EFFECTS OF DIPHENYLHYDANTOIN ON HIPPOCAMPAL EVOKED
AND DIAZEPAM RESPONSES*
W. D. MATTHEWS and J. D. CONNOK Department of Pharmacology, The Milton Umversity
College
S. Hershey of Medicine. Hershey.
Medical Center, The Pennsylvania Pennsylvania 17033. U.S.A.
State
Summary-Diphenylhydantoin and diazepam were compared with regard to their effects on responses to single stimuli. and on post-tetanic potentiation of evoked potentials within the hippocampus of anaesthetized rats. fnterhippocdmpaf afferents to pyramidal cells were stimulated electrically, while extracellular field potentials were recorded at successive depths within the contralateral hippocampus. Recording electrodes penetrated perpendicularly from a surf&cc point homotopic to the site of stimulation. The anticonvulsants (40 mg/kg diphenylhydantoin. 5 mgikg diazepam). 30 min after intravenous infusion, depressed post-tetanic potentiation in the stratum radiatum. At these doses responses to single stimuli were essentially unaltered. Doses nearer the toxic range (80 mg/kg diphenylhydantoin. 10 mg/kg diazepam) also reduced post-tetanic potentiation in the stratum radiatum, but increased (up to 150%) post-tetanic potentiation recorded from granule cells in the area dentatd. The results indicate that diphenylhydantoin and diazepam have: (1) selective actions on hippocampal post-tetanic potentiation which are site and dose dependent; (2) qualitatively similar end-effects on post-tetanic potentials in two hippocampal subregions; (3) minimal effects on responses to single stimuli at doses which depress or enhance hippocampal post-tetanic potentiatton.
‘Xphenylhydantoin (Dilantin; DPH; 5,5-diphenyl-2.4.midazolidinedione) and diazepam (Valium; 7-chlorol,3-dihydro-l-mkthyl-5-phenyl-2H-l,4-benzodiazepin?-one) are used clinically to control fhe symptoms of convulsions. Both drugs are effective in terminating status epilepticus (NICOL, TUTTON and SMITH, 1969; WALLIS, KUTT and MCDOWELL, 1968) and in the ‘:hronic management of some forms of seizures (AIRD and WOODBURY,1974). The chemical structures of the compounds appear unrelated in two-dimensional representation. However, CAMERMANand CAMERMAN (1970) in calling attention to similarities in their spatial conformation, suggested that the compounds might exert anti-epileptic effects through interactions with the same unspecified receptor in the brain. Experiments on the responses in spinal and peripheral nerves of laboratory animals seem to indicate that the anticonvulsant action of DPH is related to reduction of post-tetanic potentiation (WOODBURY, 1968). Post-tetanic potentiation is presumed to be a critical event in the spread of excitatory impulses from epileptogenic focal neurones, and to reinforce focal discharges by positive feedback (WO~DBURY, 1968). At doses which have little effect on responses to single stimuli, DPH decreases post-tetanic potentiation in the spinal cord. autonomic ganglion and neuromuscular junction (ESPLIN, 1957; RAINES and STANDAERT,1966). Thus, ESPLIN (1957) proposed that DPH, by reducing post-tetanic potentiation, prevents the spread of abnormal electrical activity from an epi*This research was supported in part Research Grant NS08884 from the National Neurological Diseases and Stroke.
by USPHS Institute of 181
leptic focus to normal brain tissue. Diazepam also depresses post-tetanic potentiation in sympathetic ganglia without affecting the usual responses to single stimuli (SURIA and COSTA, 1973). Diverse effects on electrical activity in limbic system nuclei have been described for diazepam (OLDS and OLDS, 1969; GUERRERO-FIGUEROA.GALLANT, GUERRERO-FIGUERO and GALLANT 1973) but its mechanism of action in seizure control remains obscure. Our experimental goals were to determine if DPH and diazepam modify evoked responses and posttetanic potentiation in the hippocampus, a brain area relevant to epileptogenesis. The hippocampus was chosen as a test site because it has a low threshold for seizure development (GREEN, 1964) and because hippocampal pathways serve as routes for the spread of seizure activity to other brain regions (AJMONEMARSAN and STOLL, 1951). Moreover, DPH and diazepam alter the discharge patterns of hippocdmpal neurones (OLDS and OLDS, 1969; IZQUIERD~ and NASELLO, 1970). In our study, the effects of systemic injections of the drugs were measured by changes in field potentials, since the laminar cytological organization within the hippocampus lends itself to field potential analysis (ANDERSEN, 1959). METHODS Drug effects on hippocampal field potentials were studied in 70 adult rats (2oo-450 g) anaesthetized with urethane (1.5 g/kg, i.p.). The calvarium and overlying cortex were removed bilaterally; exposed hippocampal surfaces were covered with a pool of warm (37°C) mineral oil. Rectal temperature was monitored with
182
W. D. MATTHEWSand J. D. CONNOH
a thermistor probe and maintained at 37 i: 0.5”C by warm water circulated through coiled tubing placed beneath the animal. All solutions were administered through a cannula in a femoral vein by an infusion pump. A tracheal cannula maintained an unobstructed airway. Single stimuli (1 Hz, 0.2 msec duration, twice threshold voltage, or 1@50 V) were delivered through a bipolar stimulating electrode on the surface of the dorsal hippocampus above CA1 pyramidal cells. Post-tetanic potentiation was elicited by trains of stimuli (l&30 Hz, 3-5 set train duration, same voltage and pulse duration as single stimuli). Extracellular field potentials were recorded in the contralateral hippocampus by glass microelectrodes (4 pm tip dia, l.sl.5 MR, filled with 4 M sodium chloride) positioned homotopic to the site of stimulation, and advanced through the CA1 pyramidal cell layer to the granule cell layer of the area dentata. The circuit was completed through a disk electrode in a saline-soaked pad placed against the oral mucosa. A dual beam storage oscilloscope amplified and displayed the signals. Data were recorded on magnetic tape for computer analysis. Diphenylhydantoin sodium (Sigma Chemical Co.) or diazepam (kindly provided by HoffmanLaRoche, Inc.) was dissolved in a vehicle consisting of propylene glycol (400 ml), sodium benzoate (48.8 g) benzoic acid (1.2 g), benzyl alcohol (15 ml), ethyl alcohol (100 ml) and distilled water (sufficient to make a litre). The pH was adjusted to 11.0 with 1~ sodium hydroxide. Drugs were dissolved in the vehicle on the day of the experiment. The doses utilized were anticonvulsant (20 and 40 mg/kg DPH; 2.5 and 5 mg/kg diazepam) against the tonic phase of maximal electroshock, or near the concentrations that cause ataxia (80 mg/ kg DPH; 10 mg/kg diazepam) in rodents (SWINYARD and CASTELLION, 1966; TOMAN, SWINYARD and GWDMAN, 1946). Control infusions consisted of a volume of vehicle equivalent to the amount needed to dissolve the highest dose of each drug. In preliminary experiments, a rate of intravenous infusion of vehicle plus drug which produced minimal and transient changes in mean blood pressure, heart rate and respiration was determined. This information was obtained from 12 anaesthetized rats whose femoral arteries were cannulated and connected to a pressure transducer. Mean blood pressure and heart rate before and during infusion were recorded continuously with a polygraph. A gas analyzer monitored respiratory rate and % CO2 in expired air. Field potentials were averaged by a Technical Instruments CAT 1000. Values for the amplitudes of potentials were obtained by measuring, on the analog printout, vertical heights from baseline to peak deflection. The magnitude of post-tetanic potentiation was expressed as a ratio of the mean amplitude of the first 5 potentials after the train to the mean amplitude of 5 responses just prior to the train. The average magnitude of 335 post-tetanic potentiations just
