The effects of SP-111, a water-soluble THC derivative, on neuronal activity in the rat brain

Brain Research, 139 (1978) 263-275 © Elsevier/North-Holland Biomedical Press

263

THE EFFECTS OF SP-Ill, A WATER-SOLUBLE THC DERIVATIVE, ON NEURONAL ACTIVITY IN THE RAT BRAIN

MENAHEM SEGAL

Isotope Department, lYeizmann Institute of Science, Rehovot (Israel) (Accepted April 27th, 1977)

SUMMARY

The effects of A1-THC and of SP-111, a water-soluble A1-THC derivative, on spontaneous cellular activity and responsiveness to afferent stimulation were studied in the hippocampus of the awake rat. The effects of SP-111 on spontaneous and neurotransmitter-induced changes in neuronal activity in anesthetized rat cerebellum and hippocampus were studied with the method of microiontophoresis. When applied parenterally into the awake rat, SP-111 caused behavioral changes similar to those seen after a A1-THC injection. In addition, it caused a reduction of spontaneous activity of cells in the hippocampus with a single-spike firing pattern without affecting firing of bursting neurons. Furthermore, the averaged evoked field responses to commissural stimulation were reduced by a third up to 2-3 h after the injection. High phoretic currents (100 nA) of SP-111 reduced spontaneous activity of cerebellar and hippocampal neurons. Lower currents potentiated cerebellar inhibitory responses to iontophoretically applied norepinephrine. Even lower currents (50 nA) potentiated responses to iontophoretically applied GABA in the cerebellum. The most potent effect of SP-111 was the antagonism of aspartate-induced excitation of cells in the hippocampus. It is suggested that SP-111 antagonizes an acidic amino acid neurotransmitter in several synapses of the rat hippocampus, and that it may also potentiate the efficacy of transmission in GABAergic and noradrenergic synapses.

INTRODUCTION

Despite the widespread interest in the cannabinoids, there have been relatively few studies concerning the physiological actions of these drugs in the brain. Boyd et alp first reported that Al-tetrahydrocannabinol (A1-THC) injected into primates causes the appearance of epileptic discharges. Similarly Mark Sega114 applied A1-THC topically into rabbit hippocampus and found generation of epileptic discharges. On

264

SP-111

Z~-THC /--x

-)'-o~c5% Fig. 1. Chemical structure of SP-I 11 and the parent compound Al-tetrahydrocannabinol (A1-THC) (see ref. 22).

the other hand, Feeney et al. s found that A1-THC elevates the threshold of afterdischarges to weak electrical stimulation in cat hippocampus. Boyd et al. 4 agree that evoked potentials are depressed in monkey's cortex. Recently, though, Vardaris et al. 21 suggested that A1-THC enhances postsynaptic excitability and attenuates recurrent inhibition in paralyzed rat hippocampus. Differences among the reports could be due to routes of administration (i.p. vs. local), preparations, pathways and observed time courses of effects (3 min vs. 3.5 h). Further studies of the cannabinoids were hampered by the fact that THC compounds are water-insoluble. A water-soluble derivative of A1-THC, SP-111 (Fig. 1), first described by Zitko et al. 22 has behavioral effects comparable to those of A1-THC (see ref. 7) and is therefore an ideal substitute for the latter in the study of the physiological action of the cannabinoids in the brain. The present report summarizes results of experiments on the effects of SP-111, applied parenterally, on spontaneous hippocampal unit activity and responses to afferent stimulation in the awake rat and on the effects ofiontophoretically applied SP-111 on hippocampal and cerebellar neuronal activity and responsiveness to application of putative neurotransmitters in the urethane-anesthetized rat. METHODS

Adult (200-300 g) male Wistar rats of a local breeding colony were used. Two types of experiments were performed using, respectively, the anesthetized and awake rat preparations. Rats prepared for the chronic experiments were implanted according to previously described methods 18. Briefly, each rat was implanted with 2-3 62 #m wires in each hemisphere. Some rats were also implanted with a pair of stimulating electrodes in one hippocampus. Responses to stimulation of these electrodes were recorded in the contralateral hemisphere. The effects of the drugs on the spontaneous activity of single cells in area CA1 of the hippocampus and on responses to interhemispheric stimulation were measured separately in two groups of rats. After termination of the experiments the rats were perfused with a mixture of glutaraldehyde (2 ~ ) and paraformaldehyde (3 ~ ) prepared in phosphate buffer, and the brains sectioned on a freezing stage for localization of electrode placements. Further details of the methodology are presented elsewhere 15-17. Rats prepared for the iontophoresis experiments were anesthetized with urethane (1.2 g/kg with supplementary injections when needed) and placed in a stereotaxic

