0306-4522/90 $3.00+ 0.00
Neuroscience Vol.38,No. 3,pp.621-627,1990
Pergamon Press plc
Printed in Great Britain
Q 1990 IBRO
TETANUS TOXIN BLOCKS INHIBITION OF GRANULE CELLS fN THE DENTATE GYRUS OF THE URETHANE-ANAESTHETIZED RAT L. E. SUNDSTR~~M and J. H. MELLANBY* University of Oxford, Department of Experimental Psychology, South Parks Road, Oxford OX1 3UD, U.K. Abstract-Field potentials of dentate granule cells in response to stimulation of the perforant path have been studied before and after injecting tetanus toxin (200 mouse LD,, or phosphate-buffered saline in controls) into the hilus of the dentate gyrus of rats under urethane anaesthesia. Within 1 h of toxin injection, the population spike, but not the slope of the excitatory postsynaptic potential, had increased markedly in amplitude and double or treble population spikes appeared in response to perforant path stimulation. Both paired pulse inhibition (15ms interval between conditioning and test stimuli) and commissural inhibition (IO-ms interval) were substantially reduced by the toxin. Neither multiple spikes nor the reduction in inhibition were seen in controls. Apparent inhibition of the excitatory postsynaptic potential, seen with paired stimuli to the perforant path, was not affected by the toxin. At later times after the injections, a progressive increase in the size of the spikes was seen in the controls while in the toxin animals there was often a secondary decrease in size. It is concluded that tetanus toxin can block both feed-back and feed-forward inhibitory components acting on dentate granule cells. The results are discussed with respect to the role of inhibitory processes in the control of epileptogenesis.
Although the animals recover from the overt epilepsy and their electroencephalograms apparently return to normal within about six weeks,” there are very long-lasting, if not permanent, electrophysiological a presynaptic block of inhibition in the spinal cord abnormalities in the hippocampus: both excitation and brainstem.30g49The spastic paralysis characteristic and inhibition may be impaired up to a year after of the disease, tetanus, is thought to be the result In parallel with these physioof the loss of inhibitory control over excitation of toxin injection. 8*2’,34 logical changes, the animals also show behavioural motoneurons. Experimentally, tetanus toxin has been injected directly into the brain and shown to deficits in learning and memory (e.g. Refs 8, 31). block inhibition in the cerebellum and substantia In the present work, the acute effects of the nigra.‘2s’3 Local injections of a minute amount of the toxin have been studied on field potentials recorded toxin into various areas of the brain leads to the from the granule cell layer of the dentate gyrus in establishment of a chronic excitatory focus.2s~29~3’ response to orthodromic stimulation via the perforant Although the overall effect of the toxin in the CNS path. Inhibition has been investigated by preceding is excitatory its action is not specific to inhibitory the stimulus with a conditioning shock either to the transmission: with larger doses and/or longer time perforant path (paired pulse inhibition) or to the it can also block excitatory synapses both centrally contralateral hilus (commissural inhibition). Both and peripherally.3~7~‘5~‘s’22~z4~35~42 The toxin does not feed-back and feed-forward inhibition of granule cells apparently cause death of either hippocampal pyraare well established in the hippocampus.5*6s9J4Firing midal cells or dentate granule cells around its site of granule cells in response to stimulation of the of injection29 and since it does not produce any perforant path activates feed-back inhibition via reduction in glutamate decarboxylase levels in the basket cells, which lie within and below the granule hippocampus4’ it probably does not kill GABAergic cell layer and produce GABA-mediated inhibition on inhibitory cells either. the granule cell soma, and probably also feed-forward When injected into the hippocampus or amygdala, inhibitory components.4~‘6~37~47 Commissural stimutetanus toxin produces a chronic epileptic syndrome lation activates a form of feed-forward inhibition in which the rats show epileptiform activity in the which may also be in part produced via basket electroencephalogram and have intermittent spontancells.“J9 eous motor fits for a period of about three weeks.29*3i In the present experiment, an insulated stainless steel cannula containing the tetanus toxin in solution was used as a recording electrode. It was possible to *To whom correspondence should be addressed. locate the cannula, and hence also the subsequent Abbreviations: EPSP, excitatory postsynaptic potential; PBS, phosphate-buffered saline. toxin injection, in the region just below the granule Tetanus toxin is an exceptionally potent protein neurotoxin whose lethal action in mammals seems to result from its ability to enter the CNS and cause
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cell body layer.27 The toxin is well known to bind rapidly and tightly to nervous tissue36,48and hence it is likely that it would remain well localized to the injection site. Excitation and inhibition of granule cells were investigated before and at intervals after either toxin or buffer (control) injections for periods up to I h. Some of these results have already been presented in abstract form.33.43 EXPERIMENTAL
MELLANBY (ii)
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PROCEDURES
Tetanus toxin was kindly provided by Dr R. 0. Thomson, Wellcome Biotech., Beckenham, Kent. It was assayed by the method of Mellanby et ~1.)~and contained about 2,000,000 mouse LD$mg prOkitI. Male Sprague-Dawley rats bred in the department weighing between 350 and 580 g were anaesthetized by intraperitoneal injection of 1.2g/kg Urethane (20% w/v, 6 ml/kg; Sigma UUOO) and placed in a Kopf stereotaxic instrument. A bipolar concentric stimulating electrode (SNE 100, Rhodes electrodes, U.S.A.) was stereotaxically placed in the entorhinal area at 0.2 mm posterior and 4.4 mm lateral to lambda (atlas of K&rig and Klipell) and 2.2 mm below dura. The recording cannula was lowered into the hippocampus at coordinates 3.6 mm posterior and 1.8 mm lateral from bregma while monitoring field potentials evoked from stimulating the perforant path. The depths of the recording cannula and stimulating electrode was adjusted to maximize the population spike (just below the granule cell body layer). A second stimulating electrode was placed in the contralateral hilus of the dentate gyms to stimulate the commissural fibres at a position homotopic to the recording electrode as described by Douglas et al. I4The recording cannula was made from 22-gauge and 30-gauge stainless steel needles (Cooper Needleworks, U.K.). The smaller 30-gauge needle (300 pm exterior diameter) was insulated to the tip with epoxy resin. Half a microlitre of sodium phosphate-buffered saline (PBS; pH 7.4; 0.3 OsM) or 0.5 ~1 PBS containing 200 mouse LD% tetanus toxin was injected through the cannula using a microdialysis infusion pump (Carnegie Medicin, Sweden) and the effects on field potentials were monitored for up to 7 h. The field potentials were amplified, stored and analysed on-line using a microcomputer system.M (Where multiple population spikes occurred, measurements were taken from the first of the test spikes.) Field potentials were measured in response to single stimuli to the perforant path, (r3OV increasing in 2-V steps (stimulus-response curve). Paired pulse inhibition was elicited by preceding a fixed amplitude test
J-L-
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Fig. 1. Granule cell responses before (i) and after (ii) tetanus toxin injection (200 mouse LD&. (A-E) Obtained from different rats. [A(ii) and C(ii)] Five hours after toxin injection; [B(ii) and D(ii)] 3 h after; [E(ii)] 7 h after. Granule cell field potentials were elicited by a 12-V, 0. I-ms stimulus to the perforant path. Calibrations: vertical 10 mV; horizontal 10 ms. stimulus to the perforant path by 15 ms with a conditioning stimulus to the ipsilateral perforant path increasing in 2-V steps from 2 to 20 V; commissural inhibition was measured in a similar way by preceding the perforant path stimulus by 10 ms with a stimulus to the contralateral hilus. This protocol wasrepeatedfive timesineachratusingadifferent (4,8,12,16, 20 V) test stimulus. Recordings of stimulus-response curves,
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Fig. 2. Paired pulse inhibition of the or tetanus toxin, (2OOmouse LD&. followed 15 ms later by test stimulus before; (iii) control: 1 h after; (iv)
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I
population spike. Typical examples of the effect of buffer (control) Conditioning stimulus to the perforant path (given at 0, 12V) (given at 0, 12 V) to perforant path. (i) Control: before; (ii) toxin: toxin: 1 h after. Calibrations: vertical 10 mV; horizontal 10 ms.
