Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by UNIV WINDSOR on 11/09/14 For personal use only.
cerebral cortical neanrones to antative neurotrans Dcyirrrmerzt cf Armaesrhesia Resecarch, 1lfc6ill University, Montreal, F.Q., Canada H 3 6 I Y B Received May 28, 1979 NESTOMOS, J. No, and NISTRI,A. 1979. EfTects sf micrsisntsphoretically applied flurazepam on responses of cerebral cortical neurones to putative neurotransmitters. Can. J. Physiol. Pharmacsl. 57, 1324-1 329. Utilizing standard rnicroiontophoretic techniques and recording extracellularly in cats, we studied the effects of Blurazepam, a water-soluble benzodiazepine, on the spike activity of single cerebral neurolaes and its interactions with several excitatory and inhibitory putative neurotransmitters. Large iontc~phoreticdoses (5-30 nA, 0. I A4 solution) of flurazepam induced a depression of spike amplitude. Smaller doses (less than 5 nA, 0.1 M solution or 20-50 nA, 20 rnM in 0.16 '44NaCl) seduced the excitation procluced by glutamate, aspartate, and homocysteate, but antagonism of acetylcholine-evoked excitations required large flurazepam doses (up to 30 nA, 0.1 A4 solution). Even lower doses of flurazepa~n( < 10 nA, 20 miM in 0.16 A4 NaCl) enhanced the inhibitory effect of r-aminobutyric acid (GABA) but aastagoasized that of 5-hydroxytryptamirte, and had no eirect on dopamine-induced inhibition of firing. Hence, only GARA-evoked inhibitions were significantly potentiated by flurazepam. These results demonstrate the multiple possible interactions between a benzodiazepine and different putative neurotransmitters in the maminalian cerebral cortex.
Methods Experiments were carried out on 14 cats (2-3 kg of either In the brain, benzodiazepi~seagents appear to bind The animals were anaesthetized with intraperitoneal to membrane sites distinct from those binding krsown sex). alloharbitone, inhaled methoxyflurnne, or intravenous chlonetarotrans~nitters (Braest rup and Squires 1977, ralose, and usually breathed spontaneously. When the results 1 978; MtjhIer and Okada 1 977). Benzodiazepines obtained under these three types of anaesthetics were comalso enhance the effects of iontophoretically applied pared. we noted no difference in the observed rsponse.;. ReGABA on cortical neurones (Kszhechkin and Os- cordings were obtained from the pericruciate cortex (preaasd post-cruciate), the surface of which was continuously trovskaya 1977; Zakusov et al. 7975; Zakusov et ak. irrigated with artificial cerebrospinal fluid at 37°C. A pres1977). This enhancement is by no means restricted sure foot applied on the cortical area helped to minimize to the cortex as it can be demonstrated in the brain- pulsations. After the pia was carefully opened, a rnultistern (Dray and Straugkan 1976), cuneate nucleus barrelled glass microelectr-ode waq advanced into the cortex an electronically driven micromanipulator, in steps of (Pok and Haefely l W 6 ) , dorsal raphe nucleus with 2-4 pm. The central barrel of the microelectrode contained ( Gallager 1978), and spinal cord (Choi et al. 1977; 2 M WaCl to record extracellular action potentials which Macdonald and Barker 1977; Nistri and Constanti were amplified, gated, electronically counted. and displayed l!Xi"i'a). In the present study we try to answer the on an oscilloscope and a pen recorder. The outer barrels question whether in the mammalian cerebral cortex contained the following: GARA ( 1 M , pH 4 ) , flurazepam almane@, Hoffman La Roche Ltd.) (either the action of benzodazepines is selectively exerted 0.1 A f in distilled water at p H 3.5 o r 20 mrM in 0.16 iW towards GABA responses or whether it can involve NaCI, same pH), sodium L-glutanaate (1 -a.p H 81, sodium other putative transmitters. Flurazepam was the DI,-homocysteate (0.15 M, pH 8 ) , sodium L-aspartate 4 8 M , benzodiazepine lased in our experiments because its pH 8B, acetylcholine chloride ( 1 34, pH 41, 5HT (creatine'ne sulphate salt; 40 mM, pH 5 ) , D h (HCI salt: 0.4 M, p H 5 1 , water solukflity allows it to be used for iontophoretic NaC1 (0.2 34 for current controls). All these drugs were applications. ejected isntophoretically by a constant current unit (conABRREVI.BTBBNS: GARA, y-aminobutyric acid; 5HT, 5hydrsxytryptamine; DA, dopamine. 'Present address: Department of Pharmacology, St. Barthslslmew's Hospital Medical School, University of London, Gharterhorase Square: London EClM 6BQ. En&land.