265 frame. A 2-mm round hole was drilled in the skull above the dorsal hippocampus or in the posterior bone above the cerebellum. The dura was removed and the cortex covered with warm 3 ~ agar. Activity of cells in the pyramidal layer of area CA1 of the dorsal hippocampus and of Purkinje cells in the cerebellum was recorded. The cells were identified on the basis of previously established criteriag, 17. Three of the 4 outer barrels of the 5-barrel pipette were filled with the following compounds: acetylcholine-HC1 (2.5 M, Calbiochem), 1-aspartate (0.1 M, Merck, pH 8.0), gamma aminobutyric acid (GABA, 0.1 M ,Sigma), 5-hydroxytryptamine hydrochloride (5-HT, 0.1 M, Merck), norepinephrine (NE, 0.5 M, Sigma), 1-[4-(morpholino)butyryloxy]-3-n-pentyl-6,6,9trimethyl-10a, 6a, 7,8-tetrahydrodibenzo[fl,a] pyran hydrobromide (SP-I 11, 0.1 M, a gift from Dr. M. Segal, Hadassa Medical Center, Jerusalem). The following drugs were administered parenterally: cannabidiol (CAN, 1-10 mg/kg i.p.), Al-tetrahydrocannabinol (A1-THC, 1-10 mg/kg i.p., a gift from Mr. A. Raz, The Weizmann Institute) and 6-hydroxydopamine hydrobromide (6-OHDA, 2 × 300/~g intracisternal injection). The methods of recording, criteria for data selection and analys is areas previously stated15,17. RESULTS L Studies in the awake rat (A) Behavioral effects. The behavioral syndrome caused by injection ofSP-111 was similar to that caused by injection of A1-THC (see refs. 7 and 22). Both drugs caused ataxia when injected with a dose of 10-20 mg/kg (i.p.). This ataxia, often accompanied by a cataleptic state, was evident 5-15 min after the injection and lasted for 1-3 h. Typically, the rat was hyperexcitable and reacted with an augumented startle response to a strong external stimulus. This behavior was not seen after injection of cannabidiol, a non-psychoactive component of hashish. (B) Cannabinoid effects on spontaneous hippocampal cellular activity. The effects of A1-THC (10 cells), cannabidiol (6 cells) and SP-111 (5 cells) on spontaneous activity of hippocampal CA 1 neurons were tested in the awake rat. Among these, simultaneous recording of two cells (from two different channels) were done in three rats, while all other cells were recorded individually (one cell per rat). Cannabidiol (10 mg/kg) did not change the spontaneous activity of the cells measured within 0.5-1 h of its application. Injection of A1-THC reduced spontaneous activity of 7 neurons by 50-80 within 10-60 min of its administration while three cells were not affected. Interestingly, the affected neurons were of the single-spike type (theta cells 13) while the non-affected neurons were of the bursting type a3. Similar results were seen with SP-111 (Fig. 2); three theta cells reduced their firing rates while two bursting cells were unaffected, and perhaps a slight increase in their firing rates could be monitored. These effects lasted for 4-8 h in most rats and were correlated with the behavioral effects of the drugs. (C) SP-111 effects on responses to afferent stimulation. The responses to interhemispheric stimulation were measured in 9 rats 3-10 days after the implantation of the stimulating and recording electrodes in the hippocampus. Initially, the stimulation