before drug or vehicle administration represented control (untreated) post-tetanic potentiation. The magnitude of post-tetanic potentiation and single stimuli responses after vehicle or vehicle plus drug was expressed as a percentage of untreated values, Means and standard errors for these percentages were determined, and the effects of a drug or vehicle alone on post-tetanic potentiation and responses to single stimuli were compared statistically by Student’s t test. Probabilities less than 0.05 were considered statistically significant. RESULTS
Rate of infusion Vehicle infusions produced slight hypotension and bradycardia. effects not obtained with saline (Fig. 1). Diazepam, 4 mg/kg at the three rates shown in Figure 1. depressed mean blood pressure by 2(&25x1. Bradysignificant (P < 0.05) at an cardia became administration rate of 360 &min. None of the infusion rates of vehicle alone or vehicle plus diazepam affected respiration significantly. Values in Figure 1 represent maximal changes in the parameters measured. Depression of mean blood pressure and heart rate was transient, beginning during the infusion and returning to pre-infusion values within 5 min. An infusion rate of 71 &min for diazepam in a concentration of 5 mg/ml was chosen. For similar reasons, an infusion rate of 143 $/min was used for DPH (40 mg/ml). Laminar analysis of rat hippocarnpus Figure 2 depicts the results of a typical laminar analysis. Panel A is a schematic diagram of the cellular elements in the CA1 region and the area dentata. The pattern of field potentials at various depths is 0 HEART RATE mBLOOD PRESSURE 0 RESPIRATION RATE
i
”
360 Soline
360
Vehicle
71
_
143
360
Vehicle t Diozepom INFUSION RATE (~l/min)
Fig. I. Effects of intravenous diazepam and its vehicle on vital functions in the rat. A constant volume (0.8 ml/kg) of saline. propylene glycol vehicle, or vehicle -t diarepam (4 mg/kg) was infused at the rates given on the abscissa. The effects on heart rate. mean blood pressure. and respiratory rate are shown by bars representing the means of six experiments. Asterisks indicate a statistically significant change from untreated control (P < 0.05by paired t-test).
Anticonvulsants
and hippocampal
183
PTP POSfTrOl”
Pretmi”
Elecrrode P051+10”,
Pm
230
A\
-
L-J---
i!
A ‘Lf-----
P
ISOPo+e”+,(l,
0
F‘lg. 2. Laminar analysis of rat hippocampus A: Schematic c!iagram of major cell types encountered in vertical Fenetration by a microelectrode through CA1 region of kippocampus and area dentata. (P) CA1 pyramidal cell klody, (a) axon. (b.d.) basal dendrite, (a.d.) apical dendrite, (:om.) afferent commissural fibres from contralateral CA1 pyramidal cells. (G) granule cell of area dentata. Depth below alveus scaled on the right. B: Field potentials, in hippocampal stimuli, were response to contralateral recorded at the depths indicated. Positivity upward. C: f\mplitude of the field potentials in millivolts, plotted against recording electrode depth. Amplitudes were mea:.ured at a time post-stimulus indicated by the vertical dashed line in B.
in B. Electrodes passing through the stramonophasic positive potentials with negative inflections on the rising phase. An iso2otential point invariably appeared just beneath the ,;tratum pyramidale. In the stratum radiatum a large negative wave superimposed on a positive wave was recorded. The negative component had its peak amplitude in the proximal apical dendrites. Single stimuli evoked a monophasic positive wave in the area dentata. The isopotential depth from the alveal surface varied by + 50 pm from animal to animal. Tissue swelling during the experiments made absolute depth measurements unreliable. Therefore. the position of the tip of the recording electrode within hippocampal laminae was always referenced to the distance from the isopotential point. A representative summary of the amplitude of the waves at various depths within the hippocampus is given in Figure 2C. The positive wave in the stratum oriens reached maximum amplitude about 150 pm beneath the surface of the hippocampus. The isopotential point occurred at a depth of 250 pm. The negative wave in the stratum radiatum became maximal at 400 pm. The granule cell potential (depth of 800 ‘urn in Fig. 29) had a shorter latency than potentials recorded in strata associated with pyramidal cells or presented
I urn oriens recorded
their
processes.
Diphenylhydantoin and diazrpam on hippocumpal postManic potentiation After repetitive stimulation of commissural afferents, post-tetanic potentiation of field potentials within the hippocampus was observed at all recording
Fig. 3. Laminar analysis of post-tetanic potentiation. The position of the recording electrode within the hippocampus is given relative to the isopotential point (designated by 0). Field potentials at each depth are given in the ‘pretrain’ column. The ‘posttrain’ column shows potentiated responses after a train of stimuli (10 Hz for 3 set, twice threshold voltage). Note post-tetanic potentiation was greatest in the apical dendrites and the area dentata.
sites (Fig. 3). Post-tetanic potentiation was largest and most reproducible at 200 pm (stratum radiatum) and 600 pm (area dentata) below the isopotential point, so these two regions were chosen to study drug actions. Diphenylhydantoin depressed, in a dose-dependent manner, post-tetanic potentiation in the stratum radiatum. (Fig. 4). Depression was not observed in the area dentata; rather, dose-dependent increases in post-tetanic potentiation were produced by the drug (Fig. 4). Diphenylhydantoin (20 mg/kg) did not facilitate granule cell post-tetanic potentiation. However, 40 mg/kg caused a 40% increase in post-tetanic potentiation. Infusions of 80 mg/kg resulted in a 150% enhancement of post-tetanic potentiation. Diazepam
c
radlatum
i
200
1
q Stratum
250
0
Area
dent&a
1
Diphenylhydantoin,
80
m/kg
Fig. 4. Diphenylhydantoin effects on hippocampal posttetanic potentiation. The ordinate represents the percent of control (untreated) post-tetanic potentiation (PTP) observed in the stratum radiatum or area dentata, 30 min after intravenous infusion of vehicle or diphenylhydantoin. The heights of the bars represent mean responses; vertical lines are standard errors (6 animals at each dose). A statistically significant (P < 0.05 by Student’s t-test) change from vehicle is denoted by an asterisk.
