EFFECTS of BENZODIAZEPINES and I'entobarbital on the EVOKED POTENTIALS in TIE CAT BRAIN

EFFECTS OF BENZODIAZEPINES AND i'ENTOBARBITAL ON THE EVOKED POTENTIALS IN TIE CAT BRAIN

Toshiro TSUCHIY,-y and Sunlio KITA«AWA Institute j Piolo,('ieal Science, Pharmaccutica,.) Division, Su uiir'mo Chemical Co.. LO., Tu;aracida, Hro o 665, Japan

Accepted ",larch 11, 1976

Ah-tract--The sites of action of benodiazepincs, diazepam and ID-540 [7-chloro-5 (~~ t':uorophcn l) 1 methyl d,3-ddtvdro-2H-1,4-benmdiazepin-:-one) on the central nervous system v, are examined and compared with those of itoharbital using evoked po~cntials recorded on the limbic `o stem and hypothalamu : in the cat brain. Ben zodiazepuies a,fceted th,e sariou.s neuronal connections of the intra-limbic, linlbic hypothalamic and mi:.lbrain-limbic systems: c;peciall) the , mypdala (AM'iG)-, ven tromedial h~poti,,.ilaiutis (V%1i-) and central gra) matter (St C)-hippocampal (HIPP) evoked potentials were attemated, v ereas the AMYG-\ s1H, VNIH-AMYG and the septum (SP)-VS1H e` a' ed pieanliad v ere facilitated. Pentobarbital selectively attenuated the SCC-, V,NIH and :ASI i'C liiPP evolved pu entials, or facilitated the V\1H-r N!'i G and the SP-, 1!PP evoked p uentials. Both F enzodiazepines and pen tobarbital affect_(.1 three afferent iiippocarnpai neuronal connections, areas of the retic~_ilo h~ pothalamie Systems regu'.atiug hippocampal actin i.. , while only benzodiaze pines affected the r1caromal infiuc ,,:e of the anlygdaloid and septai areas on the hypo thalamus.

[li'eets of benzodiazepines and barbiturates on behavior and electrical activity as observed in brain structures under naturally occurring and stimulated conditions have been reported. Both citlordiazepoxide and Phenobarbital raised the threshold of hissing induced by hypothalamic stimulation in the cat (1) and unit activity in the hippocampus was selec tively inhibited by clilordiazcpoxide and diazepam, whereas pentobarbital showed a selective inhibition cf the reticular formation (2). Jalfre et al. (3) reported quantitatise methods to differentiate benzodiazepine from other psychotropic drags (,neprobamat,, chlorpromazine, amitriptyline, benzoctanline and hexobarbital), though their study dealt with only limited amygdala-hippocampal evoked potential at the intralimbie structure. In our present worl:, effects of benzodiazepines and pentobarbital were investigated qualitatively and quantitatively on several neuronal connections from limbic structure, bypothal