Granule cells of the urethane-anaesthetized
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paired pulse inhibition and commissural inhibition were made I, 3, 5 and 7 h after injecting toxin or buffer. Percentage inhibition was calculated with respect to a mean of six baseline responses to the test shock delivered alone (three before and
three after the series preceded by the conditioning stimuli). RESULTS
In these experiments, 22 rats were used, half of which received injections of buffer and half about 200 (mouse) LDw tetanus toxin into the region of the cell body layer of the dentate granule cells. In nine out of the 11 rats given tetanus toxin, a double or treble population spike was generated in response to a single perforant path stimulus but this was never seen in any of the controls. Field potential responses from five of the toxin-injected rats before and at various times after the toxin injection are shown in Fig. 1 which illustrates the multiple spiking. Paired pulse (15 ms separation; Fig. 2) and commissural (10 ms separation; Fig. 3) inhibition were effective in all the rats before injection and were maintained in the control rats after the buffer injection for the duration of the experiment (up to 7 h). In Figs 4 and 5, the mean values calculated for the percentage inhibition in the control and toxin-treated rats are compared. Whereas before the injections, both groups of rats showed around 90% or greater Mean
Stimulus
Response
MELLANBY
inhibition with both types of conditioning stimulation, 1.5 h after the toxin injection the inhibition in both cases was significantly reduced. In this experimental protocol, commissural inhibition was always investigated 30 min after the paired pulse stimulation series, and hence it is not possible to say whether one or other inhibition was blocked first. By 3-3.5 h after injection, the means for both types of inhibition were reduced substantially. [Paired pulse inhibition was also investigated at 25 ms separation and similar results (not shown) were obtained.] With paired pulse stimulation of the perforant path, a reduction of the slope of the population excitatory postsynaptic potential (EPSP) accompanied the inhibition of the spike and amounted to a maximal reduction of 40%. No comparable inhibition of the population EPSP occurred with commissurally activated inhibition of the population spike. With paired pulse stimulation of the perforant path, the inhibition of the EPSP appeared to increase 1 h after toxin injection. This was not a true increase in inhibition but was related to the increase in the size of the conditioning population spike since when the inhibition was expressed relative to the size of the conditioning spike it was unchanged. By 1 h after the toxin injection, there was a marked increase in the size of the orthodromically activated
Curves
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Fig. 6. Effect of tetanus toxin on the size of the orthodromically activated population spike. The dentate granule cell population spikes were elicited by stimulation of the perforant path before and 1 h after local injection of 200 (mouse) LD~ tetanus toxin (or buffer in controls).
+
Fig. 7. Appearance of an excitatory response of granule cells to stimulation of contralateral hilus 2 h after tetanus toxin. (i) (For comparison.) Response to 20-V stimulus to perforant path. (ii) Response to 20-V stimulus to contralateral hilus. (iii) Response to 30-V stimulus to contralateral hilus. Calibrations: vertical 10 mV; horizontal 10 ms.
Granule cells of the urethane-anaesthetized
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et ~1.~~have recorded feed-forward (i.e. in the absence of a population spike) paired pulse inhibition of the extracellularly recorded EPSP at longer separations (>50ms). Also, Oliver and Miller’s records3’ show what could be feed-back or feed-forward inhibition (with a population spike) of the extracellularly recorded EPSP at 20 ms separation. The mechanism and/or the site of generation of the early inhibition of the EPSP seen in the present experiments must be different from that for inhibition of the population spike since it is unaffected by the toxin at a time when inhibition of the spike is blocked. It is likely that the toxin remains mainly localized at or below the cell body layer where it is injected since it is known to bind rapidly and tightly to nervous tissue. Thus a dendritic or presynaptic site of generation of this “inhibition” is supported. The pronounced increase in the size of population spikes during the first hour after toxin injection was not accompanied by an increase in the EPSP slope. This apparent increase in excitability of the granule cells could result from a block of tonic inhibition or of feed-forward inhibitiongJ6 (or of course from changes in the intrinsic properties of the granule cells). A similar increase in spike size occurred more slowly in controls, becoming evident at times later than 2-3 h after the buffer injections. Such an increase in response in control animals has been seen in other experiments (not shown here) where either a DISCUSSION small wire or glass micropipette recording electrode The results show clearly that within 1-3 h of the was used, and also in experiments where very local injection, under urethane anaesthesia, of about infrequent test stimulations were made. It thus seems 200 (mouse) LD, of tetanus toxin in the dentate hilus, likely that this increase is related to the prolonged there is a marked reduction (and in many cases procedure under urethane anaesthesia rather than to complete abolition) of both paired pulse and commisthe nature of the recording cannula or the injection surally activated inhibition of the granule cell popuof fluid from it or to the multiple electrical stimulation spike. At the same time, the cells develop lations. This phenomenon has previously been redouble or treble population spikes in response to a ported by others.’ single stimulus to the perforant path and in at least one In contrast to the findings in controls, in half of case, an excitatory response to commissural stimuthe toxin-injected animals, there was a late (3+ h) lation occurred where previously there had been only decrease in both the population spike and the EPSP. inhibition apparent. It seems therefore that the toxin This suggests a decrease in excitatory drive and could can block both recurrent and feed-forward inhibition be a result of the ability of the toxin, given a longer of the granule cell population spike. Feed-back inhitime (or a higher dose) to block excitation as well bition involves basket cells and since these employ as inhibition in the mammalian CNS.23,24 Since, GABA as a transmitter it would be expected to be however, the excitatory synapses from the perforant blocked by tetanus toxin. Any component of feedpath are on the dendrites of the granule cells, this forward inhibition which also employs basket cells explanation would require that some toxin leaked would therefore also be blocked. It is, however, likely back up the needle track .3’ It has been shown prethat other types of interneuronsm are also involved viously in the spinal cord in uivo that, with longer time in commissurally activated feed-forward inhibition. or high doses, tetanus toxin can block excitation as We have not yet investigated whether tetanus toxin well as inhibition23 and recently published experiments also blocks the late (GABA,)-mediated inhibition of in vitro on hippocampal slices have shown a similar granule cell~.~~*~* effect.‘O The apparent inhibition of the population EPSP Multiple population spikes occurred exclusively in which we observed with paired pulse stimulation at the toxin-injected group of rats. The ability of tetanus 15ms pulse interval could be either presynaptic toxin to generate multiple population spikes has pre“habituation” of the excitatory [email protected]’or viously been demonstrated in vitro in CA 1 pyramidal could result from inhibition acting on the dendrites,4x38 cells in hippocampal slices” in response to stimulation shunting the EPSP, as recorded at the soma. Rausche of the Schaffer collaterals. As many as 10, and spike. Mean stimulus-response curves obtained before and 1 h after either control or toxin injections are shown in Fig. 6. It can be seen that in the toxin-injected group there was a marked increase in the size of response to all stimulus strengths. A much less pronounced increase was seen in the control group. There was no concomitant increase in the EPSP. After the first hour from the injection, the controls usually showed a steady increase in spike size (but not EPSP slope) which was maintained for as long as 7 h. In contrast, in the toxin rats there was sometimes a marked decrease in the response at longer times and this decrease involved both the size of the population spike and the slope of the population EPSP. In four out of the eight toxin rats in which the experiment was carried on for 7 h, there was such a reduction. However, in one rat there was no change in spike size and in three others there was an increase; but in two of these there was no block of inhibition either and so perhaps the toxin had not been successfully injected. In the third, inhibition was blocked and in addition multiple spiking occurred. An interesting chance observation was made in one of the toxin rats: 2 h after the injection, commissural stimulation apparently produced a granule cell population response, and with the largest stimulus (30V) a double population spike was elicited (Fig. 7).