184
W. D. MATTHEWSand J. D.
Vehicle
5 Dlazepam,
IO
mdkg
Fig. 5. Diazepam effects on hippocampal post-tetanic potentiation. Ordinate represents percent of control (untreated) post-tetanic potentiation in the stratum radiatum or area dentdta 30 min after intravenous infusion of vehicle or diazepam. The heights of the bars represent mean responses; vertical lines are the standard errors (6 animals at each dose). Statistically signilicant (P -c 0.05 by Student’s t-test) changes from vehicle are denoted by asterisks.
reduced post-tetanic potentiation in the stratum radiatum at 5 and 10 mg/kg doses (Fig. 5). At 10 mg/kg, diazepam increased granule cell post-tetanic potentiation by SSo/;, (Fig. 5). The effects of lower doses of diazepam (2.5 and 5 mg/kg) on granule cell post-tetanic potentials were not statistically significant. Diphenylhydantoin and diuzepam ou stimuli
~q~on.srs
to
single
Diphenylhydantoin (20 and 40 mg,kg) had negligible effects on the amplitudes or latencies of field potentials evoked in the stratum radiatum or the area dentata by 1 Hz stimuli. In some instances, the highest dose of DPH (80 mg/kg) reduced field potential amplitudes by as much as 20%, but this response was not sufficiently consistent to be of statistical significance. Infusions of diazepam (25, 5. 10 mg/kg) did not depress the amplitudes of the field potentials elicited by single stimuli at either recording site.
DISCUSSION
The task of obtaining quantitatively reproducible field potentials is made somewhat easier if the experimental animals are anaesthetized. Presumably, general anaesthetic agents exert some effects of their own in setting the level of neurone excitability and by interacting with other drugs. Although the rats in this study were anaesthetized with urethane, laminar analyses within the hippocampus and posttetanic potentiation of the evoked waves were highly reproducible. Moreover, dose-dependent changes in electrical activity were elicited by systemic injections of DPH or diazepam after reasonable latencies. However, it seems probable that the parameters of stimu-
CONNOR
lation and the effective doses of the drugs would be different in unanaesthetized animals. The propylene glycol vehicle, when given intravenously. depressed the cardiovascular system. The extent of hypotension and bradycardia in the rat was similar to that reported for the cat (LOUIS, KUTT and MCDOWELL. 1967; SHARER and KUTT, 1971). These effects were enhanced by diazepam; thus, a slow rate of infusion was chosen to minimize adverse cardiovascular responses. Drug actions were evaluated 15 min or more after the end of the infusion, at which time blood pressure and heart rate had returned to preinjection values. For these reasons. the effects of the anticonvulsants on hippocampal potentials cannot be explained simply by diminished perfusion of the brain. However. when a drug is given systemically and responses are measured in a specific region of the brain. a possibility exists that the effects are secondary to an action of the drug at another brain site. In these studies we cannot eliminate this possibility. Field potentials in rat hippocampus in response to commissural stimuli resemble those of other species (CRAGG and HAMLYN, 1955; ANDERSEN, 1960; EIDELBERG, 196 1; FUJITA and SAKATA. 1962). The maximum amplitude of the negative potential in the stratum radiatum was recorded near the termination of the afferent commissural fibres from contralateral pyramidal cells. ANDERSEN(1960) interpreted this negative wave as a summated excitatory postsynaptic potential (EPSP) elicited in the apical dendrites of CA1 pyramidal cells by a direct commissural volley. We also obtained an evoked potential in the granule cells of the area dentata upon stimulation of the contralateral hippocampus. BLACKSTAD (1956) and RAISMAN, COWMAN and POWELL (1965) demonstrated interhippocampal fibres to the area dentata which terminate within a well defined zone in the dendritic field of the granule cell layer. The source of these commissural fibres is. however, unknown. The short latency (cu. 5 msec) of the evoked potential in the granule cells during contralateral hippocampal stimulation may be taken tentatively as electrophysiological evidence for a direct commissural connection between these regions. The field potential of the granule cells exhibited the largest post-tetanic potentiation after repetitive stimulation. This large posttetanic potentiation might be explained by the high density of granule somata. Alternatively, volleys arriving through the commissural pathway may have a high probability for activating a large percentage of the total number of synapses within a discrete zone of the granule cells (BLACKSTAD, 1956). The observations that DPH and diazepam depress post-tetanic potentiation in the stratum radiatum without affecting significantly the responses to single stimuli agree with what has been reported for diphenylhydantoin in the spinal cord, autonomic ganglion and neuromuscular junction (ESPLIN, 1957; RAINES and STANDAERT.1966), and for diazepam in
185
Anticonvulsants and hippocampal PTP amphibian ganglia (SUKIA and COSTA. 1973). An unexpected observation was the lack of depression of posttetanic potentiation in the granule cell region at doses of the drugs which clearly depress post-tetanic potentiation in apical dendrites. Moreover, high doses of DPH and diazepam increased granule cell posttetanic potentiation. The results indicate that DPH and diazepam exert selective actions on hippocampal post-tetanic potentiation which are lamina and dosedependent. This selectivity. which was not evident in studies on non-cerebral tissues (WOODBURY, 1968) may imply that post-tetanic potentiation in the stratum radiatum is mechanistically different from that n the granule cells. Commissural afferents to the proximal apical dendrites of CA1 pyramidal ceils apparently originate ‘rom contralateral CA1 neurones. However, the neur,0nal source of commissural afferents to granule cells IS unknown. Perhaps the neurohumoral agents which mediate commissural transmission in these two areas differ. From work with peripheral nerve. post-tetanic potentiation is thought to be a presynaptic phenomenon. a consequence of increased probability of transmitter release by an impulse after rapid, repetitive stimulation of presynaptic elements. Diphenylhydantoin and diazepdm may depress the increased release of certain neurotransmitters, but not others. Compounds that diminish the release of neurotransmitter by post-train impulses would also depress posttetanic potentiation. However. post-tetanic potentiation in brain is probably generated by mechanisms quite different from those which elicit post-tetanic potentiation in peripheral nerves. Brain post-tetanic potentials in response to tetanizing stimuli may be the result of recruitment of active fibres which are normally outside the fringe of excitation by single. isolated stimuli (ESPLIN and ZABLOCKA, 1969). Depression of the post-tetanic potentiation of the negative extracellular potential in pyramidal cell apical dendrites lends indirect. pharmacological support for the hypothesis of ANDEKSEN(1960) that this wave represents a summated EPSP. It would seem reasonable that anti-epileptic agents, such as DPH and diazepam might reduce or prevent the potentiation of EPSPs after a burst of rapid stimuli. The polarity of the field potentials in the area dentata was positive. Positive slow waves have been reported in the granule cell body layer in response to entorhinal stimulation. A late portion of the positive wave was correlated with intracellularly recorded inhibitory postsynaptic potentials (IPSPs. LIIMO, 197 1; ANDERSEE, HOLMQVIST and VOORHOEVE. 1966). Perhaps the positive slow wave we observed after commissural stimulation represents granule cell IPSPs. Additional correlative information on the relationship between extracellular field potentials and synaptic events in the hippocampus would permit a more definitive analysis of the mechanisms of action of these compounds. Doses of DPH and diazepam utilized in this study were within the range of doses which protect rodents
against the tonic phase of maximal electroshock siezures (BANZIGER and HANE, 1967). Qualitatively similar patterns of action of DPH and diazepam against hippocampal post-tetanic potentials were obtained in our experiments, i.e. both compounds depressed posttetanic potentiation in the stratum radiatum, while either not affecting or enhancing post-tetanic potentiation in the area dentata. In these test situations in rim, DPH and diazepam cause similar end-effects on complex systems. However, there is no direct evidence that the drugs elicit their end-effects by the same molecular mechanisms. REFERENCES AIRD, R. B. and WOODBURY. D. M. (1974). The pharmacological approach. In: Thp Manayrment of Epilepsy, (KUGELMASS, I. N., Ed.) pp. 149-238, Charles C. Thomas, Springfield, Illinois. AJMONE-MARSAN, C. and STOLL. J.. JK. (1951). Subcortical connections of the temporal pole in relation to temporal lobe seizures. Arc/~ N>urof. ‘Psyhiut. 66: 669-686. ANDERSON.P. (1959). Interhinuocamoal immdses. I. Origin, course and histribution in cat. raGbit anh rat. Acta phvsiol. .scund. 47: 63-90. ANDERSE~, P. (1960). Interhippocampal impulses. II. Apical dendritlc activation of CA1 neurons. Acttr physiol. scrrnd.