$1
structed by Mr. J . Knowles, Ejectronics Laboratory, McIntyre Building, McGill University) which provides continuous monitoring of barrel resistance, a high driving voltage ( u p to 250 %1), and current compelasaton. Retaining currents o f about 80 nA were used to minimize drug leakage from the electrodes. A n Ag-AgG1 ground electrode was inserted in the neck muscles.
0008-4ai2/79/1 a 1324-06$01. O C B ~ O 8979 National Research Council of Canada/Conseil national de recherches du Canada
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by UNIV WINDSOR on 11/09/14 For personal use only.
COMMUNICATION
FLU
2nA
FIG. 1. Pen recorder tracmgs of firing responses of single cortical neurones induced by ghtaanate (GEU) and aspartate (ASP) in (A) or by glutamate and homocysteate (HOhl) in (B) (ditkrent neurone from (A)). Note depression of these responles in the presence of flurazepam and the slow recovery time. The ekfect of iontophoretically applied GAWA and flurazepam on responces of a cortical neurone to repeated administrations of glutamate (60 nAg is \hswn in (C). Note the inhibitory action of GABA which is dependent on the iontophoretic dose. Flurazepam (6 nA, 24) rnM in 0.16 M NaCl solution) has little effect on glutarnate responses, but clearly enhances the inhibitory action of GABA (6 nA).
Results these responses was seen. The spike amplitude sf the Efiects of Flurazepam on Responses evoked by Ex- amino acid response was progressively decreased and temporarily suppressed, suggesting a depdarizacitatory A rmho A cicls or A cetylcholine Although spontaneously active neurones were tion block or a local anaesthetic effect of flurazepam. frequently encountered during a microelectrode pen- As the latency of the amino acid respcmses was etration into the cortex, the vast majority of neurones slightly reduced by fluaazepam, a depolarizing action (at depths between 0.8 and 1.5 mm) was recorded of flurazeparn (eventually leading to blocking of firfollowing repeated (or constant) iontophoretic ap- ing) seems more likely. In order to avoid these problenns, much smaller plications of excitants such as glutamate, aspartate, or homocysteate. Ejecting iontophoretic currents Wurazeparra currents had to be used (less than 5 nA were adjusted so that closely reproducible firing rates through 0.1 M solution or by utilizing 20 m M BHurazwerc observed. i n this way we avoided the inevitable epam in 0. % 6 M NaCL) ; when these precautions were fluctuations in spike activity of spontaneously active taken, the action of glutannate (and other excitants) cells sftcn found during prolonged recordings. When was apparently diminished by Murazepam in a reapplied in relatively large doses (5-30 nA from 0.1 versible manner with no depression of the spike conh4 solutions), flurazepam frequently depressed the figuration. Pen recorder tracings of neuroamal firing spike amplitude and then blocked spontaneous spike rates evokcd by glutarnate and aspartatc (in A ) or activity. When similar applications were performed in glutanlate and hornocysteatc (in B ) are shown in conjunction with repeated administrations of excita- Fig. 1 : flurazepam (0. I h4 solution) inhibited these tory amino acids, which werc giving stable submaxi- responses within 20-30 s, an aff'ect that had a rather ma1 responses, temporary or reversible depression of slow recovery (up to 20 min). In many instances,
CAW. J. PHYSIOL. PHARMACBL. VOL. 57, 8979
TABLEI. Interactions of flurazepam with various excitants or inhibitors of cortical neurones
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by UNIV WINDSOR on 11/09/14 For personal use only.
Number of cells tested Glutamate Hornocysteate Aspartate Acetylcholine Spontaneous GABA
5HT DA
No change
Increased *
Excitatory responses in the presence of flurazepam 28 15 2 6 6 4 1 Inhibitory responses in the presence of flurazeyam 16 16 10 1 2 12
7
3
Decreased* 28 13
6 6 3
7 2
*The terms "increased" or "decreased" indicate a change of more than 10% in the overall tesponse in pen recorder tracings (duration multiplied by half-peak amplitude).
