Conditioning of the Microcirculation in the Rat L. KATO, B. G6zsY AND MxaCZL LvMmvx

Institut de Microbiologie et d'Hygijne de l'Universit~ de Montrdal, and H6spital des Laurentides I'Annonciation, P. Q., Canada Abstract-The serotonin-eatecholamine balancing mechanism maintains normal vascular tonus and permeability of the mieroeirculation. Serotonin provokes local vasodilatation and increases capillary permeability in the skin of rats. Released or injected catecholamines inhibit the serotonin-indueed peripheral hemodynamic alterations. Following eleelxoeonvulsivetreatment (ECT) (US), there is a catecholamine release and the microcirculatory response to serotonin does not occur. When the administration of a visual stimulus (in six consecutive occasions) proceedingly coincided with the administration of the ECT, it was noted that the light became an effective stimulus for this system. Light alone is an indifferent stimulus to catecholamine release. Thus, the inhibition of the serotonin-induced vasodilatation by ECT (via catecholamine release) became eonditioned to the visual-conditional stimulus. Since the net effect of extrinsic serotonin depends on free cateeholamines at the vascular bed, the intensity of the serotonin-induced vascular response reflects alterations in the chemical balancing mechanism. This mechanism can be conditioned with the present technique. The result indicates that there is central nervous system mediation. Tim MICROCIRCULATIONIS A biological system which is chemically mediated; the release of the chemical mediators can be local or via the central nervous system (CNS). The dilator amines, histamine ( H ) and serotonin (5-HT) and the constrictor catecholamines, dopamine and norepinephrine are released from the perivascular mast cells and nerve endings. T h e constrictor amines released from the adrenals similarly contribute to the microcirculation. It is generally agreed that peripheral functions or pathological alterations which are solely peripheral in origin, cannot be conditioned. In this presentation a technique is described which demonstrates the conditioning of the capillary tonus and permeability, a mechanism which is in part centrally mediated. Methods and Materials Sprague-Dawley rats of both sexes, weighing 110-140 g were used. The animals were kept at constant temperature and humidity and fed standard Purina C h o w diet, with water ad libitum. The animals were kept in a quiet room with dim daylight illumination. This investigation was supported in part by grants from the Ministry of Health of the Province of Quebec (Federal Provincial Health Research grants). 90

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The rats were randomly divided into three groups. The two control groups were composed of 36 rats, and the experimental group had 70 rats. 1. The first control group received no treatment. 2. The second group received ECT as the unconditional stimulus (US) twice daily at approximately the same time in the morning and afternoon for three consecutive days. Electroshock was given by an apparatus specially constructed for the rat. Electrodes were placed on the ears of the animals and the ECT was a 60-cycle alternating current, 50 ma, with each electrical impulse of 0.2 seconds' duration. 3. Acquisition of the capillary conditioned response to a formerly indifferent stimulus was attempted in the third group of rats. The animals were presented first with the conditional stimulus (CS). This was a visual stimulus of three seconds" duration from a 500W lamp at a distance of 18 inches above the head of the rat. The rat was placed in a carton box, with the electrodes attached to both ears. The CS was followed immediately by the administration of the US as in Group 2. Treatment was given twice daily for three days. Both the CS and the US were given individually to each rat in a separate room. Convulsions lasted for 180 • 60 seeonds. Rats were permitted to recuperate from the shock in a carton box in another room. On the evening of the third day the abdomen of 30 rats in each group was shaved with an electric razor and treated with a chemical depilator (NEET). Rats were then divided into subgroups a, b, and c in each group. The conditioning was tested on the morning of the fourth day using the following procedure: Vasodilatation (Vd) and increased capillary permeability (Icp) was provoked by injecting 0.2 ~g 5-HT (serotonin creatine sulfate) in 0.1 ml physiological saline solution intradermally into the depilated abdominal skin. Immediately thereafter, 0.5 ml India ink was injected intravenously into the tail vein. (30 ml Gunther's ink was suspended in 70 ml 3% gelatin in distilled water). One hour later the rats were killed and the carbon deposition at the site of serotonin injection was estimated as "per cent vascular response" as described by GSzsy and Kato (1961). The diameter and intensity of the '%lack spot" were considered to be the visible sign of the intensity of the serotonin-induced Vd and Icp. This capillary response was provoked in separate groups of three (see Fig. 1) rats five minutes before and 10, 30 or 60 minutes after both the CS and US.