48: 178-m208. ANDERSEN. P.. HOLMQVIST,B. and VOORHOEVE,P. E. (1966). Entorhmal activation of dentate granule cells. .4cm phy\iol. .sctr,~d. 66: 44X-460. BANZIGEK. R. and HANE. D. (1967). Evaluation of a new convulsant for anticonvulsant screening. Archs int. Pharmcody. ThGr. 167: 245-249. BLACKSTAD, T. W. (1956). Commissural connections of the hippocampal region in the rat, with special reference to their mode of termination. J. conlp. Neural. 105: 417536. CAMERMAN,A. and CAMEKMAN,N. (1970). Diphenylhydantoin and diazepam: molecular structure similarities and steric basis of anticonvulsant activity. Scimcr. N.Y. 168: 1457-1458. CRAGC;, B. C. and HAMLYN. L. H. (1955). Action potentials of the pyramidal neurons in the hippocampus of the rabbit. J. Phy.sio/., Land. 129: 60%627. EII)ELBERG, E. (1961). Hippocampal dendritic responses in rabbits. J. Neuroph_vsiol. 24: 521-533. ESPLIN, D. W. (1957). Effects of diphenylhydantoin on synaptic transmission in cat spinal cord and stellate ganglion. J. Pharmnc. up. Thu. 120: 301-323. ESPLIK, D. W. and ZABLOCKA. B. (1969). Effects of tetanization on transmitter dynamics. Epilepsia 10: 193-210. FUJITA. Y. and SAKATA. H. (1962). Electrophysiological properties of CA1 and CA2 apical dendrites of rabbit hippocampus. J. Nrurophrsiol. 25: 209-222. GREEN. J. D. (1964). The hippocampus. Ph!,sio/. Rec. 44: 561k608. GUERRERO-FIGUEROA, R., GALLANT, D. M., GUERREROFIGUEROA, C. and GALLANT, J. (1973) Electrophysiological analysis of the action of four benzodiazepine derivatives on the central nervous system. In: The Bmmdiazepines, (GARATTINI, S.. MUSSINI, E. and RANDALL, L. 0. Eds.). pp. 489-512, Raven Press, New York. IZQUIERDO, I. and NASELLO, A. G. (1970). Pharmacological evidence that hippocampal facilitation, post-tetanic potentiation and seizures may be due to a common mechanism. E:pl Neural. 27: 399409. LoMO. T (1971).Patterns of activation in a monosynaptic cortical pathway: the perforant path input of the dentate area of the hippocampal formation. E.xp/ Bruin Rrs. 12: 18-45.
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LOUIS, S., KUTT, H. and MCDOWIILL, F. (1967). The cardiocirculatory changes caused by intravenous Dilantin and its solvent. At,?. Hr~rr J. 74: 523-529. NICOL. C. F., TUTTON. J. C. and SMITH, B. H. (1969). Parenteral diazepam in status epilepticus. Nr,uroloq~‘, hfinrzrup. 19: 332-343. 0~1)s. M. E. and OLDS. J. (1969). Effects of anxiety-relicving drugs on unit discharges in hippocampus, reticular mid-brain, and pre-optic area in the freely moving rat. I~rt. J. Newopharwc 8: X7-103. RAINES. A. and STANIIAERT, F. G. (1966). Prc- and post.junctional effects of diphenylhydantoin at the cat soleus neuromuscular junction. J. Phurr~~crc. rp. The-. 153: X-366. RAISMAX G.. COWMAN, W. M. and POWELL, T. P. S. (1965). The extrinsic affcrent. commissural and association tibers of the hippocampus. Brtrin 88: 963-996. H. (1971). Intravenous and KUTT, SHARER, L. administration of diazepam. Arch. Neural. 24: 169-175.
SL:KIA, A. and COSTA. E. (1973). Benrodiazepines and posttctanic potentiation in sympathetic ganglia of the bullfrog. Bruin Res. _W 235 239. SWINYARU, E. A. and CASTELLIOX. A. W. (1966). Anticonvulsant properties of some ben7odiazepines. J. Phurwc. e-p. Thu. 151: 369~-375. TOMAN, J. E. P.. SWINYAKII. E. A. and GOOINAN. L. S. (1946). Properties of maximal seizures and their alteration b) anticonvulsant drugs and other agents. J. ,Ymrophy~iol. 9: 23 1 239. WALLIS. W.. KUTT. H. and McDowt~r,, F. (1968). Intravenous diphenylhydantoin in treatment of acute repctitive seizures. Nmdoyy, Mir~nctrp. 18: 5 13-528. WOODR~RY, D. M. (1968). Mechanisms of action of anticonvulsants. In: Basic !Mcchtrr~isn~t of thr Ep~/rpsir.s. (JASPER, H.. WARD, A. and PoI+. A. Eds.). pp. 647-681, Little. Brown, and Co.. Boston.