VI.-\TLRIALS AND METHODS Twenty-five adult cats of either sex, weighing between 2.3 and 3.5 kg, were prepared under ethyl ether anesthesia. The trachea and femoral vein were cannulated for artificial respiration and drug injection, respectively. After incision of the dura, coaxial electrodes (11M1odelB-9005 Sin-Ei) were positioned into the basolateral nucleus of the right amygdala (ANIYG, Fr. 10.0, L. 11.0, H. --5.5), the ipsilateral dorsal hippocampus (HIPP or dHIPP, Fr. 4.5, L. 5.0, H. 8.0), the ipsilateral ventral hippocampus (vHIPP, Fr. 6.5, L. 11.0, H. 2.0), the ipsilateral ventromedial hypo thalamus (VMH, Fr. 11.5, L. 1.3, H. -6.0), the ipsilateral septum (SP, Fr. 14.5, L. 1.0, H. 4.5), the ipsilateral central gray matter (SGC, Fr. 3.0, L. 1.5, H. 1.0) and the ipsilateral piriform cortex (PIR, Fr. 9.5, L. 11.0, H. -8.5), the atlas of Jasper and Ajmone-Marsan (4) serving as a guide. Immobilization of the cat was maintained by 5 mg/kg gallamine triethiodide (teisan) given i.p. every 2 hours. All wound edges were infiltrated with lidocaine hydrochloride (Xylocaine®) repeatedly through the experiment. Artificial positive pressure respiration (25 times/min) was maintained constant throughout the experiment, and body temperature was sustained with a warming-pan. The combination of the stimulation and recording sites was as follows: AMYG-HIPP, AMYG-VMH, VMH-HIPP, VMH-AMYG, SGC-HIPP, SP-HIPP, SP-VMH, AMYG vHIPP and AMYG-PIR. The stimuli consisted of 0.3 msee rectangular pulses of 0.8 mA which were delivered to each site every second by a Nihon-Kohden MSE-3 Stimulator. Responses from each ipsilateral recording site were fed into a San-Ei model IA52 electroencephalograph pre amplifier and analyzed by ATAC 501-20 (Nihon-Koden) computer of average transients. Analysis time was 102.4 cosec. Data summed for 100 responses were displayed on the screen of the ATAC and photographed by a Nihon-Kohden model PC-2B continuous recording camera or recorded by X-Y recorder (Watanabe). The parameter of each evoked potential was a peak latency of the first wave, either negative or positive, and peak to peak amplitude. The drugs were diazepam and ID-540 [7-chloro-5-(o-fluorophenyl)-1-methyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one] (5) which were synthesized on our laboratory, and pentobarbital (Nembutal®). Benzodiazepines were dissolved in a 50'110PEG (polyethylene glycol '400) saline solution. ID-540 and diazepam were injected i.v. starting with 0.1 mg/kg, and the dose was increased successively to 0.3, 1 and 5 mg/ kg at an interval of 25-30 nmin. Pentobarbital were injected with 1, 3, 10 and 30 mg/kg at the same interval. The solvent for benzodiazepines was injected by the same route and interval as control. Only one substance was tested with cumulative doses in each experiment. The values of the amplitude and latency of the evoked potentials were calculated from 3 recordings in the control (pre-drug) or the test period 3 hours after the initial anes thetization with ether. The effect of each dose of the drugs or solvent PEG on the two parameters was expressed as percentages of the mean control (pre-drug) value. Five cats were used for each drug or solvent. Student's t-test was used for the comparison of the action between the drugs and solvent at identical doses. The electrode positions were histologically verified after termination of the experiments.

RESULTS Effects of ben:odiaZcpines on neuronal connections Benzodiazepines strongly inhibited the amygdala-hippocampal evoked potential which was characterized by a positive wave with the maximal amplitude at 35 to 50 msec followed by a negative wave. The effects of diazepam and ID-540 were a dose-dependent decrease of the peak to peak amplitude and slight increase of the positive wave peak latency (Fig. 1). When the evoked potentials were recorded simultaneously on the piriform cortex, ventral

FIG. 1. Effects of diazepam (v), ID-540 (F]), solvent PEG (~) and pentobarbital (7) on the amygdala-hippocampal evoked potential. * : significantly defferent from solvent (P<0.05). An example of the evoked potential is shown in the upper part. The horizontal line is a peak latency and the vertical line is a peak to peak amplitude. Positivity down. Calibration: 10 msec and 100 yV.

FIG. 2. Evoked potentials in dorsal (upper trace), ventral (middle trace) hippocampus and piriform cortex (lower trace) were elicited by stimulation of the amygdala. A: control, B: effect of ID-540 I mg/kg. Note disappearence of the amygdala dorsal hippocampal evoked potential, reduction of the amygdala-ventral hip pocampal evoked potential and slight reduction of the amygdala-piriform cortical evoked potential. Positivity down. Calibration: 50 msec and 100 yV. and dorsal hippocampus by the amygdaloid stimulation, the following amplitude reductions were found as compared with control at 1 mg/kg (Fig. 2): amygdala-piriform cortical evoked potential, 90%; amygdala-ventral hippocampal evoked potential, 70%; and amygdala dorsal hippocampal evoked potential, 30%. Both compounds attenuated the amplitude of the ventromedial hypothalamus and central gray matter-hippocampal evoked potentials as interpreted by the negative-positive wave (Table 1, Figs. 3, 4). The strong decrease in

TABLE 1. Similar effects of diazepam, ID-540 and pentobarbital on the amplitude of the ventromedial hypothalamus-hippocampal and -amygdaloid evoked potentials and the septum-hippocampal evoked potential