population
626
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SU~DSTR~~ and J. H. MELLANBV
occasionally 50 population spikes were observed in the CA1 in response to stimulation, This contrasts with the present observations in dentate gyrus where only double or treble spikes were seen. Disinhibition by other means, e.g. ~nici~lin or bicuculline application, also reveals that CA1 and CA3 pyramidal cells have a greater inherent tendency to fire repetitively when disinhibited than do granule cells of the dentate gyrus.‘6.S0 In our previous experiments where tetanus toxin has been used to produce a chronic epileptiform focus in the hippo~mpus (e.g. see Refs 8,29), we have used a much lower dose of toxin (S-10 mouse LD5~, compared with 200 mouse LD,, in the present acute experiments). However, in an original pilot experiment,28 a dose comparable with that used here produced motor fits within a day and major convulsions the day after. it would appear that the Mock of inhibition seen here is the first step in the chain of
events which leads to the development of fits. With much lower doses, where fits take several days to appear, presumably the block of inhibition is of slower onset. Continuous electroencephaIographic
recordings with electrodes implanted in the hippocampus have revealed that the earliest abnormal electrical output is of short bursts of spiking.” These lengthen and over the next day or two lead up to seizure discharges fasting i or 2min which may be accompanied by the motor components of a typical “limbic” fit (complex partial seizures). CONCLUSION
While it has been shown in u&o in ~p~rnpal slices that seizure activity can occur on activation of excitation in the absence of any reduction of inhibition,” the present in uioo results give an example of a situation where block of inhibition and the production of multiple spikes occur simultaneously and are probably causally related. .&knowledgements---We are grateful for the financial support of the British Epilepsy Research Foundation, CIBAGEIGY pharmaceuticals and St Hildas College, Oxford. We would like to thank Dr T. V. P. Bliss for teaching us the recording techniques and K.E. Sundstrom for help in designing the recording apparatus.
REFERENCES 1. Abraham W. C. and Mason S. E. (1988) Effects of the NMDA receptor/channel
antagonists CPP and MK801 on hippocampal field potentials and long-term potentiation in anaesthetized rabbits. Brain Res. 462, 40-.46. 2. Alger B. E. and Teyler Y. J. (1976) Long term and short term plasticity in the CAI, CA3 and dentate regions of the rat hippocampal slice. Brain Res. 110, 463-480. 3. Ambache N., Morgan R. S. and Payling Wright Ci. (1948) The action of tetanus toxin on the rabbit’s iris. J. Physio(., Land. 107,45-53. 4. Andersen P., Dingledine R., Gjerstad L., Langmoen I. A. and Mosfeldt Laursen A. M. (1980) Two different responses of hippocampal pyramidal cells to application of gamma-aminobutyric acid. J. Physiol., Land. 305, 279-296. 5. Andersen P., Eccles J. C. and Loyning Y. (1963) Recurrent inhibition in the hippocampus with identification of the inhibitory cell and its synapses. Nature 1% 540-542. 6. Andersen P., Eccles J. C. and Liiyning Y. (1964) Pathway of postsynaptic inhibition in the hippocampus. J. Neurophysiol. 27, 608-619. 7. Bevan S. and Wendon L. M. B. (1984) A study of the action of tetanus toxin on rat soleus neuromuscular junctions. J. Physiol., Land. t48, J-17.
8. Brace H. M., Jefferys J. G. R. and Mellanby J. H. (1985) Long-term changes in hippocampd physiology and learning ability of rats after intrahippocampal tetanus toxin, J. Physiol., Lond. 368, 343-357. 9. Buzsaki G. and Eidelberg E. (1981) Commissural projections to the dentate gyrus of the rat: evidence for feed-forward inhibition. Brain Res. 230, 346-350. 10. Calabresi P., Beneditti M., Mercuri N. B. and Bernardi G. (1989) Selective depression of synaptic transmission by tetanus toxin: a comparative study on bippocampal and neostriatal slices. Neuroscience 30, 663-670. 11. Colljn~~dge G. L. and Herron C. E. (1985) Effects of tetanus toxin on GABA synapses in the mamm~ian central nervous system. In Proceedings the Seventh International Conference on Tetanus (eds Nistico G., Maestroani P. and Pizzura M.), pp. 127-142. Rome. 12. Curtis D. R., Felix D., Game C. J. A. and McCulloch R. M. (1973) Tetanus toxin and the synaptic release of GABA.
of
Brain Res. 51, 358-362. 13. Davies J. and Tongroach P. (1979) Tetanus toxin and synaptic inhibition in the substantia nigra and striatum of the rat. J. Physiol., Lond. 290, 23-36. 14. Douglas R. M., McNaughton B. L. and Goddard G. V. (I983) Commissural inbibition and facilitation of granule ceil discharge in fascia dentata. J. camp. Neural. 219, 285-294.