AND DIAZEPAM RESPONSES*
W. D. MATTHEWS and J. D. CONNOK Department of Pharmacology, The Milton Umversity
College
S. Hershey of Medicine. Hershey.
Medical Center, The Pennsylvania Pennsylvania 17033. U.S.A.
State
Summary-Diphenylhydantoin and diazepam were compared with regard to their effects on responses to single stimuli. and on post-tetanic potentiation of evoked potentials within the hippocampus of anaesthetized rats. fnterhippocdmpaf afferents to pyramidal cells were stimulated electrically, while extracellular field potentials were recorded at successive depths within the contralateral hippocampus. Recording electrodes penetrated perpendicularly from a surf&cc point homotopic to the site of stimulation. The anticonvulsants (40 mg/kg diphenylhydantoin. 5 mgikg diazepam). 30 min after intravenous infusion, depressed post-tetanic potentiation in the stratum radiatum. At these doses responses to single stimuli were essentially unaltered. Doses nearer the toxic range (80 mg/kg diphenylhydantoin. 10 mg/kg diazepam) also reduced post-tetanic potentiation in the stratum radiatum, but increased (up to 150%) post-tetanic potentiation recorded from granule cells in the area dentatd. The results indicate that diphenylhydantoin and diazepam have: (1) selective actions on hippocampal post-tetanic potentiation which are site and dose dependent; (2) qualitatively similar end-effects on post-tetanic potentials in two hippocampal subregions; (3) minimal effects on responses to single stimuli at doses which depress or enhance hippocampal post-tetanic potentiatton.
‘Xphenylhydantoin (Dilantin; DPH; 5,5-diphenyl-2.4.midazolidinedione) and diazepam (Valium; 7-chlorol,3-dihydro-l-mkthyl-5-phenyl-2H-l,4-benzodiazepin?-one) are used clinically to control fhe symptoms of convulsions. Both drugs are effective in terminating status epilepticus (NICOL, TUTTON and SMITH, 1969; WALLIS, KUTT and MCDOWELL, 1968) and in the ‘:hronic management of some forms of seizures (AIRD and WOODBURY,1974). The chemical structures of the compounds appear unrelated in two-dimensional representation. However, CAMERMANand CAMERMAN (1970) in calling attention to similarities in their spatial conformation, suggested that the compounds might exert anti-epileptic effects through interactions with the same unspecified receptor in the brain. Experiments on the responses in spinal and peripheral nerves of laboratory animals seem to indicate that the anticonvulsant action of DPH is related to reduction of post-tetanic potentiation (WOODBURY, 1968). Post-tetanic potentiation is presumed to be a critical event in the spread of excitatory impulses from epileptogenic focal neurones, and to reinforce focal discharges by positive feedback (WO~DBURY, 1968). At doses which have little effect on responses to single stimuli, DPH decreases post-tetanic potentiation in the spinal cord. autonomic ganglion and neuromuscular junction (ESPLIN, 1957; RAINES and STANDAERT,1966). Thus, ESPLIN (1957) proposed that DPH, by reducing post-tetanic potentiation, prevents the spread of abnormal electrical activity from an epi*This research was supported in part Research Grant NS08884 from the National Neurological Diseases and Stroke.
by USPHS Institute of 181
leptic focus to normal brain tissue. Diazepam also depresses post-tetanic potentiation in sympathetic ganglia without affecting the usual responses to single stimuli (SURIA and COSTA, 1973). Diverse effects on electrical activity in limbic system nuclei have been described for diazepam (OLDS and OLDS, 1969; GUERRERO-FIGUEROA.GALLANT, GUERRERO-FIGUERO and GALLANT 1973) but its mechanism of action in seizure control remains obscure. Our experimental goals were to determine if DPH and diazepam modify evoked responses and posttetanic potentiation in the hippocampus, a brain area relevant to epileptogenesis. The hippocampus was chosen as a test site because it has a low threshold for seizure development (GREEN, 1964) and because hippocampal pathways serve as routes for the spread of seizure activity to other brain regions (AJMONEMARSAN and STOLL, 1951). Moreover, DPH and diazepam alter the discharge patterns of hippocdmpal neurones (OLDS and OLDS, 1969; IZQUIERD~ and NASELLO, 1970). In our study, the effects of systemic injections of the drugs were measured by changes in field potentials, since the laminar cytological organization within the hippocampus lends itself to field potential analysis (ANDERSEN, 1959). METHODS Drug effects on hippocampal field potentials were studied in 70 adult rats (2oo-450 g) anaesthetized with urethane (1.5 g/kg, i.p.). The calvarium and overlying cortex were removed bilaterally; exposed hippocampal surfaces were covered with a pool of warm (37°C) mineral oil. Rectal temperature was monitored with
182
W. D. MATTHEWSand J. D. CONNOH
a thermistor probe and maintained at 37 i: 0.5”C by warm water circulated through coiled tubing placed beneath the animal. All solutions were administered through a cannula in a femoral vein by an infusion pump. A tracheal cannula maintained an unobstructed airway. Single stimuli (1 Hz, 0.2 msec duration, twice threshold voltage, or 1@50 V) were delivered through a bipolar stimulating electrode on the surface of the dorsal hippocampus above CA1 pyramidal cells. Post-tetanic potentiation was elicited by trains of stimuli (l&30 Hz, 3-5 set train duration, same voltage and pulse duration as single stimuli). Extracellular field potentials were recorded in the contralateral hippocampus by glass microelectrodes (4 pm tip dia, l.sl.5 MR, filled with 4 M sodium chloride) positioned homotopic to the site of stimulation, and advanced through the CA1 pyramidal cell layer to the granule cell layer of the area dentata. The circuit was completed through a disk electrode in a saline-soaked pad placed against the oral mucosa. A dual beam storage oscilloscope amplified and displayed the signals. Data were recorded on magnetic tape for computer analysis. Diphenylhydantoin sodium (Sigma Chemical Co.) or diazepam (kindly provided by HoffmanLaRoche, Inc.) was dissolved in a vehicle consisting of propylene glycol (400 ml), sodium benzoate (48.8 g) benzoic acid (1.2 g), benzyl alcohol (15 ml), ethyl alcohol (100 ml) and distilled water (sufficient to make a litre). The pH was adjusted to 11.0 with 1~ sodium hydroxide. Drugs were dissolved in the vehicle on the day of the experiment. The doses utilized were anticonvulsant (20 and 40 mg/kg DPH; 2.5 and 5 mg/kg diazepam) against the tonic phase of maximal electroshock, or near the concentrations that cause ataxia (80 mg/ kg DPH; 10 mg/kg diazepam) in rodents (SWINYARD and CASTELLION, 1966; TOMAN, SWINYARD and GWDMAN, 1946). Control infusions consisted of a volume of vehicle equivalent to the amount needed to dissolve the highest dose of each drug. In preliminary experiments, a rate of intravenous infusion of vehicle plus drug which produced minimal and transient changes in mean blood pressure, heart rate and respiration was determined. This information was obtained from 12 anaesthetized rats whose femoral arteries were cannulated and connected to a pressure transducer. Mean blood pressure and heart rate before and during infusion were recorded continuously with a polygraph. A gas analyzer monitored respiratory rate and % CO2 in expired air. Field potentials were averaged by a Technical Instruments CAT 1000. Values for the amplitudes of potentials were obtained by measuring, on the analog printout, vertical heights from baseline to peak deflection. The magnitude of post-tetanic potentiation was expressed as a ratio of the mean amplitude of the first 5 potentials after the train to the mean amplitude of 5 responses just prior to the train. The average magnitude of 335 post-tetanic potentiations just