266

Fig. 2. Effects of SP-111 on spontaneous activity of two neurons in the awake rat hippocampus. The two neurons (A and B) were recorded simultaneously from two different channels in the same rat. Top traces are specimen records of the two neurons and their corresponding interspike-interval histograms (ISH). Note the differences in the time scale between the two cells and the shape of their spikes. A has spikes with a constant size, whereas B fires in bursts, which consist of spikes with decreasing sizes. The ISHs sum up 1024 intervals in 0.5 msec bins each. The absence of counts in the first bin in each ISH indicates a refractory period. Such a 'refractory bin' is not present when the recording is taken of more than one cell in a channel. The bottom two traces (A and B) are I-min exerpts of a continuous record taken over 8 h of recording. As in Figs. 1 4 , the cellular activity is integrated over 1-sec intervals. Note the gradual reduction in spontaneous activity in A and a recovery within 5 8 h.

w a s a d j u s t e d so as t o p r o d u c e a s t a b l e r e s p o n s e w i t h t h e l o w e s t p o s s i b l e c u r r e n t . T h e t y p i c a l s t i m u l a t i o n p a r a m e t e r s w e r e 0.1 m s e c , 3 - 5 V ( 3 0 - 5 0 # A ) a p p l i e d o n c e e v e r y 2 sec. A v e r a g e s o f 1 6 - 3 2 s t i m u l i w e r e t a k e n w i t h a n O r t e c S i g n a l A v e r a g e r a n d p l o t t e d o n a c h a r t r e c o r d e r . T h e m a g n i t u d e o f t h e r e s p o n s e a t t h e p e a k w a s m e a s u r e d f o r all t h e a v e r a g e s (Fig. 3). P o s s i b l e c h a n g e s in e x c i t a b i l i t y w e r e m e a s u r e d w i t h t h e t w i n - p u l s e t e c h n i q u e ; a s e c o n d (test) p u l s e w a s a p p l i e d 0, 15, 30, 60 o r 90 m s e c a f t e r t h e first

267

B.

A.

o.7.5

1

15 C-T

30 60 INTERVAL(reset)

90

0.1mV

30msec

Fig. 3. Effects of SP-111 on evoked hippocampal responses to interhemispheric stimulation in the awake rat. A: magnitude (measured as the highest deflection from the zero line) of the averaged response at various conditioning-test intervals (in msec) before, 10 min after and 3 h after an intraperitoneal injection of SP-111 (10 mg/kg). Note that 10 rain after SP-111 injection there is no change in the initial (conditioning) potential but there is a large reduction in the response to the test pulse. The opposite is true when the test is made 3 h after the injection. B: a specimen record of the response to a twin-pulse interhemispheric stimulation with 30-msec interpulse interval, before (thick line) and 3 h after (thin line) injection of SP-111. Negativity up. The recording electrode was localized in stratum radiatum of area CA1 of the dorsal hippocampus.

(conditioning) pulse, and changes in the magnitude of the responses to the test pulse relative to the response to the conditioning pulse were measured. The following changes in the responses to the interhemispheric stimulation were f o u n d within 1-2 h after a parenteral (10 mg/kg i.p.) injection o f SP-111: (1) The magnitude o f the response to a single interhemispheric stimulation was reduced by an average o f 31 ~ in all of the 9 rats tested (range ---- 10~-53~o). This effect was not apparent when the response was measured some 10 min after the injection o f the drug (Fig. 3). It was evident when the behavioral effects (see below) o f SP- 111 were taking place, i.e. within 1-3 h after the injection, disappearing some 6-12 h later. (2) The responses to the test pulse relative to the conditioning pulse could be classified into two types, depending on the electrode placements: (a) cases where the responses to the test pulse were larger than the responses to the conditioning pulse, indicating a facilitatory process and (b) cases where the responses to the test pulse were smaller than the responses to the conditioning, indicating a postsynaptic inhibitory process. This was usually the case when the recording electrode was placed in the pyramidal layer. W h e n the predrug responses to the test pulse were larger than the predrug responses to the conditioning pulse (as is the case in Fig. 3), there was a reduction in the test responses after SP-111 (10-20 ~o), although not to the same extent as the reduction observed in the response to the conditioning pulse relative to predrug response. On the other hand, in cases where the predrug responses to the test pulse were smaller (40-50 ~ ) relative to the responses to the conditioning pulse, this reduction

268 in response was n o t observed after the d r u g treatment, i.e. the responses to the test pulse were no longer smaller (80-90 ~ ) than the responses to the c o n d i t i o n i n g pulse after SP- 11 l. This indicates a r e d u c t i o n in the effectiveness o f the p o s t s y n a p t i c inhibitory process.