the flurazepam-induced depression could not be counteracted by increasing the amino acid ejecting currents. In addition, this kind of flurazepam-induced depression did not show selectively towards any of the three amino acids which were applied in such a manner that submaximal matched responses were always obtained. It may be mentioned here that Zakusov et a/. (1997) found no interaction of intravenous diazepam with glut amate responses of cortical neurones. However, the experimental conditions used by these workers (e.g., intravenous diazepan1 given to unanaesthetized rabbits) are clearly different from those of our study. Another effect noted by us during this type of flurazepam application was that neurones which initially showed a spontaneously bursting spike activity appeared to fire in a steady manner following iontophoretic application of flurazepam. This effect was usually reversible within 2-3 min from the end of flu raze pan^ application. A number of neurones firing spontaneously at low rates were excited by iontophoretically applied acetylcholine. The acetylcholine responses were typically slower in onset than the responses to excitatory amino acids and often had a phase of transient firing depression preceding an excitatory phase outlasting the application period -(a. IgrwpviC a n d 76ilEs 1963 ) . These acetylcholine responses were also antagonized by flurazepam, though relatively large doses (up to 38 nA, 8.1 M solution) were required to achieve such depressions without a concomitant reduction in spike size.
during sustained application of GABA (cf., for example, effects of 6 nA GABA in Fig. 1@) ; a transient rebound of spike activity was often seen shortly after the end of GABA application. Flurazepana (6 nA through 20 m M in 0.16 ha NaCl solution) had little detectable effect on glutamate-evoked responses but clearly enhanced the peak depression due to GABA (Fig. 1C) without altering the fading of the GABA response. In ~ r d e to r examine the specificity of flurazepam actions on GABA-evoked inhibitions, inhibitory responses induced by monoamines were also studied in the same neurones. Both 5HT and DA reversibly inhibited neuronal firing, although these substances were apparently weaker than GABA (as judged on the basis of the iontophoretic current used) and depressed only two-thirds of the neurones examined (the action of GABA was ubiquitous). The majority of DA responses were unaffected by the presence of flurazepam (see Table 1 ) ; in the remaining cases either a slightly increased inhibition due to reduction of fading (cf. Fig. 2 F and G ) or some antagonism was noted. Flurazepam (5 nA through 20 lnlM in 8.16 M NaCl solution) often antagonized (in a reversible manner) the inhibitory responses to 5HT (Tab@ 1 ): I t i s shown-in Fig, 2 t h a t i r x the s a m e &xrone flurazepam could potentiate GABA, antagonize 5HT, and prolong the action of DA; a larger flurazegam dose reduced glutamate-evoked responses (but not spike amplitude). A summary of the action of flurazepam on responses to iontophoretically applied transmitters is given in Table I.
Eflects of Fkurazeparn on Inhibitory Responses ind~lcedby GABA, 5 H T , and D A Iontophoretica~lyapplied GABA depressed the neuronal firing induced by glutamate or homocysteate. Such a depression had a rapid onset and faded
Discussion It is not totally surprising to observe a variety of flurazepam effects, related to the dose administered. Large doses (presumably achieved with large cur-
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by UNIV WINDSOR on 11/09/14 For personal use only.
COMMUNICATION
G -?I
1327
GLU
F L U 5nA
GABA OnA ,120
F kbl 5nA c GABA 0 n A
FIG. 2. Effects of different inhibitory substances on firing responses of a single cortical neurone to glutamate (GLTJ, 19 nA). (A-C) Flurazepam (FLU, 5 nA from a 20 m21 in 0.16 kfNaCl solution) or GABA applied by removing its
retaining current have a slight inhibitory effect which is greater when the two substances are simultaneously applied. (19) Increasing iontophoretic doses of 5HT inhibit glutamate responses; this inhibition ic reversed by concomitant application of flurazepam (5 nA) in (E). (F) Inhibition evoked by DA (5 nA); note fading of this eflect. Flurazepam (5 nA) in (G) eliminates this fading. (H) Depressant effect of a larger flurazepam (20 I ~ solution M in 0.16 hf NaCl) iontophoretic dose (20 nA) on glutamate responses.
rents through concentrated solutions) apparently interfered with the spike-generating mechanisms of the neurones as shown by the depression of spike amplitude and the lowering of the threshold for depolarization block (cf. also Curtis et al. 1976) . In view of the large currents needed we also consider the depression of acetylcl~olineresponscs a rather unspecific effect of flurazepam. When smaller doses of flurazepam are used, responses to excitatory amino acids could be reversibly diminished, with no change in spike amplitude. However, if we consider as the most powerful effects of flurazepam those elicited by the smallest iontophoretic currents, it is evident that the main action of this benzsdiazepine was an enhancement of neuronal inhibitions, particularly those evoked by GABA. It is interesting to compare the GABA-enhancing potency of flurazepam under in vivo and in vitro conditions. This might disclose whether the present results of in vivo iontophoretic experiments were obtained with flurazepam doses similar to those used previously (Nistri and Constanti 1978a) in vitro.