before drug or vehicle administration represented control (untreated) post-tetanic potentiation. The magnitude of post-tetanic potentiation and single stimuli responses after vehicle or vehicle plus drug was expressed as a percentage of untreated values, Means and standard errors for these percentages were determined, and the effects of a drug or vehicle alone on post-tetanic potentiation and responses to single stimuli were compared statistically by Student’s t test. Probabilities less than 0.05 were considered statistically significant. RESULTS
Rate of infusion Vehicle infusions produced slight hypotension and bradycardia. effects not obtained with saline (Fig. 1). Diazepam, 4 mg/kg at the three rates shown in Figure 1. depressed mean blood pressure by 2(&25x1. Bradysignificant (P < 0.05) at an cardia became administration rate of 360 &min. None of the infusion rates of vehicle alone or vehicle plus diazepam affected respiration significantly. Values in Figure 1 represent maximal changes in the parameters measured. Depression of mean blood pressure and heart rate was transient, beginning during the infusion and returning to pre-infusion values within 5 min. An infusion rate of 71 &min for diazepam in a concentration of 5 mg/ml was chosen. For similar reasons, an infusion rate of 143 $/min was used for DPH (40 mg/ml). Laminar analysis of rat hippocarnpus Figure 2 depicts the results of a typical laminar analysis. Panel A is a schematic diagram of the cellular elements in the CA1 region and the area dentata. The pattern of field potentials at various depths is 0 HEART RATE mBLOOD PRESSURE 0 RESPIRATION RATE
i
”
360 Soline
360
Vehicle
71
_
143
360
Vehicle t Diozepom INFUSION RATE (~l/min)
Fig. I. Effects of intravenous diazepam and its vehicle on vital functions in the rat. A constant volume (0.8 ml/kg) of saline. propylene glycol vehicle, or vehicle -t diarepam (4 mg/kg) was infused at the rates given on the abscissa. The effects on heart rate. mean blood pressure. and respiratory rate are shown by bars representing the means of six experiments. Asterisks indicate a statistically significant change from untreated control (P < 0.05by paired t-test).
Anticonvulsants
and hippocampal
183
PTP POSfTrOl”
Pretmi”
Elecrrode P051+10”,
Pm
230
A\
-
L-J---
i!
A ‘Lf-----
P
ISOPo+e”+,(l,
0
F‘lg. 2. Laminar analysis of rat hippocampus A: Schematic c!iagram of major cell types encountered in vertical Fenetration by a microelectrode through CA1 region of kippocampus and area dentata. (P) CA1 pyramidal cell klody, (a) axon. (b.d.) basal dendrite, (a.d.) apical dendrite, (:om.) afferent commissural fibres from contralateral CA1 pyramidal cells. (G) granule cell of area dentata. Depth below alveus scaled on the right. B: Field potentials, in hippocampal stimuli, were response to contralateral recorded at the depths indicated. Positivity upward. C: f\mplitude of the field potentials in millivolts, plotted against recording electrode depth. Amplitudes were mea:.ured at a time post-stimulus indicated by the vertical dashed line in B.
in B. Electrodes passing through the stramonophasic positive potentials with negative inflections on the rising phase. An iso2otential point invariably appeared just beneath the ,;tratum pyramidale. In the stratum radiatum a large negative wave superimposed on a positive wave was recorded. The negative component had its peak amplitude in the proximal apical dendrites. Single stimuli evoked a monophasic positive wave in the area dentata. The isopotential depth from the alveal surface varied by + 50 pm from animal to animal. Tissue swelling during the experiments made absolute depth measurements unreliable. Therefore. the position of the tip of the recording electrode within hippocampal laminae was always referenced to the distance from the isopotential point. A representative summary of the amplitude of the waves at various depths within the hippocampus is given in Figure 2C. The positive wave in the stratum oriens reached maximum amplitude about 150 pm beneath the surface of the hippocampus. The isopotential point occurred at a depth of 250 pm. The negative wave in the stratum radiatum became maximal at 400 pm. The granule cell potential (depth of 800 ‘urn in Fig. 29) had a shorter latency than potentials recorded in strata associated with pyramidal cells or presented
I urn oriens recorded
their
processes.
Diphenylhydantoin and diazrpam on hippocumpal postManic potentiation After repetitive stimulation of commissural afferents, post-tetanic potentiation of field potentials within the hippocampus was observed at all recording
Fig. 3. Laminar analysis of post-tetanic potentiation. The position of the recording electrode within the hippocampus is given relative to the isopotential point (designated by 0). Field potentials at each depth are given in the ‘pretrain’ column. The ‘posttrain’ column shows potentiated responses after a train of stimuli (10 Hz for 3 set, twice threshold voltage). Note post-tetanic potentiation was greatest in the apical dendrites and the area dentata.
sites (Fig. 3). Post-tetanic potentiation was largest and most reproducible at 200 pm (stratum radiatum) and 600 pm (area dentata) below the isopotential point, so these two regions were chosen to study drug actions. Diphenylhydantoin depressed, in a dose-dependent manner, post-tetanic potentiation in the stratum radiatum. (Fig. 4). Depression was not observed in the area dentata; rather, dose-dependent increases in post-tetanic potentiation were produced by the drug (Fig. 4). Diphenylhydantoin (20 mg/kg) did not facilitate granule cell post-tetanic potentiation. However, 40 mg/kg caused a 40% increase in post-tetanic potentiation. Infusions of 80 mg/kg resulted in a 150% enhancement of post-tetanic potentiation. Diazepam
c
radlatum
i
200
1
q Stratum
250
0
Area
dent&a
1
Diphenylhydantoin,
80
m/kg
Fig. 4. Diphenylhydantoin effects on hippocampal posttetanic potentiation. The ordinate represents the percent of control (untreated) post-tetanic potentiation (PTP) observed in the stratum radiatum or area dentata, 30 min after intravenous infusion of vehicle or diphenylhydantoin. The heights of the bars represent mean responses; vertical lines are standard errors (6 animals at each dose). A statistically significant (P < 0.05 by Student’s t-test) change from vehicle is denoted by an asterisk.