II. Iontophoretic studies The activity o f 58 cerebellar Purkinje cells a n d 47 h i p p o c a m p a l p y r a m i d a l cells was recorded in 15 rats. SP-11 l h a d a small inhibitory action (reduction o f firing by 2 0 - 3 0 ~ ) on s p o n t a n e o u s activity o f the recorded neurons. This effect was evident when m o d e r a t e (50 nA) to high (100 hA) ejection currents were used. When low currents (0-40 nA) were used there were no significant effects o f the drug t o w a r d s s p o n t a n e o u s activity o f the r e c o r d e d neurons. The effects o f S P - I I 1 could not be m i m i c k e d by the mere ejection o f N a + or C l - i o n s from the balance barrel. The effects o f n o r e p i n e p h r i n e (NE) were tested on a total o f 41 neurons in the cerebellum (Cb) a n d 19 neurons in the h i p p o c a m p u s (Fig. 4). As previously r e p o r t e d 9,17, N E causes depression o f s p o n t a n e o u s activity o f neurons in b o t h structures. In 15 C b neurons, the concurrent a p p l i c a t i o n o f SP-111 with m o d e r a t e to high currents p o t e n t i a -

Fig. 4. Potentiation by SP-III of the action of norepinephrine (NE) towards cerebellar Purkinje cellular activity. NE was applied in pulses of constant duration (bars of 10 sec) and interpulse intervals, with the designated current (in nA). SP-111 was ejected in one continuous pulse which overlapped some of the NE pulses. Spontaneous activity was integrated over 1-sec intervals and the results plotted on a chart recorder. Note the relatively larger effect of SP-111 in its second application on the same neuron (bottom trace). Calibration : abscissa, time, ordinate, spikes per sec.

269

Fig. 5. Comparison of the effects of SP-111 on GABA- and NE-induced inhibition of spontaneous cerebellar activity. Note that a much smaller (10 nA vs. 100 nA) current is needed to produce GABA inhibition than NE inhibition. SP-111 affects both the latency and magnitude of the cellular responses to GABA without affecting the responses to NE. ted cellular responses to NE, while there was no effect of the drug in 18 of the cells (Fig. 5) and, in three more, SP- 111 appeared to have a partial antagonistic action towards the effects of NE. The activity of 8 cerebellar cells in 6-OHDA treated rats was recorded as well, in order to test the possibility that the effects of SP-111 are exerted presynaptically. It was assumed that, if the drug modifies a reuptake mechanism 10, it is expected not to have an effect in rats devoid of their NE terminals after being treated with 6-OHDA. Although no careful statistical comparisons of the data were performed, in none of the 8 cells tested did SP-111 appear to interact with the effects of NE. The effects of SP-111 towards NE-induced inhibition of spontaneous activity in the hippocampus were somewhat different from those observed in the cerebellum. In only three of 19 cells tested was there an apparent interaction between NE and SP-111 to produce an enhanced inhibitory response to NE (see ref. 14). In 16 other cells there was no effect of the drug towards NE inhibition when moderate to high (100 nA) currents for ejection of SP-111 were used. G A B A was tested on 9 cells in the cerebellum. The spontaneous activity of all neurons was inhibited during the iontophoretic administration of GABA. SP-111, when applied with a moderate (30-50 nA) ejection current, caused a marked potentiation of cellular responses to GABA (Fig. 5). The action of SP-111 towards G A B A inhibition was more effective and was produced with lower currents than those producing NE inhibition in the cerebellum. Serotonin (5-HT) was tested on 9 cells in the hippocampus. All of them reacted by inhibition to the application of 5-HT. SP-111, when ejected with moderate (50 nA) currents potentiated the effects of 5-HT in only three of the neurons, while in the other 6 cells there was no apparent interaction between 5-HT and SP-111 (Table I).