With bath applications of flurazepam to frog spinal neurones in vitro, a 2.5 pM concentration was found to potentiate selectively the action of GABA (Nistri and Constanti 1978a, 1978b). It is difficult to know the actual concentration ( C ) of Blurazepam attained in viva with iontophoretic ejections. An approximation may be obtained from the diffusion equation adopted for the case of iontophoretic release from a "point source" for a given time ( t ) (Jaeger 1965 ) . This equation can provide the concentration of the substance (C) 1 s ( t ) after the application ( t , ) has been stopped. This approach is perhaps more meaningful as we are dealing with a nonequilibrium distribution of iontophoretic substances and therefore we can only estimate the final concentration which builds up during the whole application. C
=
erfc -i-
.
4rrmDr(
-
2(0:)1/
-
-
erfc
z[o(r
I
r,)11/3
where rn (the rate of iontophoretic release per unit time) IT/Fn ( I current. T = transport number, F = Faraday constant, n = valency). We have not
1328
CAW. J. PHYSIOL. PHARMACOL. VQL. 57, 1979
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measured the value of T for flurazeparn although it is likely to be similar to that of other polycyclic agents such as chlorpromazinc, haloperidol, and imiprarnine, whose transport numbers range betwcen 0.04 and 8.08 (Zieglg5nsberger et aE. 1974). If we assume an average of 0.05 for flurazeparn, we shall have to divide this by 80 as we used a 165 niM NaCB solution and therefore 20 rnM Aurazepan~may be expccted to carry only about ,l,,th of the current Wowing through the solution. Hence, the effective transport number may be as low as 0.005. By assurning this value and a 5-nA current, we can directly substitute the other values into the above equation if we consider that r (distance from the electrode tip) is 50 pm, the diffusion coefficient (D)is B0-%crraVs (cf. Curtis pt al. 1960), and tl is 135 s (cf. Fig. 1C). The estimated concentration is then 3 pM, a value close to the effective dose in in vitro experiments on the frog spinal cord. In practice, the concentration attained in vivo might have been even smaller as both T and r are probably overestimated. Our present results thus confirm the high sensitivity of responses elicited by GABA to bath-applied flurazepam (Nistri and Constaisti 1978a, 19778). The enhancement of GABA effects by flurazepam was undoubtedly occurring at a postsynaptic level (either on GABA receptors or ionophores) (Costa and Guidottj 1979) ; howcvcr, some facilitation of endogenous GABA release rnight also play a role in this phenomenon (Ncstoros and Nistri 1978). Additional useful data on flurazepam-GABA interactions might be provided by studies with GABA receptor antagonists; unfortunately the poor antagonistic properties of bicuculline and picrotoxin in the cerebral cortex (Gndfriand ct al. 1978; Hill et al. 1973) would not allow us to reach reliable conchsions. A novel finding of the present study was the antagonism by flurazepam of 5HT-evoked corticaZ inhibitions contrasted by the relative insensitivity of DA-evoked inhibitions to iontophoretically applied flurazepam. This indicates that the observed potenAaticm aF GABA iffhibitions wasquite-specific a n d not extended to other chemically evoked inhibitory responses. Furthermore, this effect of flurazeparn on 5HT responses is in keeping with behavioural observations on the interactions of benzodiazepines with central 5HT-containing neurones (Stein et ak. 1973). In conclusion, cortical inhibitions are more sensitive than cortical excitations to flurazepam. Among the inhibitory responses, those induced by GABA are particularly susceptible to this benzodiazepine, which probably acts by increasing the effectiveness
of GABA on postsynaptic sites (receptors or ionophores). However, other inhibitory responses (e.g., due to 5HT) may be affected by flurazepam in concentrations which still leave the excitatory responses unchanged. Acknowledgments Wc thank Professor K. KrnjeviC for helpful discussions and suggestions and Mr. N. Schestakowich for skilful photography. Flurazcpam hydrochloride was kindly donated by Hoffman La Roche Ltd., Montreal. The authors were Fellows of the Medical Research Council of Canada. These results will be submitted to McGill University by J.N.N. as part of a Ph.D. thesis. BRAESIRUP, C., and 5yum~~s, R. F. 1977. Specific benzodiazepine receptors in rat brain characterized by highafinity (%) diazepam binding. Proc. Natl. Acad. Sci. U.S.A. 94, 3805-3809. 1978. Pharmacological characterization of benzodiazepine receptors in the brain. Eur. J. Pharmacol. 48, 263-270. CHOI, D. W., FARB,D. H., and Fnscnr~~c~, G. D. 1977.