15. Duchen L. W. and Tonge D. A. (1973) The effects of tetanus toxin on neuromuscular transmission and on the morphology of motor endplates in slow and fast skeletal muscle of the mouse. J. Physioi., Land. 228, 157-172. 16. Fricke R. and Prince D. A. (1984) Electrophysiology of dentate granule cells. J. Neurophysiol. 51, 195-209. 17. Frotscher M. and Zimmer J. (1983) Commissural fibres terminate on non-pyramidal neurons in the guinea pig hippocampus-a combined golgi/E.M. study. Brain Res. 265, 289-293. 18. Hatxrmann F., Dreyer F. and Bigalke H. (1980) Tetanus toxin blocks neuromuscular transmission in uirm like botulinum A toxin. ~~nyn-Schm~edeberg’s Arch. Pharmac. 311 33-40. 19. Hawkins C. A. and Mellanby J. H. (1987) Limbic epilepsy induced by tetanus toxin: a lon~tudinal ele~troen#phalographic study. Epiiepsia 28, 431-444. 20. Higashima M. (1988) Inhibitory processes in development of seizure activity in hippocampal slices. Expl Brain Res. 12, 37-44. 21, Jefferys J. G. R. and Williams S. F. (1987) Physiological and behavioural consequences of seizures induced in rats by intrahippocampal tetanus toxin. Brain 110, 517-532.
Granule cells of the urethane-anaesthetized
rat
627
22. Kaeser H. E. and Saner A. (1970) The effect of tetanus toxin on neuromuscular transmission. Eur. Neural. $193-205. 23. Kanda K. and Takano K. (1983) Effects of tetanus toxin on the excitatory and inhibitory postsynaptic potentials in the cat motoneurone. J. Physiol., Land. 335, 319-334. 24. Kryzhanovsky G. N. (1960) Functional changes in the central nervous system in tetanus and the mode of action of the tetanus toxin, III: the central and peripheral action of the tetanus toxin. Bull. exp. Biol. Med. 49, 34-39. 25. Kryzhanovsky G. N., Rekhtman M. B. and Konikov B. A. (1976) Experimental photogenic epilepsy. In The Nervous System, Vol. 4, p. 363. ICRS, Medical Science, Moscow. 26. McNaughton B. L. (1980) Evidence for two physiologically distinct perforant path-ways to the fascia dentata. Bruin Res. 199, l-19. 27. McNaughton B. L. and Barnes C. A. (1977) Physiological identification and analysis of dentate granule cell responses to stimulation of the medial and lateral perforant paths in the rat. J. camp. Neural. 175, 430-454. 28. Mellanby J. H. and George G. (1975) Cephalic tetanus in rats. In Proceedings of the Fourth International Conference on Teronus, Foundation Merieux, Lyon, pp. 201-204. 29. Mellanby J. H., George G., Robinson A. and Thompson P. A. (1977) Epileptiform syndrome in rats produced by injecting tetanus toxin into the hippocampus. J. Neural. Neurosurg. Psychiat. 40, 404-414. 30. Mellanby J. H. and Green G. (1981) How does tetanus toxin act? Neuroscience 6, 281-300. 31. Mellanby J. H., Hawkins C. A., Mellanby H., Rawlins J. N. P. and Impey M. E. (1984) Tetanus toxin as a tool for studying epilepsy. J. Physiol., Paris 79, 207-215. 32. Mellanby J. H., Mellanby H., Pope D. and Van Heyningen W. E. (1968) Ganglioside as a prophylactic agent in experimental tetanus in mice. J. gen. Microbial. 54, 161-168. 33. Mellanby J. H. and Sundstriim L. E. (1989) Tetanus toxin and inhibitory mechanisms in the dentate gyrus of the anaesthetized rat. J. Physiol., Land. 413, 33~. 34. Mellanby J. H., Sundstrom L. E. and Kindling (1988) Behaviour and memory, In The Clinical Relevance of Kindling. (eds Bolwig T. G. and Trimble M. R.), pp. 103-113. John Wiley, Chichester. 35. Mellanby J. H. and Thompson P. A. (1972) The effects of tetanus toxin at the neuromuscular junction in the gold fish. J. Physiol. Lond. 224, 407-419. 36. Mellanby J. H. and Whittaker V. P. (1968) The fixation of tetanus toxin by synaptic membranes. J. Neurochem. 15, 205-208. 37. Oliver M. W. and Miller J. J. (1985) Alterations of inhibitory processes in the dentate gyrus following kindling-induced epilepsy. Expi Brain Res. 57, 443-447. 38. Rausche G., Sarvey J. M. and Heinemann U. (1988) Lowering extracellular calcium reverses paired pulse habituation into facilitation and removes a late IPSP. Neurosci. Left 88, 275-280. 39. Seress L. and Ribak C. E. (1984) Direct commissural connections to the basket cells of the hippocampal dentate gyrus: anatomical evidence for feed-forward inhibition. J. Neurocytol. 13, 215-225. 40. Somogyi P., Nunzi M. G., Gorio A. and Smith A. D. (1983) A new type of specific intemeuron in the monkey hippocampus forming synapses exclusively with the axon initial segments of pyramidal cells. Brain Res. 259, 137-142. 41. Stanley M. and Mellanby J. H. (1989) Glutamic acid decarboxylase (GAD) in rat hippocampus after recovery from tetanus toxin-induced epileptiform syndrome. Neurosci. Left. Suppl. 36, S55. 42. Sugimoto N., Matsuda M., Ohunuki Y., Nakayama T. and Imai K. (1982) Neuromuscular blocking in acutely tetanus intoxicated mice. Biken J. 25, 21-28. 43. Sundstrom L. E. and Mellanby J. H. (1989) Acute effects of tetanus toxin on granule cells of the dentate gyrus of the rat in vivo. In Proceedings of the The Eighth International Conference on Tetanus (eds Nistico G., Bizzini B., Bytchenko
B. and Thiau R.), pp. 149-153. Pythagora Press, Rome. 44. Sundstriim L. E. (1988) An electrophysiological study of tetanus toxin induced hippocampal epilepsy. D. Phil Thesis, Oxford University. 45. Takano K., Kirchner F., Gremmelt A., Matsuda M., Ozutsumi K. and Sugimoto N. (1989) Blocking effects of tetanus toxin and its fragment (A-B) on the excitatory and inhibitory synapses of the spinal motoneuron in the cat. Toxicon 27, 385-392. 46. Teyler T. J. and Alger B. E. (1976) Monosynaptic in vitro. Brain Res. 115, 413-425.