270 TABLE I

SP-1 II interactions with putative transmitters NE

GA BA

5HT

A SP

A Ch

Cerebellum block no effect potentiate

3 23 15

0 0 3

-

8 I 0

0 0 0

H i p p o c a m p u s block no effect potentiate

0 16 3

-

6 3

18 3 0

0 5 0

Fig. 6. Responses o f a hippocampal neuron (A) and a cerebellar neuron (B) to aspartate (ASP) and the antagonism of this response by SP-I 11. Aspartate has a more potent action in the hippocampus, where the spontaneous activity is relatively low, than in the cerebellum. Also, SP-I 11 has a more potent antagonistic action in the hippocampus. A and B were recorded with the same 5-barrel pipette in the same rat.

271

:

05 sec

t

:

i

ASP

!

It

BEFORE

DURING 20 nA SP 111

AFTER

5 Sec

ACH

ASP

20

10

Fig. 7. Comparison of the effects of acetylcholine (ACh) and aspartate (ASP) on hippocampal neuronal activity and the selective antagonism by SP-111 towards aspartate excitation. The top trace is a specimen record of a response of a cell to the iontophoresis of ASP. Spike at the onset of the ASP current is a switch artifact. The bottom three traces are poststimulus time histograms summing up 5 repetitions of responses of the neuron to 20 nA of ACh and 10 nA of aspartate each. The responses to ASP are completely antagonized during SP-111, whereas the responses to ACh are not changed. A m o n g the excitatory agents tested, a s p a r t a t e p r o d u c e d s h o r t latency excitation o f 21 h i p p o c a m p a l a n d 9 cerebellar neurons. Unlike its interaction with the i n h i b i t o r y agents, SP-111 a n t a g o n i z e d the excitatory action o f a s p a r t a t e in 18 o f the 21 h i p p o c a m p a l a n d 8 o f the 9 cerebellar n e u r o n s (Fig. 6). This a n t a g o n i s m was p r o d u c e d even when very low currents (0-10 nA) o f SP-111 were used, especially with h i p p o c a m p a l cells. The a c t i o n o f SP-111 a p p e a r e d to be fairly specific as there were no effects o f the d r u g t o w a r d s A C h - i n d u c e d excitation in 5 cells tested simultaneously with a s p a r t a t e a n d A C h in the h i p p o c a m p u s (Fig. 7).

272

comm rad

~sP?

al Fig. 8. A schematic diagram of the proposed effects of SP-111 in the hippocampus. The large pyramidal neuron in stratum pyramidale (pyr) sends an axon collateral which innervates the interneuron in stratum oriens (or). This in turn projects a GABA-containing terminal on the pyramidal cell. The hippocampus receives a commissural (comm) connection which terminates in stratum radiatum (rad). It is suggested that the hippocampal pyramidal neurons use an acidic aminoacid, glutamate or aspartate (ASP) as a neurotransmitter and that SP-111 antagonizes responses to the activation of these synapses. DISCUSSION The present studies demonstrate distinct effects of SP-I 11, a water-soluble A 1T H C derivative towards cellular activity in two structures of the rat brain, the hippocampus (HPC) and cerebellum (Cb). These two structures were selected for study because the recorded populations of neurons there are physiologically identifiable, and because of accumulated evidence for the presence there of several putative neurotransmitters 9,17. It was found that, when applied iontophoretically with high currents, SP-l 11 had a small direct inhibitory action towards spontaneous cellular firing rates. Also, a marked potentiation of neuronal responses to concurrent application of norepinephrine (NE) was recorded in the Cb. This effect might have been mediated by a blockade of a reuptake mechanism, as suggested previously 19. Indeed, desmethylimipramine, a drug which blocks N E reuptake mechanism, has also been reported to potentiate effects of N E in the cerebellum 9. Alternately, a blockade of the enzyme mono-amino-oxidase (Shurr, private communication) can also account for the observed effects. Hence, when NE-containing terminals were destroyed with 6 - O H D A , the effects of SP-111 towards N E inhibition were no longer evident. Interestingly, there was no apparent effect of SP-11 ! towards N E inhibition in the hippocampus, although both structures receive similar noradrenergic input. The action of N E is different in the two structures, the time course o f N E action in the hippocampus being much longer than in the cerebellum, indicating a possible difference in the reuptake mechanisms. This