FIG. 3. Effects of pentobarbital (0), diazepam (Q), ID-540 (F-) and solvent PEG (C) on the central gray matter-hippocampal evoked potential. See details as in Fig . 1. Fin. 4. Evoked potential elicited on the hypothalamus-limbic and efferent septal neuronal connections. A: the hippocampal (upper trace) and the amygdaloid (lower trace) evoked potentials by stimulation of the ventrornedial hypothalamus. B: the amygdala-ventromedial hypothalamic evoked potential. C: the hippocampal (upper trace) and the ventromedial h%pothalamic (lower trace) evoked potentials by stimulation of the septum. The horizontal line is a peak latency and the vertical line is a peak to peak amplitude. Positivity down. Calibration: 10 msec and 100 p V. amplitude of the central gray matter-hippocampal evoked potential was dose-dependent, and the amplitude was decreased by 50°io after the injection of diazepam at 5 mg/kg. The degree of inhibition was the same as for the amplitude of the amygdala-hippocampal evoked potential (Figs. 1, 3). The amplitude of the ventromedial hypothalamus-hippocampal evoked potential remained unchanged by diazepam up to a dose of 0.3 mg, kg, but at 1 mg/kg, it was reduced by 60",'). ID-540 reduced the amplitude of both hippocampal evoked potentials less dose-dependently than diazepam, especially for the amplitude of the central gray matter hippocampal evoked potential (Fig. 3). Other evoked potentials, i.e., the amygdala and septum-ventromedial hypothalamic, ventromedial hypothalamus-amygdaloid and septum-hippocampal evoked potentials, were

Ft(;. 5. Effects of diazepam ID-540 C), solvent PEG ( ) and pentobarbital (0) on the amplitude of the amygdala-ventromedial hypothalamic evoked po tential. *: significantly different from solvent (P<0.05). A measured amplitude is shown in Fig. 4. B. Flo. 6. Effects of diazepam (Z-1), ID-540 (:--), solvent PEG ()) and pentobarbital (7) on the amplitude of the first wave at the septum-ventromedial hypothalamic evoked potential. *: significantly different from solvent (P70.05). A measured amplitude is shown in Fig. 4, C lower trace. also changed by cumulative doses of benzodiazepines. Effect of benzodiazepines on these evoked potentials was observed with an increase in the amplitude, which after injection of these agents was seen to increase by 140-1500"' at the maximal dose of 5 mg,/kg, however, the latency did not change. Diazepam and ID-540 exerted the same potency on these evoked potentials (Figs. 5, 6 and Table 1).

Effects of pentobarbital on neuronal connections The amplitude of the hippocampal evoked potential by stimulation of the central gray matter was markedly inhibited by pentobarbital. The dose-dependency of the inhibition was marked, and at a subanesthetic dose of 10 mg; kg, the amplitude was about 50% that of control; at anesthetic dose of 30 mg/kg, it was less than 10° (Fig. 3). Pentobarbital also attenuated the ventromedial hypothalamus-hippocampal evoked potential with a sub anesthetic dose (Table 1). With pentobarbital, there was a tendency toward increase in the negative wave peak latency of the two hippocampal evoked potentials recorded with stimu lation of the midbrain and hypothalamus, however, such was not statistically significant. Among other evoked potentials reduced by pentobarbital, the amygdala-hippocampal evoked potential was inhibited without an increase of positive wave peak latency, and the amplitude was reduced by 56.1 ±21.3 j; at anesthetic dose (Fig. 1). Figures 5 and 6 show examples of the evoked potentials in which the amplitude was slightly reduced by pen tobarbital. On the other hand, the amplitudes of the ventromedial hypothalamus-amygdaloid and septum-hippocampal evoked potentials were increased by pentobarbital (Table 1). The ventromedial hypothalamus-amygdaloid evoked potential varied markedly in terms of the dose of the agent given, comparable to the evoked potential in the central gray matter hippocampal connection.