habituation in the vertebrate forebrain: the dentate gyrus examined
47. Thalmann R. H. and Ayala G. F. (1982) A late increase in potassium conductance follows synaptic stimulation of granule neurons of the dentate gyrus. Neurosci. Lett. 29, 243-248. 48. Van Heyningen W. E. (1974) Gangliosides as membrane receptors for tetanus toxin, cholera toxin and serotonin. Nature 249,415-417. 49. Wellhijner H. H. (1982) Tetanus neurotoxin. Rev. Physiol. Biochem. Pharmac. 93, l-68. 50. Wheal H. V., Ashwood T. J. and Lancaster B. (1984) A comparative in vitro study of the kainic acid lesioned and bicuculline treated hippocampus. In The Electrophysiology of Epilepsy (eds Schwarzkroin P. A. and Wheal H. V.), pp. 173-200. Academic Press, London. 51. White W. F., Nadler J. V. and Cotman C. W. (1979) An analysis of short term plasticity at the perforant path-granule cell synapse. Bruin Res. 178, 41-53. (Accepted
29 January 1990)
Neuroscience Vol.38,No. 3,pp.621-627,1990
Pergamon Press plc
Printed in Great Britain
Q 1990 IBRO
TETANUS TOXIN BLOCKS INHIBITION OF GRANULE CELLS fN THE DENTATE GYRUS OF THE URETHANE-ANAESTHETIZED RAT L. E. SUNDSTR~~M and J. H. MELLANBY* University of Oxford, Department of Experimental Psychology, South Parks Road, Oxford OX1 3UD, U.K. Abstract-Field potentials of dentate granule cells in response to stimulation of the perforant path have been studied before and after injecting tetanus toxin (200 mouse LD,, or phosphate-buffered saline in controls) into the hilus of the dentate gyrus of rats under urethane anaesthesia. Within 1 h of toxin injection, the population spike, but not the slope of the excitatory postsynaptic potential, had increased markedly in amplitude and double or treble population spikes appeared in response to perforant path stimulation. Both paired pulse inhibition (15ms interval between conditioning and test stimuli) and commissural inhibition (IO-ms interval) were substantially reduced by the toxin. Neither multiple spikes nor the reduction in inhibition were seen in controls. Apparent inhibition of the excitatory postsynaptic potential, seen with paired stimuli to the perforant path, was not affected by the toxin. At later times after the injections, a progressive increase in the size of the spikes was seen in the controls while in the toxin animals there was often a secondary decrease in size. It is concluded that tetanus toxin can block both feed-back and feed-forward inhibitory components acting on dentate granule cells. The results are discussed with respect to the role of inhibitory processes in the control of epileptogenesis.
Although the animals recover from the overt epilepsy and their electroencephalograms apparently return to normal within about six weeks,” there are very long-lasting, if not permanent, electrophysiological a presynaptic block of inhibition in the spinal cord abnormalities in the hippocampus: both excitation and brainstem.30g49The spastic paralysis characteristic and inhibition may be impaired up to a year after of the disease, tetanus, is thought to be the result In parallel with these physioof the loss of inhibitory control over excitation of toxin injection. 8*2’,34 logical changes, the animals also show behavioural motoneurons. Experimentally, tetanus toxin has been injected directly into the brain and shown to deficits in learning and memory (e.g. Refs 8, 31). block inhibition in the cerebellum and substantia In the present work, the acute effects of the nigra.‘2s’3 Local injections of a minute amount of the toxin have been studied on field potentials recorded toxin into various areas of the brain leads to the from the granule cell layer of the dentate gyrus in establishment of a chronic excitatory focus.2s~29~3’ response to orthodromic stimulation via the perforant Although the overall effect of the toxin in the CNS path. Inhibition has been investigated by preceding is excitatory its action is not specific to inhibitory the stimulus with a conditioning shock either to the transmission: with larger doses and/or longer time perforant path (paired pulse inhibition) or to the it can also block excitatory synapses both centrally contralateral hilus (commissural inhibition). Both and peripherally.3~7~‘5~‘s’22~z4~35~42 The toxin does not feed-back and feed-forward inhibition of granule cells apparently cause death of either hippocampal pyraare well established in the hippocampus.5*6s9J4Firing midal cells or dentate granule cells around its site of granule cells in response to stimulation of the of injection29 and since it does not produce any perforant path activates feed-back inhibition via reduction in glutamate decarboxylase levels in the basket cells, which lie within and below the granule hippocampus4’ it probably does not kill GABAergic cell layer and produce GABA-mediated inhibition on inhibitory cells either. the granule cell soma, and probably also feed-forward When injected into the hippocampus or amygdala, inhibitory components.4~‘6~37~47 Commissural stimutetanus toxin produces a chronic epileptic syndrome lation activates a form of feed-forward inhibition in which the rats show epileptiform activity in the which may also be in part produced via basket electroencephalogram and have intermittent spontancells.“J9 eous motor fits for a period of about three weeks.29*3i In the present experiment, an insulated stainless steel cannula containing the tetanus toxin in solution was used as a recording electrode. It was possible to *To whom correspondence should be addressed. locate the cannula, and hence also the subsequent Abbreviations: EPSP, excitatory postsynaptic potential; PBS, phosphate-buffered saline. toxin injection, in the region just below the granule Tetanus toxin is an exceptionally potent protein neurotoxin whose lethal action in mammals seems to result from its ability to enter the CNS and cause
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cell body layer.27 The toxin is well known to bind rapidly and tightly to nervous tissue36,48and hence it is likely that it would remain well localized to the injection site. Excitation and inhibition of granule cells were investigated before and at intervals after either toxin or buffer (control) injections for periods up to I h. Some of these results have already been presented in abstract form.33.43 EXPERIMENTAL
MELLANBY (ii)
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PROCEDURES
Tetanus toxin was kindly provided by Dr R. 0. Thomson, Wellcome Biotech., Beckenham, Kent. It was assayed by the method of Mellanby et ~1.)~and contained about 2,000,000 mouse LD$mg prOkitI. Male Sprague-Dawley rats bred in the department weighing between 350 and 580 g were anaesthetized by intraperitoneal injection of 1.2g/kg Urethane (20% w/v, 6 ml/kg; Sigma UUOO) and placed in a Kopf stereotaxic instrument. A bipolar concentric stimulating electrode (SNE 100, Rhodes electrodes, U.S.A.) was stereotaxically placed in the entorhinal area at 0.2 mm posterior and 4.4 mm lateral to lambda (atlas of K&rig and Klipell) and 2.2 mm below dura. The recording cannula was lowered into the hippocampus at coordinates 3.6 mm posterior and 1.8 mm lateral from bregma while monitoring field potentials evoked from stimulating the perforant path. The depths of the recording cannula and stimulating electrode was adjusted to maximize the population spike (just below the granule cell body layer). A second stimulating electrode was placed in the contralateral hilus of the dentate gyms to stimulate the commissural fibres at a position homotopic to the recording electrode as described by Douglas et al. I4The recording cannula was made from 22-gauge and 30-gauge stainless steel needles (Cooper Needleworks, U.K.). The smaller 30-gauge needle (300 pm exterior diameter) was insulated to the tip with epoxy resin. Half a microlitre of sodium phosphate-buffered saline (PBS; pH 7.4; 0.3 OsM) or 0.5 ~1 PBS containing 200 mouse LD% tetanus toxin was injected through the cannula using a microdialysis infusion pump (Carnegie Medicin, Sweden) and the effects on field potentials were monitored for up to 7 h. The field potentials were amplified, stored and analysed on-line using a microcomputer system.M (Where multiple population spikes occurred, measurements were taken from the first of the test spikes.) Field potentials were measured in response to single stimuli to the perforant path, (r3OV increasing in 2-V steps (stimulus-response curve). Paired pulse inhibition was elicited by preceding a fixed amplitude test
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Fig. 1. Granule cell responses before (i) and after (ii) tetanus toxin injection (200 mouse LD&. (A-E) Obtained from different rats. [A(ii) and C(ii)] Five hours after toxin injection; [B(ii) and D(ii)] 3 h after; [E(ii)] 7 h after. Granule cell field potentials were elicited by a 12-V, 0. I-ms stimulus to the perforant path. Calibrations: vertical 10 mV; horizontal 10 ms. stimulus to the perforant path by 15 ms with a conditioning stimulus to the ipsilateral perforant path increasing in 2-V steps from 2 to 20 V; commissural inhibition was measured in a similar way by preceding the perforant path stimulus by 10 ms with a stimulus to the contralateral hilus. This protocol wasrepeatedfive timesineachratusingadifferent (4,8,12,16, 20 V) test stimulus. Recordings of stimulus-response curves,
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Fig. 2. Paired pulse inhibition of the or tetanus toxin, (2OOmouse LD&. followed 15 ms later by test stimulus before; (iii) control: 1 h after; (iv)
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I
population spike. Typical examples of the effect of buffer (control) Conditioning stimulus to the perforant path (given at 0, 12V) (given at 0, 12 V) to perforant path. (i) Control: before; (ii) toxin: toxin: 1 h after. Calibrations: vertical 10 mV; horizontal 10 ms.