cerebral cortical neanrones to antative neurotrans Dcyirrrmerzt cf Armaesrhesia Resecarch, 1lfc6ill University, Montreal, F.Q., Canada H 3 6 I Y B Received May 28, 1979 NESTOMOS, J. No, and NISTRI,A. 1979. EfTects sf micrsisntsphoretically applied flurazepam on responses of cerebral cortical neurones to putative neurotransmitters. Can. J. Physiol. Pharmacsl. 57, 1324-1 329. Utilizing standard rnicroiontophoretic techniques and recording extracellularly in cats, we studied the effects of Blurazepam, a water-soluble benzodiazepine, on the spike activity of single cerebral neurolaes and its interactions with several excitatory and inhibitory putative neurotransmitters. Large iontc~phoreticdoses (5-30 nA, 0. I A4 solution) of flurazepam induced a depression of spike amplitude. Smaller doses (less than 5 nA, 0.1 M solution or 20-50 nA, 20 rnM in 0.16 '44NaCl) seduced the excitation procluced by glutamate, aspartate, and homocysteate, but antagonism of acetylcholine-evoked excitations required large flurazepam doses (up to 30 nA, 0.1 A4 solution). Even lower doses of flurazepa~n( < 10 nA, 20 miM in 0.16 A4 NaCl) enhanced the inhibitory effect of r-aminobutyric acid (GABA) but aastagoasized that of 5-hydroxytryptamirte, and had no eirect on dopamine-induced inhibition of firing. Hence, only GARA-evoked inhibitions were significantly potentiated by flurazepam. These results demonstrate the multiple possible interactions between a benzodiazepine and different putative neurotransmitters in the maminalian cerebral cortex.
Methods Experiments were carried out on 14 cats (2-3 kg of either In the brain, benzodiazepi~seagents appear to bind The animals were anaesthetized with intraperitoneal to membrane sites distinct from those binding krsown sex). alloharbitone, inhaled methoxyflurnne, or intravenous chlonetarotrans~nitters (Braest rup and Squires 1977, ralose, and usually breathed spontaneously. When the results 1 978; MtjhIer and Okada 1 977). Benzodiazepines obtained under these three types of anaesthetics were comalso enhance the effects of iontophoretically applied pared. we noted no difference in the observed rsponse.;. ReGABA on cortical neurones (Kszhechkin and Os- cordings were obtained from the pericruciate cortex (preaasd post-cruciate), the surface of which was continuously trovskaya 1977; Zakusov et al. 7975; Zakusov et ak. irrigated with artificial cerebrospinal fluid at 37°C. A pres1977). This enhancement is by no means restricted sure foot applied on the cortical area helped to minimize to the cortex as it can be demonstrated in the brain- pulsations. After the pia was carefully opened, a rnultistern (Dray and Straugkan 1976), cuneate nucleus barrelled glass microelectr-ode waq advanced into the cortex an electronically driven micromanipulator, in steps of (Pok and Haefely l W 6 ) , dorsal raphe nucleus with 2-4 pm. The central barrel of the microelectrode contained ( Gallager 1978), and spinal cord (Choi et al. 1977; 2 M WaCl to record extracellular action potentials which Macdonald and Barker 1977; Nistri and Constanti were amplified, gated, electronically counted. and displayed l!Xi"i'a). In the present study we try to answer the on an oscilloscope and a pen recorder. The outer barrels question whether in the mammalian cerebral cortex contained the following: GARA ( 1 M , pH 4 ) , flurazepam almane@, Hoffman La Roche Ltd.) (either the action of benzodazepines is selectively exerted 0.1 A f in distilled water at p H 3.5 o r 20 mrM in 0.16 iW towards GABA responses or whether it can involve NaCI, same pH), sodium L-glutanaate (1 -a.p H 81, sodium other putative transmitters. Flurazepam was the DI,-homocysteate (0.15 M, pH 8 ) , sodium L-aspartate 4 8 M , benzodiazepine lased in our experiments because its pH 8B, acetylcholine chloride ( 1 34, pH 41, 5HT (creatine'ne sulphate salt; 40 mM, pH 5 ) , D h (HCI salt: 0.4 M, p H 5 1 , water solukflity allows it to be used for iontophoretic NaC1 (0.2 34 for current controls). All these drugs were applications. ejected isntophoretically by a constant current unit (conABRREVI.BTBBNS: GARA, y-aminobutyric acid; 5HT, 5hydrsxytryptamine; DA, dopamine. 'Present address: Department of Pharmacology, St. Barthslslmew's Hospital Medical School, University of London, Gharterhorase Square: London EClM 6BQ. En&land.
