2 Bowery, N.G., Hudson, A.L. and Price, W., GABAA and GABAB receptor site distribution in the rat central nervous system, Neuroscience, 20 (1987) 365 383. 3 Collingridge, G.L., Thompson, P.A., Davies, J. and Mellanby J., In vitro effect of tetanus toxin on GABA release from rat hippocampal slices, J. Neurochem., 37 (1981 ) 1039-1041. 4 Curtis, D.R., Felix, D., Game, C.J.A. and McCullogh, R.M., Tetanus toxin and the synaptic release of GABA, Brain Res., 51 (1973) 358-362. 5 Davies, J. and Tongroach, P., Tetanus toxin and synaptic inhibition in the substantia nigra and striatum of the rat, J. Physiol. (Lond.), 290 (1979) 23-26. 6 De Sarro, G.B., Bagetta, G. and Nistic6, G., Antagonism by drugs enhancing GABAergic transmission of central effects of tetanus toxin. In G. Nistic6, P. Mastroeni and M. Pitzurra (Eds.), Seventh International Conference on Tetanus, Gangemi, Rome, 1985, pp. 142-156. 7 Dutar, P. and Nicoll, R.A., Pre- and postsynaptic GABAB receptors in the hippocampus have different pharmacological properties, Neuron, 1 (1988) 585-591. 8 Gawade, S., Bon, C. and Bizzini, B., The use of antibody Fab fragments specifically directed to two different complementary parts of the tetanus toxin molecule for studying the mode of action of the toxin, Brain Res., 334 (1985) 139-146. 9 Gill, R., Foster, A.C. and Woodruff, G.N., Systemic administration of MK-801 protects against ischemia-induced hippocampal neurodegeneration in the gerbil, J. Neurosci., 7 (1987) 3343-3349. 10 Mellanby, J., George, G., Robinson, A. and Thompson, P.A., Epileptiform syndrome in rats produced by injecting tetanus toxin into the hippocampus, J. Neurol. Neurosurg. Psychiatry, 40 (1977) 404414. 11 Mellanby, J. and Green, J., How does tetanus toxin act?, Neuroscience, 6 (1981) 281-300. 12 Mellanby, J. and Thompson, P.A., Tetaus toxin in the rat hippocampus, J. Physiol. (Lond.), 269 (1977) 44-45 P.
12 13 Nadler, J.V., 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.), 260 (1976) 538 540. 14 Newberry, N.R. and Nicoll, R.A., Baclofen directly hyperpolarizes hippocampal cells, Nature (Lond.), 308 (1984) 45ff452. 15 Onodera, H., Sato, G. and Kogure, K., Lesions to Schaffer collaterals prevents ischemic death of CAI pyramidal cells, Neurosci. Lett., 68 (I 986) 169- 174. 16 Ottersen, O.P. and Storm-Mathisen, J. In A. Bj6rklund, T. H6kfelt and M.J. Kuhar (Eds.), Handbook of Chemical Neuroanatomy, Vol. 3, Elsevier, Amsterdam, 1984, pp. 141 246. 17 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1981. 18 Rothman, S.M. and Olney, J.W., Glutamate and the pathophysiology of hypoxic-ischaemic brain damage, Ann. Neurol., 19 (1986) 105- 11 I. 19 Simon, R.P., Swan, J.H., Griftith. T. and Meldrum, B.S. Blockade of N-methyl-o-aspartatc receptors may protect against ischemic damage in the brain, Science, 226 (1984) 850 852. 20 Suzuki, R., Yamaguchi, T., Li, C.L. and Klatzo, I., The effects of 5-minute ischemia in mongolian gerbils: I1. Changes of the spontaneous neuronal activity in cerebral cortex and CA~ sector of hippocampus, Acta Neuropathol., 60 (1983) 217 222. 21 Taylor, C.P., How do seizurcs begin'? Clues from hippocampal slices, Trends Neurosci . 11 (1988) 375 378.