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The remaining 24 rats in the third group were used for extinction trials. These rats received the CS twice daily (except Sunday); this involved the presentation of light for 3 seconds as during the previous days. The vascular response to 0.2 ~g 5-HT was tested on the abdominal skin of three rats, on each of the following eight days (except Sunday). Results As shown in Figure 1, serotonin (0.2 ~g), provoked a strong vascular response in all three groups of rats when the vasodilator amine was injected intradermally prior to the US and the CS, or both. The serotonin-induced Vd and Icp was assigned as 100~ vascular response, as measured by the size and intensity of carbon deposition at the site of the serotonin iniection. In all three groups the vascular response was strongly inhibited when serotonin was injected 10 or 30 minutes after the ECT (US) or the light (CS) plus ECT (US) were effected. In each group the usual maximal

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(100~;) vascular response was measured one hour after the US and the CS plus US. The CS was presented to the normal rats or to the rats which were pretreated for three days with the US alone. When serotonin was injected intradermally 10, 30 or 60 minutes later, the usual Vd and Icp occurred with full intensity. The conditionability of the microcirculatory response to serotonin is shown in Figure 1. Rats of the third group which were pretreated six times during the previous three days with the US precedingly coinciding with the CS received the fourth day the CS alone. Ten minutes later serotonin (0.2 ~g) was injected intradermally and the Vd and Icp was nearly absent. Thirty minutes after the CS administration, the vascular response to serotonin was 50g inhibited, but returned to normal after one hour. The microcirculation responded similarly to the CS, US, or to the CS and US together. One out of nine rats responded with light convulsions after the presentation of the CS. One rat responded with strong convulsions and died within 20 minutes. In all the rats of this group the peripheral vasoconstriction was so strong following the presentation of the CS (light alone), that the intravenous administration of the India ink suspension was extremely difficult or unsuccessful. Figure 2 shows results of the extinction experiment. On the fourth day rats which were pretreated for three days with the CS and US received twice daily the CS, and each day three rats were challenged with an intradermal injection of serotonin. As before, the vascular bed became conditioned to the CS and the capillary bed behaved passively to serotonin after the presentation of the CS. During eight consecutive days the vascular response to serotonin progressively returned to the normal 100~; level when presented twice daily with the CS. Thus the extinction of the conditioned response was achieved in eight days. Discussion

A physiological balancing mechanism maintaining normal peripheral vascular tonus and capillary permeability has long been suspected (GSzsy and Kato, 1966). It became evident that this mechanism is chemically mediated by the catecholamines (Willoughby and Spector, 1964) which produce vasoconstriction and inhibit Icp, while histamine and serotonin both induce Vd and Icp (Rowley and Benditt, 1956). The bioamines are locally available from the perivascular mast cells and nerve endings, but systemic stresses like toxins, shock, convulsions, emotional and psychic fae-

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FIG. 2. In the rat pretreated six times in three days with CS and US the vascular response to serotoninwas stronglyinhibited. When the CS, was presented twice daffy (1') during eight consecutive days, the vascular response (vasodffatationand increased permeability) graduafly returned to normal. Extinctionof the CR was achieved.

tors release mediators which participate in the functioning of the capillary network. Our previous results have shown that following ECT the capillary network acquired a transitory passive behavior to the vasodilator bioamines ( G6zsy, Kato, Roy, Groh and Lallonde, 1965, G6zsy et al., 1967, St-Jean, G6zsy and Kato, 1967, Trsic, Roy, G6zsy and Kato, 1967). Experimental evidence was presented by Kato, G6zsy, Roy and Groh (1965, 1967) that these alterations were independent of the local chemical mediators and that biochemical changes in the concentration of brain amines coincided with the peripheral circulatory alterations. With the help of classical Pavlovian procedures and using the presented methodology, our results showed that the capillary network can be conditioned to a formerly indifferent stimulus. The extinction of this acquired reflex was also successful. The capillary tonus and capillary endothelial filtration at any given instant is the net effect of a chemical balancing mechanism in which the catecholamines oppose the dilating effects of histamine and serotonin. Whenever the capillary bed, or more precisely the muscular elements of the minute vessels, became refractory to stimulation, the