184
W. D. MATTHEWSand J. D.
Vehicle
5 Dlazepam,
IO
mdkg
Fig. 5. Diazepam effects on hippocampal post-tetanic potentiation. Ordinate represents percent of control (untreated) post-tetanic potentiation in the stratum radiatum or area dentdta 30 min after intravenous infusion of vehicle or diazepam. The heights of the bars represent mean responses; vertical lines are the standard errors (6 animals at each dose). Statistically signilicant (P -c 0.05 by Student’s t-test) changes from vehicle are denoted by asterisks.
reduced post-tetanic potentiation in the stratum radiatum at 5 and 10 mg/kg doses (Fig. 5). At 10 mg/kg, diazepam increased granule cell post-tetanic potentiation by SSo/;, (Fig. 5). The effects of lower doses of diazepam (2.5 and 5 mg/kg) on granule cell post-tetanic potentials were not statistically significant. Diphenylhydantoin and diuzepam ou stimuli
~q~on.srs
to
single
Diphenylhydantoin (20 and 40 mg,kg) had negligible effects on the amplitudes or latencies of field potentials evoked in the stratum radiatum or the area dentata by 1 Hz stimuli. In some instances, the highest dose of DPH (80 mg/kg) reduced field potential amplitudes by as much as 20%, but this response was not sufficiently consistent to be of statistical significance. Infusions of diazepam (25, 5. 10 mg/kg) did not depress the amplitudes of the field potentials elicited by single stimuli at either recording site.
DISCUSSION
The task of obtaining quantitatively reproducible field potentials is made somewhat easier if the experimental animals are anaesthetized. Presumably, general anaesthetic agents exert some effects of their own in setting the level of neurone excitability and by interacting with other drugs. Although the rats in this study were anaesthetized with urethane, laminar analyses within the hippocampus and posttetanic potentiation of the evoked waves were highly reproducible. Moreover, dose-dependent changes in electrical activity were elicited by systemic injections of DPH or diazepam after reasonable latencies. However, it seems probable that the parameters of stimu-
CONNOR
lation and the effective doses of the drugs would be different in unanaesthetized animals. The propylene glycol vehicle, when given intravenously. depressed the cardiovascular system. The extent of hypotension and bradycardia in the rat was similar to that reported for the cat (LOUIS, KUTT and MCDOWELL. 1967; SHARER and KUTT, 1971). These effects were enhanced by diazepam; thus, a slow rate of infusion was chosen to minimize adverse cardiovascular responses. Drug actions were evaluated 15 min or more after the end of the infusion, at which time blood pressure and heart rate had returned to preinjection values. For these reasons. the effects of the anticonvulsants on hippocampal potentials cannot be explained simply by diminished perfusion of the brain. However. when a drug is given systemically and responses are measured in a specific region of the brain. a possibility exists that the effects are secondary to an action of the drug at another brain site. In these studies we cannot eliminate this possibility. Field potentials in rat hippocampus in response to commissural stimuli resemble those of other species (CRAGG and HAMLYN, 1955; ANDERSEN, 1960; EIDELBERG, 196 1; FUJITA and SAKATA. 1962). The maximum amplitude of the negative potential in the stratum radiatum was recorded near the termination of the afferent commissural fibres from contralateral pyramidal cells. ANDERSEN(1960) interpreted this negative wave as a summated excitatory postsynaptic potential (EPSP) elicited in the apical dendrites of CA1 pyramidal cells by a direct commissural volley. We also obtained an evoked potential in the granule cells of the area dentata upon stimulation of the contralateral hippocampus. BLACKSTAD (1956) and RAISMAN, COWMAN and POWELL (1965) demonstrated interhippocampal fibres to the area dentata which terminate within a well defined zone in the dendritic field of the granule cell layer. The source of these commissural fibres is. however, unknown. The short latency (cu. 5 msec) of the evoked potential in the granule cells during contralateral hippocampal stimulation may be taken tentatively as electrophysiological evidence for a direct commissural connection between these regions. The field potential of the granule cells exhibited the largest post-tetanic potentiation after repetitive stimulation. This large posttetanic potentiation might be explained by the high density of granule somata. Alternatively, volleys arriving through the commissural pathway may have a high probability for activating a large percentage of the total number of synapses within a discrete zone of the granule cells (BLACKSTAD, 1956). The observations that DPH and diazepam depress post-tetanic potentiation in the stratum radiatum without affecting significantly the responses to single stimuli agree with what has been reported for diphenylhydantoin in the spinal cord, autonomic ganglion and neuromuscular junction (ESPLIN, 1957; RAINES and STANDAERT.1966), and for diazepam in
185
Anticonvulsants and hippocampal PTP amphibian ganglia (SUKIA and COSTA. 1973). An unexpected observation was the lack of depression of posttetanic potentiation in the granule cell region at doses of the drugs which clearly depress post-tetanic potentiation in apical dendrites. Moreover, high doses of DPH and diazepam increased granule cell posttetanic potentiation. The results indicate that DPH and diazepam exert selective actions on hippocampal post-tetanic potentiation which are lamina and dosedependent. This selectivity. which was not evident in studies on non-cerebral tissues (WOODBURY, 1968) may imply that post-tetanic potentiation in the stratum radiatum is mechanistically different from that n the granule cells. Commissural afferents to the proximal apical dendrites of CA1 pyramidal ceils apparently originate ‘rom contralateral CA1 neurones. However, the neur,0nal source of commissural afferents to granule cells IS unknown. Perhaps the neurohumoral agents which mediate commissural transmission in these two areas differ. From work with peripheral nerve. post-tetanic potentiation is thought to be a presynaptic phenomenon. a consequence of increased probability of transmitter release by an impulse after rapid, repetitive stimulation of presynaptic elements. Diphenylhydantoin and diazepdm may depress the increased release of certain neurotransmitters, but not others. Compounds that diminish the release of neurotransmitter by post-train impulses would also depress posttetanic potentiation. However. post-tetanic potentiation in brain is probably generated by mechanisms quite different from those which elicit post-tetanic potentiation in peripheral nerves. Brain post-tetanic potentials in response to tetanizing stimuli may be the result of recruitment of active fibres which are normally outside the fringe of excitation by single. isolated stimuli (ESPLIN and ZABLOCKA, 1969). Depression of the post-tetanic potentiation of the negative extracellular potential in pyramidal cell apical dendrites lends indirect. pharmacological support for the hypothesis of ANDEKSEN(1960) that this wave represents a summated EPSP. It would seem reasonable that anti-epileptic agents, such as DPH and diazepam might reduce or prevent the potentiation of EPSPs after a burst of rapid stimuli. The polarity of the field potentials in the area dentata was positive. Positive slow waves have been reported in the granule cell body layer in response to entorhinal stimulation. A late portion of the positive wave was correlated with intracellularly recorded inhibitory postsynaptic potentials (IPSPs. LIIMO, 197 1; ANDERSEE, HOLMQVIST and VOORHOEVE. 1966). Perhaps the positive slow wave we observed after commissural stimulation represents granule cell IPSPs. Additional correlative information on the relationship between extracellular field potentials and synaptic events in the hippocampus would permit a more definitive analysis of the mechanisms of action of these compounds. Doses of DPH and diazepam utilized in this study were within the range of doses which protect rodents
against the tonic phase of maximal electroshock siezures (BANZIGER and HANE, 1967). Qualitatively similar patterns of action of DPH and diazepam against hippocampal post-tetanic potentials were obtained in our experiments, i.e. both compounds depressed posttetanic potentiation in the stratum radiatum, while either not affecting or enhancing post-tetanic potentiation in the area dentata. In these test situations in rim, DPH and diazepam cause similar end-effects on complex systems. However, there is no direct evidence that the drugs elicit their end-effects by the same molecular mechanisms. REFERENCES AIRD, R. B. and WOODBURY. D. M. (1974). The pharmacological approach. In: Thp Manayrment of Epilepsy, (KUGELMASS, I. N., Ed.) pp. 149-238, Charles C. Thomas, Springfield, Illinois. AJMONE-MARSAN, C. and STOLL. J.. JK. (1951). Subcortical connections of the temporal pole in relation to temporal lobe seizures. Arc/~ N>urof. ‘Psyhiut. 66: 669-686. ANDERSON.P. (1959). Interhinuocamoal immdses. I. Origin, course and histribution in cat. raGbit anh rat. Acta phvsiol. .scund. 47: 63-90. ANDERSE~, P. (1960). Interhippocampal impulses. II. Apical dendritlc activation of CA1 neurons. Acttr physiol. scrrnd.