Granule cells of the urethane-anaesthetized
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paired pulse inhibition and commissural inhibition were made I, 3, 5 and 7 h after injecting toxin or buffer. Percentage inhibition was calculated with respect to a mean of six baseline responses to the test shock delivered alone (three before and
three after the series preceded by the conditioning stimuli). RESULTS
In these experiments, 22 rats were used, half of which received injections of buffer and half about 200 (mouse) LDw tetanus toxin into the region of the cell body layer of the dentate granule cells. In nine out of the 11 rats given tetanus toxin, a double or treble population spike was generated in response to a single perforant path stimulus but this was never seen in any of the controls. Field potential responses from five of the toxin-injected rats before and at various times after the toxin injection are shown in Fig. 1 which illustrates the multiple spiking. Paired pulse (15 ms separation; Fig. 2) and commissural (10 ms separation; Fig. 3) inhibition were effective in all the rats before injection and were maintained in the control rats after the buffer injection for the duration of the experiment (up to 7 h). In Figs 4 and 5, the mean values calculated for the percentage inhibition in the control and toxin-treated rats are compared. Whereas before the injections, both groups of rats showed around 90% or greater Mean
Stimulus
Response
MELLANBY
inhibition with both types of conditioning stimulation, 1.5 h after the toxin injection the inhibition in both cases was significantly reduced. In this experimental protocol, commissural inhibition was always investigated 30 min after the paired pulse stimulation series, and hence it is not possible to say whether one or other inhibition was blocked first. By 3-3.5 h after injection, the means for both types of inhibition were reduced substantially. [Paired pulse inhibition was also investigated at 25 ms separation and similar results (not shown) were obtained.] With paired pulse stimulation of the perforant path, a reduction of the slope of the population excitatory postsynaptic potential (EPSP) accompanied the inhibition of the spike and amounted to a maximal reduction of 40%. No comparable inhibition of the population EPSP occurred with commissurally activated inhibition of the population spike. With paired pulse stimulation of the perforant path, the inhibition of the EPSP appeared to increase 1 h after toxin injection. This was not a true increase in inhibition but was related to the increase in the size of the conditioning population spike since when the inhibition was expressed relative to the size of the conditioning spike it was unchanged. By 1 h after the toxin injection, there was a marked increase in the size of the orthodromically activated
Curves
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Fig. 6. Effect of tetanus toxin on the size of the orthodromically activated population spike. The dentate granule cell population spikes were elicited by stimulation of the perforant path before and 1 h after local injection of 200 (mouse) LD~ tetanus toxin (or buffer in controls).
+
Fig. 7. Appearance of an excitatory response of granule cells to stimulation of contralateral hilus 2 h after tetanus toxin. (i) (For comparison.) Response to 20-V stimulus to perforant path. (ii) Response to 20-V stimulus to contralateral hilus. (iii) Response to 30-V stimulus to contralateral hilus. Calibrations: vertical 10 mV; horizontal 10 ms.
Granule cells of the urethane-anaesthetized
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et ~1.~~have recorded feed-forward (i.e. in the absence of a population spike) paired pulse inhibition of the extracellularly recorded EPSP at longer separations (>50ms). Also, Oliver and Miller’s records3’ show what could be feed-back or feed-forward inhibition (with a population spike) of the extracellularly recorded EPSP at 20 ms separation. The mechanism and/or the site of generation of the early inhibition of the EPSP seen in the present experiments must be different from that for inhibition of the population spike since it is unaffected by the toxin at a time when inhibition of the spike is blocked. It is likely that the toxin remains mainly localized at or below the cell body layer where it is injected since it is known to bind rapidly and tightly to nervous tissue. Thus a dendritic or presynaptic site of generation of this “inhibition” is supported. The pronounced increase in the size of population spikes during the first hour after toxin injection was not accompanied by an increase in the EPSP slope. This apparent increase in excitability of the granule cells could result from a block of tonic inhibition or of feed-forward inhibitiongJ6 (or of course from changes in the intrinsic properties of the granule cells). A similar increase in spike size occurred more slowly in controls, becoming evident at times later than 2-3 h after the buffer injections. Such an increase in response in control animals has been seen in other experiments (not shown here) where either a DISCUSSION small wire or glass micropipette recording electrode The results show clearly that within 1-3 h of the was used, and also in experiments where very local injection, under urethane anaesthesia, of about infrequent test stimulations were made. It thus seems 200 (mouse) LD, of tetanus toxin in the dentate hilus, likely that this increase is related to the prolonged there is a marked reduction (and in many cases procedure under urethane anaesthesia rather than to complete abolition) of both paired pulse and commisthe nature of the recording cannula or the injection surally activated inhibition of the granule cell popuof fluid from it or to the multiple electrical stimulation spike. At the same time, the cells develop lations. This phenomenon has previously been redouble or treble population spikes in response to a ported by others.’ single stimulus to the perforant path and in at least one In contrast to the findings in controls, in half of case, an excitatory response to commissural stimuthe toxin-injected animals, there was a late (3+ h) lation occurred where previously there had been only decrease in both the population spike and the EPSP. inhibition apparent. It seems therefore that the toxin This suggests a decrease in excitatory drive and could can block both recurrent and feed-forward inhibition be a result of the ability of the toxin, given a longer of the granule cell population spike. Feed-back inhitime (or a higher dose) to block excitation as well bition involves basket cells and since these employ as inhibition in the mammalian CNS.23,24 Since, GABA as a transmitter it would be expected to be however, the excitatory synapses from the perforant blocked by tetanus toxin. Any component of feedpath are on the dendrites of the granule cells, this forward inhibition which also employs basket cells explanation would require that some toxin leaked would therefore also be blocked. It is, however, likely back up the needle track .3’ It has been shown prethat other types of interneuronsm are also involved viously in the spinal cord in uivo that, with longer time in commissurally activated feed-forward inhibition. or high doses, tetanus toxin can block excitation as We have not yet investigated whether tetanus toxin well as inhibition23 and recently published experiments also blocks the late (GABA,)-mediated inhibition of in vitro on hippocampal slices have shown a similar granule cell~.~~*~* effect.‘O The apparent inhibition of the population EPSP Multiple population spikes occurred exclusively in which we observed with paired pulse stimulation at the toxin-injected group of rats. The ability of tetanus 15ms pulse interval could be either presynaptic toxin to generate multiple population spikes has pre“habituation” of the excitatory [email protected]’or viously been demonstrated in vitro in CA 1 pyramidal could result from inhibition acting on the dendrites,4x38 cells in hippocampal slices” in response to stimulation shunting the EPSP, as recorded at the soma. Rausche of the Schaffer collaterals. As many as 10, and spike. Mean stimulus-response curves obtained before and 1 h after either control or toxin injections are shown in Fig. 6. It can be seen that in the toxin-injected group there was a marked increase in the size of response to all stimulus strengths. A much less pronounced increase was seen in the control group. There was no concomitant increase in the EPSP. After the first hour from the injection, the controls usually showed a steady increase in spike size (but not EPSP slope) which was maintained for as long as 7 h. In contrast, in the toxin rats there was sometimes a marked decrease in the response at longer times and this decrease involved both the size of the population spike and the slope of the population EPSP. In four out of the eight toxin rats in which the experiment was carried on for 7 h, there was such a reduction. However, in one rat there was no change in spike size and in three others there was an increase; but in two of these there was no block of inhibition either and so perhaps the toxin had not been successfully injected. In the third, inhibition was blocked and in addition multiple spiking occurred. An interesting chance observation was made in one of the toxin rats: 2 h after the injection, commissural stimulation apparently produced a granule cell population response, and with the largest stimulus (30V) a double population spike was elicited (Fig. 7).