$1
structed by Mr. J . Knowles, Ejectronics Laboratory, McIntyre Building, McGill University) which provides continuous monitoring of barrel resistance, a high driving voltage ( u p to 250 %1), and current compelasaton. Retaining currents o f about 80 nA were used to minimize drug leakage from the electrodes. A n Ag-AgG1 ground electrode was inserted in the neck muscles.
0008-4ai2/79/1 a 1324-06$01. O C B ~ O 8979 National Research Council of Canada/Conseil national de recherches du Canada
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by UNIV WINDSOR on 11/09/14 For personal use only.
COMMUNICATION
FLU
2nA
FIG. 1. Pen recorder tracmgs of firing responses of single cortical neurones induced by ghtaanate (GEU) and aspartate (ASP) in (A) or by glutamate and homocysteate (HOhl) in (B) (ditkrent neurone from (A)). Note depression of these responles in the presence of flurazepam and the slow recovery time. The ekfect of iontophoretically applied GAWA and flurazepam on responces of a cortical neurone to repeated administrations of glutamate (60 nAg is \hswn in (C). Note the inhibitory action of GABA which is dependent on the iontophoretic dose. Flurazepam (6 nA, 24) rnM in 0.16 M NaCl solution) has little effect on glutarnate responses, but clearly enhances the inhibitory action of GABA (6 nA).
Results these responses was seen. The spike amplitude sf the Efiects of Flurazepam on Responses evoked by Ex- amino acid response was progressively decreased and temporarily suppressed, suggesting a depdarizacitatory A rmho A cicls or A cetylcholine Although spontaneously active neurones were tion block or a local anaesthetic effect of flurazepam. frequently encountered during a microelectrode pen- As the latency of the amino acid respcmses was etration into the cortex, the vast majority of neurones slightly reduced by fluaazepam, a depolarizing action (at depths between 0.8 and 1.5 mm) was recorded of flurazeparn (eventually leading to blocking of firfollowing repeated (or constant) iontophoretic ap- ing) seems more likely. In order to avoid these problenns, much smaller plications of excitants such as glutamate, aspartate, or homocysteate. Ejecting iontophoretic currents Wurazeparra currents had to be used (less than 5 nA were adjusted so that closely reproducible firing rates through 0.1 M solution or by utilizing 20 m M BHurazwerc observed. i n this way we avoided the inevitable epam in 0. % 6 M NaCL) ; when these precautions were fluctuations in spike activity of spontaneously active taken, the action of glutannate (and other excitants) cells sftcn found during prolonged recordings. When was apparently diminished by Murazepam in a reapplied in relatively large doses (5-30 nA from 0.1 versible manner with no depression of the spike conh4 solutions), flurazepam frequently depressed the figuration. Pen recorder tracings of neuroamal firing spike amplitude and then blocked spontaneous spike rates evokcd by glutarnate and aspartatc (in A ) or activity. When similar applications were performed in glutanlate and hornocysteatc (in B ) are shown in conjunction with repeated administrations of excita- Fig. 1 : flurazepam (0. I h4 solution) inhibited these tory amino acids, which werc giving stable submaxi- responses within 20-30 s, an aff'ect that had a rather ma1 responses, temporary or reversible depression of slow recovery (up to 20 min). In many instances,
CAW. J. PHYSIOL. PHARMACBL. VOL. 57, 8979
TABLEI. Interactions of flurazepam with various excitants or inhibitors of cortical neurones
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by UNIV WINDSOR on 11/09/14 For personal use only.
Number of cells tested Glutamate Hornocysteate Aspartate Acetylcholine Spontaneous GABA
5HT DA
No change
Increased *
Excitatory responses in the presence of flurazepam 28 15 2 6 6 4 1 Inhibitory responses in the presence of flurazeyam 16 16 10 1 2 12
7
3
Decreased* 28 13
6 6 3
7 2
*The terms "increased" or "decreased" indicate a change of more than 10% in the overall tesponse in pen recorder tracings (duration multiplied by half-peak amplitude).