DISCUSSION In the present results, three afferent pathways of the hippocampal from the amygdala, ventromedial hypothalamus and central gray matter were inhibited by diazepam and ID-540. The inhibition of the amygdala-hippocampal evoked potential was the same as that reported by Morillo (6), Morillo et al. (7) and Jalfre et al. (3). The arnygdala-hippocampal evoked potential could be mediated along multisynaptic pathways in the piriform cortex (8), and the effects with benzodiazepines and other psychotropic drugs (chlorpromazine, mepro bamate, amitriptyline, benzoctamine and hexobarbital) differed (3). Morillo (6) considered that the action of benzodiazepines on the amygdala-hippocampal evoked potential is ap parently not mediated through midbrain reticular influence and could not be attributed to inhibition of the hippocampus because the contralateral hippocampal-hippocampal evoked potential was enhanced by benzodiazepine. The influence on the diencephalic level, parti cularly on the hypothalamus, may not be related to atygdala-hippocampal excitability, since both amygdala-ventromedial hypothalamic and ventromedial hypothalamus-amygdaloid evoked potentials were facilitated by diazepam and ID-540. Thus, the site of the inhibitory action on the amygdala-hippocampal evoked potential is speculated to be the multisynaptic pathways in the piriform cortex. Our data suggest that the dorsal and ventral hippocampal and piriform cortical evoked potentials elicited by stimulation of the amygdala were at tenuated by ID-540 in the order of a longer latency which occurs in a mediated number of synapses. The septum-hippocampal evoked potential was facilitated but the amygdala-, ventro medial hypothalamus and central gray matter-hippocampal evoked potentials were inhibited by benzodiazepines. Should the septum-hippocampal connection function in relation to the hippocampus, then benzodiazepines may inhibited the afferent connections to the hippocampus. Pentobarbital has been shown to have inhibitory action on the midbrain (9-13). In the present experiment, pentobarbital strongly inhibited the reticulo-limbic activating system such as the central gray matter-hippocampal connection and affected the ventro medial hypothalamus-hippocampal connection. It has been reported that stimulation of the medial hypothalamus as well as the reticular formation induces arousal response in the hippocampus (1416). We assume that the hypothalamic-limbic activating system is in hibited by pentobarbital because of the inhibitory action on the ventromedial hypothalamus hippocampal evoked potential. Benzodiazepines appear to inhibit the two limbic activating systems from the reticular formation and the medial hypothalamus as action on the central gray matter and ventromedial hypothalamus-hippocampal evoked potentials is similar to that seen with pentobarbital, though inhibitory potency of benzodiazepines was not in creased by cumulative doses. The amygdala and septum-ventromedial hypothalamic evoked potentials were affected by an increase in the amplitude with benzodiazepines, but with pentobarbital only a slight decrease is seen (Figs. 5 and 6). We considered that the effects of benzodiazepines on the ventromedial hypothalamic evoked potentials are not indirectly affected by inhibition of the midbrain, because it has been reported that high frequency stimulation on the midbrain increased the amplitude of the ventromedial hypothalamic evoked potential by stimulation of the septum but decreased that of the evoked potential by stimulation of the amygdala (17), and pentobarbital, a depressive agent on the midbrain, decreased that of the evoked potentials by stimulation of both areas. There are data that the septal area normally acts to dampen emotional behavior displayed on the hypothalamus, while the amygdala may facilitate a hypothalamic emotional center (18). Morillo (6) suggested that inhibition of the amygdala-hippocampal evoked potential by benzodiazepines might support the theory that the amygdala exerts a facilitatory influence on the hypothalamus, thus participating in the mechanism modulating emotional output. From our present findings, the inhibition of benzodiazepines on hyperemotionality of septal-lesioned rat does not appear to be derived from the reduction of amygdaloid facili tatory influence on the hypothalamus; rather, the septal and amygdaloid influence on the hypothalamus may be somewhat enhanced by benzodiazepines. Nagy and Decsi (19) re ported that when diazepam was injected into the amygdaloid complex in the cat, the carbacol induced hypothalamic rage reaction was inhibited. It is suggested that neuronal influence of the amygdala on the hypothalamus may be enhanced by diazepam, thus modulating emotional output. From this aspect, the action of benzodiazepines differs from that of pentobarbital. Acknowledgements: We wish thank Drs. H. Yamamoto and H. Fukushima for helpful criticism of data and Mr. S. Tan-ii for his assistance with the histology.

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