population
626
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SU~DSTR~~ and J. H. MELLANBV
occasionally 50 population spikes were observed in the CA1 in response to stimulation, This contrasts with the present observations in dentate gyrus where only double or treble spikes were seen. Disinhibition by other means, e.g. ~nici~lin or bicuculline application, also reveals that CA1 and CA3 pyramidal cells have a greater inherent tendency to fire repetitively when disinhibited than do granule cells of the dentate gyrus.‘6.S0 In our previous experiments where tetanus toxin has been used to produce a chronic epileptiform focus in the hippo~mpus (e.g. see Refs 8,29), we have used a much lower dose of toxin (S-10 mouse LD5~, compared with 200 mouse LD,, in the present acute experiments). However, in an original pilot experiment,28 a dose comparable with that used here produced motor fits within a day and major convulsions the day after. it would appear that the Mock of inhibition seen here is the first step in the chain of
events which leads to the development of fits. With much lower doses, where fits take several days to appear, presumably the block of inhibition is of slower onset. Continuous electroencephaIographic
recordings with electrodes implanted in the hippocampus have revealed that the earliest abnormal electrical output is of short bursts of spiking.” These lengthen and over the next day or two lead up to seizure discharges fasting i or 2min which may be accompanied by the motor components of a typical “limbic” fit (complex partial seizures). CONCLUSION
While it has been shown in u&o in ~p~rnpal slices that seizure activity can occur on activation of excitation in the absence of any reduction of inhibition,” the present in uioo results give an example of a situation where block of inhibition and the production of multiple spikes occur simultaneously and are probably causally related. .&knowledgements---We are grateful for the financial support of the British Epilepsy Research Foundation, CIBAGEIGY pharmaceuticals and St Hildas College, Oxford. We would like to thank Dr T. V. P. Bliss for teaching us the recording techniques and K.E. Sundstrom for help in designing the recording apparatus.
REFERENCES 1. Abraham W. C. and Mason S. E. (1988) Effects of the NMDA receptor/channel
antagonists CPP and MK801 on hippocampal field potentials and long-term potentiation in anaesthetized rabbits. Brain Res. 462, 40-.46. 2. Alger B. E. and Teyler Y. J. (1976) Long term and short term plasticity in the CAI, CA3 and dentate regions of the rat hippocampal slice. Brain Res. 110, 463-480. 3. Ambache N., Morgan R. S. and Payling Wright Ci. (1948) The action of tetanus toxin on the rabbit’s iris. J. Physio(., Land. 107,45-53. 4. Andersen P., Dingledine R., Gjerstad L., Langmoen I. A. and Mosfeldt Laursen A. M. (1980) Two different responses of hippocampal pyramidal cells to application of gamma-aminobutyric acid. J. Physiol., Land. 305, 279-296. 5. Andersen P., Eccles J. C. and Loyning Y. (1963) Recurrent inhibition in the hippocampus with identification of the inhibitory cell and its synapses. Nature 1% 540-542. 6. Andersen P., Eccles J. C. and Liiyning Y. (1964) Pathway of postsynaptic inhibition in the hippocampus. J. Neurophysiol. 27, 608-619. 7. Bevan S. and Wendon L. M. B. (1984) A study of the action of tetanus toxin on rat soleus neuromuscular junctions. J. Physiol., Land. t48, J-17.
8. Brace H. M., Jefferys J. G. R. and Mellanby J. H. (1985) Long-term changes in hippocampd physiology and learning ability of rats after intrahippocampal tetanus toxin, J. Physiol., Lond. 368, 343-357. 9. Buzsaki G. and Eidelberg E. (1981) Commissural projections to the dentate gyrus of the rat: evidence for feed-forward inhibition. Brain Res. 230, 346-350. 10. Calabresi P., Beneditti M., Mercuri N. B. and Bernardi G. (1989) Selective depression of synaptic transmission by tetanus toxin: a comparative study on bippocampal and neostriatal slices. Neuroscience 30, 663-670. 11. Colljn~~dge G. L. and Herron C. E. (1985) Effects of tetanus toxin on GABA synapses in the mamm~ian central nervous system. In Proceedings the Seventh International Conference on Tetanus (eds Nistico G., Maestroani P. and Pizzura M.), pp. 127-142. Rome. 12. Curtis D. R., Felix D., Game C. J. A. and McCulloch R. M. (1973) Tetanus toxin and the synaptic release of GABA.
of
Brain Res. 51, 358-362. 13. Davies J. and Tongroach P. (1979) Tetanus toxin and synaptic inhibition in the substantia nigra and striatum of the rat. J. Physiol., Lond. 290, 23-36. 14. Douglas R. M., McNaughton B. L. and Goddard G. V. (I983) Commissural inbibition and facilitation of granule ceil discharge in fascia dentata. J. camp. Neural. 219, 285-294.