the flurazepam-induced depression could not be counteracted by increasing the amino acid ejecting currents. In addition, this kind of flurazepam-induced depression did not show selectively towards any of the three amino acids which were applied in such a manner that submaximal matched responses were always obtained. It may be mentioned here that Zakusov et a/. (1997) found no interaction of intravenous diazepam with glut amate responses of cortical neurones. However, the experimental conditions used by these workers (e.g., intravenous diazepan1 given to unanaesthetized rabbits) are clearly different from those of our study. Another effect noted by us during this type of flurazepam application was that neurones which initially showed a spontaneously bursting spike activity appeared to fire in a steady manner following iontophoretic application of flurazepam. This effect was usually reversible within 2-3 min from the end of flu raze pan^ application. A number of neurones firing spontaneously at low rates were excited by iontophoretically applied acetylcholine. The acetylcholine responses were typically slower in onset than the responses to excitatory amino acids and often had a phase of transient firing depression preceding an excitatory phase outlasting the application period -(a. IgrwpviC a n d 76ilEs 1963 ) . These acetylcholine responses were also antagonized by flurazepam, though relatively large doses (up to 38 nA, 8.1 M solution) were required to achieve such depressions without a concomitant reduction in spike size.
during sustained application of GABA (cf., for example, effects of 6 nA GABA in Fig. 1@) ; a transient rebound of spike activity was often seen shortly after the end of GABA application. Flurazepana (6 nA through 20 m M in 0.16 ha NaCl solution) had little detectable effect on glutamate-evoked responses but clearly enhanced the peak depression due to GABA (Fig. 1C) without altering the fading of the GABA response. In ~ r d e to r examine the specificity of flurazepam actions on GABA-evoked inhibitions, inhibitory responses induced by monoamines were also studied in the same neurones. Both 5HT and DA reversibly inhibited neuronal firing, although these substances were apparently weaker than GABA (as judged on the basis of the iontophoretic current used) and depressed only two-thirds of the neurones examined (the action of GABA was ubiquitous). The majority of DA responses were unaffected by the presence of flurazepam (see Table 1 ) ; in the remaining cases either a slightly increased inhibition due to reduction of fading (cf. Fig. 2 F and G ) or some antagonism was noted. Flurazepam (5 nA through 20 lnlM in 8.16 M NaCl solution) often antagonized (in a reversible manner) the inhibitory responses to 5HT (Tab@ 1 ): I t i s shown-in Fig, 2 t h a t i r x the s a m e &xrone flurazepam could potentiate GABA, antagonize 5HT, and prolong the action of DA; a larger flurazegam dose reduced glutamate-evoked responses (but not spike amplitude). A summary of the action of flurazepam on responses to iontophoretically applied transmitters is given in Table I.
Eflects of Fkurazeparn on Inhibitory Responses ind~lcedby GABA, 5 H T , and D A Iontophoretica~lyapplied GABA depressed the neuronal firing induced by glutamate or homocysteate. Such a depression had a rapid onset and faded
Discussion It is not totally surprising to observe a variety of flurazepam effects, related to the dose administered. Large doses (presumably achieved with large cur-
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by UNIV WINDSOR on 11/09/14 For personal use only.
COMMUNICATION
G -?I
1327
GLU
F L U 5nA
GABA OnA ,120
F kbl 5nA c GABA 0 n A
FIG. 2. Effects of different inhibitory substances on firing responses of a single cortical neurone to glutamate (GLTJ, 19 nA). (A-C) Flurazepam (FLU, 5 nA from a 20 m21 in 0.16 kfNaCl solution) or GABA applied by removing its
retaining current have a slight inhibitory effect which is greater when the two substances are simultaneously applied. (19) Increasing iontophoretic doses of 5HT inhibit glutamate responses; this inhibition ic reversed by concomitant application of flurazepam (5 nA) in (E). (F) Inhibition evoked by DA (5 nA); note fading of this eflect. Flurazepam (5 nA) in (G) eliminates this fading. (H) Depressant effect of a larger flurazepam (20 I ~ solution M in 0.16 hf NaCl) iontophoretic dose (20 nA) on glutamate responses.
rents through concentrated solutions) apparently interfered with the spike-generating mechanisms of the neurones as shown by the depression of spike amplitude and the lowering of the threshold for depolarization block (cf. also Curtis et al. 1976) . In view of the large currents needed we also consider the depression of acetylcl~olineresponscs a rather unspecific effect of flurazepam. When smaller doses of flurazepam are used, responses to excitatory amino acids could be reversibly diminished, with no change in spike amplitude. However, if we consider as the most powerful effects of flurazepam those elicited by the smallest iontophoretic currents, it is evident that the main action of this benzsdiazepine was an enhancement of neuronal inhibitions, particularly those evoked by GABA. It is interesting to compare the GABA-enhancing potency of flurazepam under in vivo and in vitro conditions. This might disclose whether the present results of in vivo iontophoretic experiments were obtained with flurazepam doses similar to those used previously (Nistri and Constanti 1978a) in vitro.