48: 178-m208. ANDERSEN. P.. HOLMQVIST,B. and VOORHOEVE,P. E. (1966). Entorhmal activation of dentate granule cells. .4cm phy\iol. .sctr,~d. 66: 44X-460. BANZIGEK. R. and HANE. D. (1967). Evaluation of a new convulsant for anticonvulsant screening. Archs int. Pharmcody. ThGr. 167: 245-249. BLACKSTAD, T. W. (1956). Commissural connections of the hippocampal region in the rat, with special reference to their mode of termination. J. conlp. Neural. 105: 417536. CAMERMAN,A. and CAMEKMAN,N. (1970). Diphenylhydantoin and diazepam: molecular structure similarities and steric basis of anticonvulsant activity. Scimcr. N.Y. 168: 1457-1458. CRAGC;, B. C. and HAMLYN. L. H. (1955). Action potentials of the pyramidal neurons in the hippocampus of the rabbit. J. Phy.sio/., Land. 129: 60%627. EII)ELBERG, E. (1961). Hippocampal dendritic responses in rabbits. J. Neuroph_vsiol. 24: 521-533. ESPLIN, D. W. (1957). Effects of diphenylhydantoin on synaptic transmission in cat spinal cord and stellate ganglion. J. Pharmnc. up. Thu. 120: 301-323. ESPLIK, D. W. and ZABLOCKA. B. (1969). Effects of tetanization on transmitter dynamics. Epilepsia 10: 193-210. FUJITA. Y. and SAKATA. H. (1962). Electrophysiological properties of CA1 and CA2 apical dendrites of rabbit hippocampus. J. Nrurophrsiol. 25: 209-222. GREEN. J. D. (1964). The hippocampus. Ph!,sio/. Rec. 44: 561k608. GUERRERO-FIGUEROA, R., GALLANT, D. M., GUERREROFIGUEROA, C. and GALLANT, J. (1973) Electrophysiological analysis of the action of four benzodiazepine derivatives on the central nervous system. In: The Bmmdiazepines, (GARATTINI, S.. MUSSINI, E. and RANDALL, L. 0. Eds.). pp. 489-512, Raven Press, New York. IZQUIERDO, I. and NASELLO, A. G. (1970). Pharmacological evidence that hippocampal facilitation, post-tetanic potentiation and seizures may be due to a common mechanism. E:pl Neural. 27: 399409. LoMO. T (1971).Patterns of activation in a monosynaptic cortical pathway: the perforant path input of the dentate area of the hippocampal formation. E.xp/ Bruin Rrs. 12: 18-45.
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LOUIS, S., KUTT, H. and MCDOWIILL, F. (1967). The cardiocirculatory changes caused by intravenous Dilantin and its solvent. At,?. Hr~rr J. 74: 523-529. NICOL. C. F., TUTTON. J. C. and SMITH, B. H. (1969). Parenteral diazepam in status epilepticus. Nr,uroloq~‘, hfinrzrup. 19: 332-343. 0~1)s. M. E. and OLDS. J. (1969). Effects of anxiety-relicving drugs on unit discharges in hippocampus, reticular mid-brain, and pre-optic area in the freely moving rat. I~rt. J. Newopharwc 8: X7-103. RAINES. A. and STANIIAERT, F. G. (1966). Prc- and post.junctional effects of diphenylhydantoin at the cat soleus neuromuscular junction. J. Phurr~~crc. rp. The-. 153: X-366. RAISMAX G.. COWMAN, W. M. and POWELL, T. P. S. (1965). The extrinsic affcrent. commissural and association tibers of the hippocampus. Brtrin 88: 963-996. H. (1971). Intravenous and KUTT, SHARER, L. administration of diazepam. Arch. Neural. 24: 169-175.
SL:KIA, A. and COSTA. E. (1973). Benrodiazepines and posttctanic potentiation in sympathetic ganglia of the bullfrog. Bruin Res. _W 235 239. SWINYARU, E. A. and CASTELLIOX. A. W. (1966). Anticonvulsant properties of some ben7odiazepines. J. Phurwc. e-p. Thu. 151: 369~-375. TOMAN, J. E. P.. SWINYAKII. E. A. and GOOINAN. L. S. (1946). Properties of maximal seizures and their alteration b) anticonvulsant drugs and other agents. J. ,Ymrophy~iol. 9: 23 1 239. WALLIS. W.. KUTT. H. and McDowt~r,, F. (1968). Intravenous diphenylhydantoin in treatment of acute repctitive seizures. Nmdoyy, Mir~nctrp. 18: 5 13-528. WOODR~RY, D. M. (1968). Mechanisms of action of anticonvulsants. In: Basic !Mcchtrr~isn~t of thr Ep~/rpsir.s. (JASPER, H.. WARD, A. and PoI+. A. Eds.). pp. 647-681, Little. Brown, and Co.. Boston.