15. Duchen L. W. and Tonge D. A. (1973) The effects of tetanus toxin on neuromuscular transmission and on the morphology of motor endplates in slow and fast skeletal muscle of the mouse. J. Physioi., Land. 228, 157-172. 16. Fricke R. and Prince D. A. (1984) Electrophysiology of dentate granule cells. J. Neurophysiol. 51, 195-209. 17. Frotscher M. and Zimmer J. (1983) Commissural fibres terminate on non-pyramidal neurons in the guinea pig hippocampus-a combined golgi/E.M. study. Brain Res. 265, 289-293. 18. Hatxrmann F., Dreyer F. and Bigalke H. (1980) Tetanus toxin blocks neuromuscular transmission in uirm like botulinum A toxin. ~~nyn-Schm~edeberg’s Arch. Pharmac. 311 33-40. 19. Hawkins C. A. and Mellanby J. H. (1987) Limbic epilepsy induced by tetanus toxin: a lon~tudinal ele~troen#phalographic study. Epiiepsia 28, 431-444. 20. Higashima M. (1988) Inhibitory processes in development of seizure activity in hippocampal slices. Expl Brain Res. 12, 37-44. 21, Jefferys J. G. R. and Williams S. F. (1987) Physiological and behavioural consequences of seizures induced in rats by intrahippocampal tetanus toxin. Brain 110, 517-532.
Granule cells of the urethane-anaesthetized
rat
627
22. Kaeser H. E. and Saner A. (1970) The effect of tetanus toxin on neuromuscular transmission. Eur. Neural. $193-205. 23. Kanda K. and Takano K. (1983) Effects of tetanus toxin on the excitatory and inhibitory postsynaptic potentials in the cat motoneurone. J. Physiol., Land. 335, 319-334. 24. Kryzhanovsky G. N. (1960) Functional changes in the central nervous system in tetanus and the mode of action of the tetanus toxin, III: the central and peripheral action of the tetanus toxin. Bull. exp. Biol. Med. 49, 34-39. 25. Kryzhanovsky G. N., Rekhtman M. B. and Konikov B. A. (1976) Experimental photogenic epilepsy. In The Nervous System, Vol. 4, p. 363. ICRS, Medical Science, Moscow. 26. McNaughton B. L. (1980) Evidence for two physiologically distinct perforant path-ways to the fascia dentata. Bruin Res. 199, l-19. 27. McNaughton B. L. and Barnes C. A. (1977) Physiological identification and analysis of dentate granule cell responses to stimulation of the medial and lateral perforant paths in the rat. J. camp. Neural. 175, 430-454. 28. Mellanby J. H. and George G. (1975) Cephalic tetanus in rats. In Proceedings of the Fourth International Conference on Teronus, Foundation Merieux, Lyon, pp. 201-204. 29. Mellanby J. H., George G., Robinson A. and Thompson P. A. (1977) Epileptiform syndrome in rats produced by injecting tetanus toxin into the hippocampus. J. Neural. Neurosurg. Psychiat. 40, 404-414. 30. Mellanby J. H. and Green G. (1981) How does tetanus toxin act? Neuroscience 6, 281-300. 31. Mellanby J. H., Hawkins C. A., Mellanby H., Rawlins J. N. P. and Impey M. E. (1984) Tetanus toxin as a tool for studying epilepsy. J. Physiol., Paris 79, 207-215. 32. Mellanby J. H., Mellanby H., Pope D. and Van Heyningen W. E. (1968) Ganglioside as a prophylactic agent in experimental tetanus in mice. J. gen. Microbial. 54, 161-168. 33. Mellanby J. H. and Sundstriim L. E. (1989) Tetanus toxin and inhibitory mechanisms in the dentate gyrus of the anaesthetized rat. J. Physiol., Land. 413, 33~. 34. Mellanby J. H., Sundstrom L. E. and Kindling (1988) Behaviour and memory, In The Clinical Relevance of Kindling. (eds Bolwig T. G. and Trimble M. R.), pp. 103-113. John Wiley, Chichester. 35. Mellanby J. H. and Thompson P. A. (1972) The effects of tetanus toxin at the neuromuscular junction in the gold fish. J. Physiol. Lond. 224, 407-419. 36. Mellanby J. H. and Whittaker V. P. (1968) The fixation of tetanus toxin by synaptic membranes. J. Neurochem. 15, 205-208. 37. Oliver M. W. and Miller J. J. (1985) Alterations of inhibitory processes in the dentate gyrus following kindling-induced epilepsy. Expi Brain Res. 57, 443-447. 38. Rausche G., Sarvey J. M. and Heinemann U. (1988) Lowering extracellular calcium reverses paired pulse habituation into facilitation and removes a late IPSP. Neurosci. Left 88, 275-280. 39. Seress L. and Ribak C. E. (1984) Direct commissural connections to the basket cells of the hippocampal dentate gyrus: anatomical evidence for feed-forward inhibition. J. Neurocytol. 13, 215-225. 40. Somogyi P., Nunzi M. G., Gorio A. and Smith A. D. (1983) A new type of specific intemeuron in the monkey hippocampus forming synapses exclusively with the axon initial segments of pyramidal cells. Brain Res. 259, 137-142. 41. Stanley M. and Mellanby J. H. (1989) Glutamic acid decarboxylase (GAD) in rat hippocampus after recovery from tetanus toxin-induced epileptiform syndrome. Neurosci. Left. Suppl. 36, S55. 42. Sugimoto N., Matsuda M., Ohunuki Y., Nakayama T. and Imai K. (1982) Neuromuscular blocking in acutely tetanus intoxicated mice. Biken J. 25, 21-28. 43. Sundstrom L. E. and Mellanby J. H. (1989) Acute effects of tetanus toxin on granule cells of the dentate gyrus of the rat in vivo. In Proceedings of the The Eighth International Conference on Tetanus (eds Nistico G., Bizzini B., Bytchenko
B. and Thiau R.), pp. 149-153. Pythagora Press, Rome. 44. Sundstriim L. E. (1988) An electrophysiological study of tetanus toxin induced hippocampal epilepsy. D. Phil Thesis, Oxford University. 45. Takano K., Kirchner F., Gremmelt A., Matsuda M., Ozutsumi K. and Sugimoto N. (1989) Blocking effects of tetanus toxin and its fragment (A-B) on the excitatory and inhibitory synapses of the spinal motoneuron in the cat. Toxicon 27, 385-392. 46. Teyler T. J. and Alger B. E. (1976) Monosynaptic in vitro. Brain Res. 115, 413-425.
habituation in the vertebrate forebrain: the dentate gyrus examined
47. Thalmann R. H. and Ayala G. F. (1982) A late increase in potassium conductance follows synaptic stimulation of granule neurons of the dentate gyrus. Neurosci. Lett. 29, 243-248. 48. Van Heyningen W. E. (1974) Gangliosides as membrane receptors for tetanus toxin, cholera toxin and serotonin. Nature 249,415-417. 49. Wellhijner H. H. (1982) Tetanus neurotoxin. Rev. Physiol. Biochem. Pharmac. 93, l-68. 50. Wheal H. V., Ashwood T. J. and Lancaster B. (1984) A comparative in vitro study of the kainic acid lesioned and bicuculline treated hippocampus. In The Electrophysiology of Epilepsy (eds Schwarzkroin P. A. and Wheal H. V.), pp. 173-200. Academic Press, London. 51. White W. F., Nadler J. V. and Cotman C. W. (1979) An analysis of short term plasticity at the perforant path-granule cell synapse. Bruin Res. 178, 41-53. (Accepted
29 January 1990)