273 may underly the observed difference in the efficacy of SP-111 in the two structures. The most potent effects of SP-I 11 were exerted towards aspartate-induced excitation in the hippocampus. This antagonistic action was seen even with very low ejection currents (0-10 nA) and was fairly specific; no such antagonism was seen towards ACh-induced excitation. The chronic, awake preparation allows a test of the effects of parenteral injection of A1-THC and SP-111 on cellular activity and the correlation of this activity with the behavioral action of the drugs. It was indeed observed that the two drugs had a similar cataleptoid effect towards the rats' behavior as reported earlierL Also, cannabidiol, a non-psychoactive component of hashish, did not produce these behavioral effects. Both A1-THC and SP-111 reduced spontaneous firing of cells in the hippocampus which have a characteristic single-spike firing pattern, and did not much affect the bursting-type neurons. A distinction between these two classes of neurons was already noted before by Ranck, who suggested that the bursting neurons are the pyramidal cells and that the single-spike cells are interneurons 13. The pyramidal neurons send axon collaterals to innervate the interneurons which, in turn, innervate the somata of the pyramids. This innervation was postulated to use GABA as a neurotransmitter, and its activation produces a powerful postsynaptic inhibition of pyramidal cells z. It is suggested that the observed reduction in spontaneous activity of the single-spike neurons results from a blockade of the excitatory synapse of the pyramidal neurons on the interneurons. This would be expressed in a selective depressant effect towards the interneurons and, if anything, would result in a small excitatory action towards the pyramidal cells, as was indeed observed. Another set of results of the experiments in the awake rats concerns the effects of the drugs on hippocampal evoked responses to commissural stimulation. These results can be summarized as follows: (l) SP- 111 caused a 30 700reduction in the averaged response to commissural stimulation within 2-3 h of its parenteral application; (2) this reduction was not accompanied by a proportionally similar reduction in the excitatory postsynaptic events, as measured by the twin pulse method; and (3) there was a comparable reduction in the inhibitory postsynaptic events. These results are similar to those reported earlier 21. Taken together, the results of the iontophoresis experiments along with those done in the awake rats suggest that SP- 111 and perhaps A1-THC act in the hippocampus by antagonizing an excitatory synapse of pyramidal cells on interneurons and a synapse of another branch of these neurons - - the commissural path1, 3. It is further suggested, on the basis of additional evidence, that these synapses utilize an excitatory amino acid, glutamate or aspartate as a neurotransmitter. This suggestion is based on the following: there is a growing body of evidence to suggest that the commissural path is likely to use an excitatory amino acid as a neurotransmitter6,1°-12,~5,~°; since the commissural path arises from pyramidal cells, it is likely that the cells will use the same acidic amino acid as a transmitter also in the local pathway terminating on interneurons. Fig. 7 can therefore explain the three sets of data presented above: (a) antagonism of responses to aspartate applied iontophoretically); (b) reduction in the magnitude of the response to commissural stimulation accompanied by a reduction in inhibitory pro-

274 cesses; a n d (c) a reduction in s p o n t a n e o u s firing of, presumably, interneurons. This hypothesis will also explain previous observations o n the effects of A1-THC o n hippocampal activityT,8,1L Despite the d e m o n s t r a t i o n of a specific action of a water-soluble T H C derivative towards hippocampal circuitry, the assertion that T H C exerts its behavioral effects t h r o u g h its effects o n the h i p p o c a m p u s or t h r o u g h its effect on an aspartate receptor is rather premature. F u r t h e r studies analyzing other b r a i n structures related to behavior in a similar fashion with this a n d similar control c o m p o u n d s are required before a generalization can be made. A study of other hippocampal afferents, e.g. the perforant path or the cholinergic septal afferent with a c o m b i n a t i o n of microiontophoresis a n d electrical stimulation, is i m p o r t a n t for d e t e r m i n a t i o n of the specificity of the drug effects. The results of the present study encourage such a n investigation.