With bath applications of flurazepam to frog spinal neurones in vitro, a 2.5 pM concentration was found to potentiate selectively the action of GABA (Nistri and Constanti 1978a, 1978b). It is difficult to know the actual concentration ( C ) of Blurazepam attained in viva with iontophoretic ejections. An approximation may be obtained from the diffusion equation adopted for the case of iontophoretic release from a "point source" for a given time ( t ) (Jaeger 1965 ) . This equation can provide the concentration of the substance (C) 1 s ( t ) after the application ( t , ) has been stopped. This approach is perhaps more meaningful as we are dealing with a nonequilibrium distribution of iontophoretic substances and therefore we can only estimate the final concentration which builds up during the whole application. C
=
erfc -i-
.
4rrmDr(
-
2(0:)1/
-
-
erfc
z[o(r
I
r,)11/3
where rn (the rate of iontophoretic release per unit time) IT/Fn ( I current. T = transport number, F = Faraday constant, n = valency). We have not
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CAW. J. PHYSIOL. PHARMACOL. VQL. 57, 1979
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measured the value of T for flurazeparn although it is likely to be similar to that of other polycyclic agents such as chlorpromazinc, haloperidol, and imiprarnine, whose transport numbers range betwcen 0.04 and 8.08 (Zieglg5nsberger et aE. 1974). If we assume an average of 0.05 for flurazeparn, we shall have to divide this by 80 as we used a 165 niM NaCB solution and therefore 20 rnM Aurazepan~may be expccted to carry only about ,l,,th of the current Wowing through the solution. Hence, the effective transport number may be as low as 0.005. By assurning this value and a 5-nA current, we can directly substitute the other values into the above equation if we consider that r (distance from the electrode tip) is 50 pm, the diffusion coefficient (D)is B0-%crraVs (cf. Curtis pt al. 1960), and tl is 135 s (cf. Fig. 1C). The estimated concentration is then 3 pM, a value close to the effective dose in in vitro experiments on the frog spinal cord. In practice, the concentration attained in vivo might have been even smaller as both T and r are probably overestimated. Our present results thus confirm the high sensitivity of responses elicited by GABA to bath-applied flurazepam (Nistri and Constaisti 1978a, 19778). The enhancement of GABA effects by flurazepam was undoubtedly occurring at a postsynaptic level (either on GABA receptors or ionophores) (Costa and Guidottj 1979) ; howcvcr, some facilitation of endogenous GABA release rnight also play a role in this phenomenon (Ncstoros and Nistri 1978). Additional useful data on flurazepam-GABA interactions might be provided by studies with GABA receptor antagonists; unfortunately the poor antagonistic properties of bicuculline and picrotoxin in the cerebral cortex (Gndfriand ct al. 1978; Hill et al. 1973) would not allow us to reach reliable conchsions. A novel finding of the present study was the antagonism by flurazepam of 5HT-evoked corticaZ inhibitions contrasted by the relative insensitivity of DA-evoked inhibitions to iontophoretically applied flurazepam. This indicates that the observed potenAaticm aF GABA iffhibitions wasquite-specific a n d not extended to other chemically evoked inhibitory responses. Furthermore, this effect of flurazeparn on 5HT responses is in keeping with behavioural observations on the interactions of benzodiazepines with central 5HT-containing neurones (Stein et ak. 1973). In conclusion, cortical inhibitions are more sensitive than cortical excitations to flurazepam. Among the inhibitory responses, those induced by GABA are particularly susceptible to this benzodiazepine, which probably acts by increasing the effectiveness
of GABA on postsynaptic sites (receptors or ionophores). However, other inhibitory responses (e.g., due to 5HT) may be affected by flurazepam in concentrations which still leave the excitatory responses unchanged. Acknowledgments Wc thank Professor K. KrnjeviC for helpful discussions and suggestions and Mr. N. Schestakowich for skilful photography. Flurazcpam hydrochloride was kindly donated by Hoffman La Roche Ltd., Montreal. The authors were Fellows of the Medical Research Council of Canada. These results will be submitted to McGill University by J.N.N. as part of a Ph.D. thesis. BRAESIRUP, C., and 5yum~~s, R. F. 1977. Specific benzodiazepine receptors in rat brain characterized by highafinity (%) diazepam binding. Proc. Natl. Acad. Sci. U.S.A. 94, 3805-3809. 1978. Pharmacological characterization of benzodiazepine receptors in the brain. Eur. J. Pharmacol. 48, 263-270. CHOI, D. W., FARB,D. H., and Fnscnr~~c~, G. D. 1977.