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amount of free catecholamines at the receptor sites was increased (St-Jean et al., 1967). The US in the present case was a systemic stress (ECT) which released catecholamines, thus producing an inhibition of the Vd effects of serotonin. The presented results are indirect, but they are strong evidence that in the conditioned rats, the CS released catecholamines which inhibited the Vd and permeability effects of serotinin because the catecholamines are the natural hormonal inhibitors of the dilator bioamines (Willoughby et al., 1964). Therefore, we propose that this chemical balancing mechanism acquired a conditional response to an indifferent stimulus, and after extinction of the conditioned response the same balancing mechanism functioned as in normal conditions. Gantt has shown that the effect of agents which are produced solely by action on peripheral structures without involvement of the central nervous system in their production cannot become conditional reflexes. Thus, the hyperglycemia of adrenal action, the gastric juice of histamine action, the tachycardia from adrenalin or the bradycardia of acetylcholine are not conditionable, while the same effects produced by central nervous system involvement, namely, hyperglycemia of fear, gastric juice or tachycardia to food, bradyeardia to a person, can readily become conditional reflexes. Our experiments provide evidence of the requirement of the central nervous system in formation of the conditional reflex. References

Gantt, W. H., Katzenelbogen, S., and Loucks, R. B.: An attempt to condition adrenalin hyperglycemia. Bull. ]ohns Hopkins Hosp., 60:400, 1937. Grzsy, B., and Kato, L.: Factors other than histamine affecting capillary permeability. Int. Arch. Allerg., 19:168, 1961. Grzsy, B., Kato, L., Roy, P. B., Groh, V., and Lallonde, Mi.: Investigation into the mechanism of eleetroconvulsiveshock. I. Effect of electric current on the capillary permeability and on hemodynamieal processes. Neuropsychiat., 1:623, 1965. G6zsy, B., and Kato, L.: Investigations into the balancing mechanism mainraining normal capillary tonus. Dermatologica, 132:353, 1966. Grzsy, B., Kato, L., Roy, P. B., Hosein, E. A., and Kato, G.: Effect of prolonged treatment with electroshock, metrazol, earnitine and anectine on the capillary endotheliurn. Int. 1. Neuropsychiat., 3:159, 1967. Kato, L., Grzsy, B., Roy, P. B., and Groh, V.: Investigations into the mechanism of electroshockin the rat. II. Effect of electrical stimulation on the capillary endothelium and on hemodynamie conditions in histamine and serotonin depleted rats. Int. 1. Neuropsychiat. 1:626, 1965. Kato, L., G6zsy, B., Roy, P. B., and Groh, V.: Histamine, serotonirt, epinephrine and norepinephrine in the rat brain following convulsions. Int. 1. Neuropsychiat., 3:46, 1967.

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Rowley, D. A., and Benditt, E. P.: Five-hydroxytryptamine and histamine as mediators of the vascular injury produced by agents which damage mastcells in rats. I. Exp. Med. 103:399, 1956. St-Jean, A., G6zsy, B., and Kato, L.: Potentiation of norepinephrine effects on minute vessels by imipramine and electroconvulsive treatment in rats. Arch. Int. Pharmacodgn. Th~rapie., 151:166, 1967. Trsie, J., Roy, P. B., G6zsy, B., and Kato, L.: Resistance in human skin after eleetroconvulsive treatment. Med. Pharmacol. Exp., 17:200, 1967. Willoughby, D. A., and Spector, W. C.: Adrenaline precursors in the inflammatory reaction. 1. Path. Bact., 88:159, 1964.

Pavlovian Society Meeting--1970 The 1970 Meeting of The Pavlovian Society of North America will be held on October 30-31 at the Nassau Inn, Princeton, N. J. Featured will be a panel discussion on '~ Control of Circulation" conducted by E. Cowles Andrus, former president of the American Heart Association. The panel will consist of invited experts in the field of cardiovascular physiology. Pavlovian Society members are invited to submit abstracts of papers for this meeting to: James J. Lynch The Psychiatric Institute University of Maryland School of Medicine Baltimore, Maryland 9.1201