REFERENCES 1 Andersen, P., Interhippocampal impulses. II. Apical dendritic activation of CAI neurons, Acta physiol, scand., 48 (1960) 178-208. 2 Andersen, P., Pathway of postsynaptic inhibition in the hippocampus, J. Neurophysiol., 27 (1964) 608 619. 3 Blackstad, T. W., Commissural connections of the hippocampal region in the rat with special reference to their mode of termination, J. comp. Neurol., 105 (1956) 417-537. 4 Boyd, E. S., Boyd, E. H. and Brown, L. E., Effects of Ag-tetrahydrocannabinol and pentobarbital on a cortical response evoked during conditioning, Psychopharmacology, 47 (1976) 119 122. 5 Boyd, E. S., Boyd, E. H., Muchmore, J. S. and Brown, L. G., Effects of two tetrahydrocannabinols and of pentobarbital on cortical evoked responses in the squirrel monkey, J. Pharmacol. exp. Ther., 176 (1970) 480~,88. 6 Curtis, D. R., Duggan, A. W., Felix, D., Johnston, G. A. R., Tebecis, A. K. and Watkins, J. C., Excitation of mammalian central neurons by acidic amino acids, Brain Research, 4l (1972) 283-301. 7 Dykstra, L. A., McMillan, D. E. and Harris, L. S., Effects of Ag-THC and a water soluble ester of Ag-THC on schedule-controlled behavior, Pharmacol. biochem. Behav., 3 (1975) 29-32. 8 Feeney, D. M., Wagner, H. R., McNamara, M. C. and Weiss, G., Effects of tetrahydrocannabinol on hippocampal evoked afterdischarges in cats, Exp. Neurol., 41 (1973) 357-365. 9 Hoffer, B. J., Siggins, G. R. and Bloom, F. E., Studies on norepinephrine-containingafferents to Purkinje cells of rat cerebellum. II. Sensitivity of Purkinje cells to norepinephrine and related substances administered by microiontophoresis, Brain Research, 25 (1971) 523-534. 10 Iversen, L. L. and Storm-Mathisen, J., Uptake of 3H-glutamate in excitatory nerve endings in the hippocampal formation of the rat, Acta physiol, scand., 96 (1976) 22A. 11 Johnson, J. L., Glutamic acid as a synaptic transmitter in the nervous system. A review, Brain Research, 37 (1972) 1-19. 12 Nadler, J. V., Vaca, K., 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. 13 Ranck, J. B., Studies on single neurons in dorsal hippocampal formation and septum in unrestrained rats. 1. Behavioral correlates and firing repertoires, Exp. NeuroL, 41 (1973) 461 555. 14 Segal, M., Central implantation of cannabinoids: induction of epileptiform discharges, Europ. J. Pharmacol., 27 (1974) 40-45. 15 Segal, M., Glutamate antagonists in rat hippocampus, Brit. J. Pharmacol., 58 (1976) 341-345. 16 Segal, M., Effects of brainstem priming stimulation on hippocampal responses to interhemispheric stimulation in the awake rat, Exp. Brain Res., in press. 17 Segal, M. and Bloom, F. E., The action of norepinephrine in the rat hippocampus. I. lontophoretic studies, Brain Research, 72 (1974) 79-97. 18 Segal, M. and Bloom, F. E., The action of norepinephrine in the rat hippocampus. IV. The effects of locus coeruleus stimulation on evoked hippocampal unit activity, Brain Research, 107 (1976) 513-525.

275 19 Sofia, R. D., Ertel, R. J., Dixit, B. N. and Barry, H., The effects of Al-tetrahydrocannabinol on the uptake of serotonin b2, rat brain homogenates, Europ. J. PharmacoL, 16 (1971) 257-259. 20 Spencer, H. J., Gribkoff, V. K., Cotman, C. W. and Lynch, G. S., GDEE antagonism of iontophoretic amino acid excitations in the intact hippocampus and in the hippocampal slice preparation, Brain Research, 105 (1976) 471~,81. 21 Vardaris, R. M., Weisz, D. J. and Teyler, T. J., Delta-9-tetrahydrocannabinol and the hippocampus: parametric features of the effects on CA1 field potentials, Neurosci. Abstr., 2 (1976) 377. 22 Zitko, B. A., Howes, J. A., Razdan, R. K., Dalzell, B. C., Dalzell, H. C., Sheehan, J. C., Pars, H. G., Dewey, W. L. and Harris, L. S., Water soluble derivatives of Al-tetrahydrocannabinol, Science, 177 (1972) 442444.