An apparent genetic relationship between appetitive and aversive effects of lateral hypothalamic stimulation in the mouse

Physiology & Behavior, Vol. 25, pp. 357-361. PergamonPress and BrainResearch Publ., 1980. Printedin the U.S.A.

An Apparent Genetic Relationship Between Appetitive and Aversive Effects of Lateral Hypothalamic Stimulation in the Mouse PIERRE CAZALA AND ANNE-MARIE GARRIGUES

Laboratoire de Psychophysiologie, Universit~ de Bordeaux I, Institut de Biologie animale A v e n u e des Facult~s, 33405 Talence Cedex France R e c e i v e d 21 J a n u a r y 1980

CAZALA, P. AND A.-M. GARRIGUES. An apparent genetic relationship between appetitive and aversive effects of lateral hypothalamic stimulation in the mouse. PHYSIOL. BEHAV. 25(3) 357-361, 1980.--Three experiments were performed with BALB/c and DBA/2 mice strains implanted in the ventral part of the lateral hypothalamus (LH). Delay times in approach and escape reactions caused by continuous electrical stimulation of the LH were determined in a shuttle box. At most applied current intensities, BALB/c mice triggered and interrupted stimulation more rapidly than did DBA/2 animals. In a second experiment, intracranial self-stimulation behavior (ICSS) triggered by brief (200 msec) stimulation was studied in a lever box. In this experimental situation, the response rate of the BALB/c strain was much higher than that of the DBA/2. Finally, the effects of a fragmented stimulation, reproducing the lever pressing ICSS rhythm were studied in the shuttle box on the same animals. It was seen that BALB/c mice triggered stimulation more rapidly, as in the first experiment, and that they also remained stimulated for a much longer time than DBA/2 at the highest current intensities applied, as in the second experiment. These results suggest that approach responses are related to the onset of stimulation while escape reactions are provoked by the lengthening of the stimulation. A close relationship was seen between the intensity of the approach reaction and that of the escape reaction in the same strain. Self-stimulation

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E L E C T R I C A L stimulation of brain regions capable of producing intracranial self-stimulation behavior (ICSS) causes an animal first to trigger, and then to rapidly interrupt this stimulation under certain experimental conditions. According to some workers, the switch-off response of the stimulation could result from an adaptation of the nervous elements involved [ l l , 12, 14], while others believe that it could be motivated by a change of the stimulation effect: the initial rewarding effect subsequently becoming an aversive effect If, 5, 16, 17, 18, 19, 22]. Substantiation of the "aversion" theory involves, among other things, determining if the triggering and the arrest of central stimulation result from the activation of one or of two different anatomical substrates. No satisfactory answer to this question has apparently yet been advanced. Thus, the positive correlation between the intensities of the approach and escape reactions demonstrated in the ventro-median hypothalamus by Ball 14] and in the lateral hypothalamus (LH) by ourselves [6], suggests that these two behavioral responses are determined by activation of a single substrate. Nevertheless, numerous data have shown that it is possible to independently manipulate one or the other of these two components of LH stimulation effects [2, 3, 13, 21]. This suggests that these two components result from the activation of different nervous elements. We believe that a genetic analysis could possibly enable us to resolve the problem posed by the above discordant

Mouse

data. We previously reported that a 200 msec stimulation applied in the LH led to the appearance of an ICSS behavior in mice, but that the characteristics of this phenomenon varied considerably from one strain to another [7]. Using various hybrid and recombinant groups, we also demonstrated that the difference in the rates of ICSS observed between BALB/c and DBA/2 mice was determined by the action of a small number of genetic factors. On the other hand the differential sensitivity to seizures interrupting ICSS behavior in these two lines proved to be under a genetic control which was both different from and more complex than that of the differential ICSS response frequencies [9,10]. Using this genetic tool, it was conceivable that we might find a dissociation of switch ON and switch O F F responses. If these two phenomena truly correspond to the involvement of different nervous elements, then they are each probably under distinct genetic control. The demonstration of such a partitioning would be a strong argument in favor of the "aversion" theory. We report here the comparison of the intensities of approach and escape reactions triggered by LH stimulation in BALB/c and DBA/2 mice, respectively. METHOD

Animals Twenty-one BALB/c Orl (N= 10) and D B A / 2 0 r l (N= 11)

C o p y r i g h t © 1980 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/80/090357-05502.00/0

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male mice were used. The animals were operated upon when nine weeks old.

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Sul',~Jely Under deep sodium thiopental anesthesia (DBA/2 130 mg/kg, BALB/c 90 mg/kg) the animals were stereotaxically implanted with a bipolar electrode made of two tightly twisted strands of 0.09 mm platinum wire. As in our previous experiments, the electrode was implanted in the ventral part of LH. We used the following stereotaxic coordinates: BALB/c: antero-posterior distance (AP) referring to interaural line + 1.90 mm; lateral distance (L) referring to sagittal line _ 1.00 mm; vertical coordinate (V) taken from the surface of the skull + 5.60 mm. DBA/2: AP+2.00 ram; L _+ 1.0 mm; V+5.30 mm.

Apparatus and Experimental Procedure Three experiments were performed. EXPERIMENT 1: SWITCHON--SWITCHOFF UNDER CONTINUOUS STIMULATION Animal behavior was studied in a 40×8× 12 cm shuttlebox. A photoelectric cell was placed 7.5 cm from each end of the cage. By interrupting one photobeam, the animal learned to trigger a continuous sine wave (100 Hz) stimulation, which stopped when the animal interrupted the beam at the other end of the cage. Automatic equipment recorded the times during which the animal remained stimulated (ON duration) and nonstimulated (OFF duration) with a precision of 0.01 sec. Daily sessions lasted 10 rain. No stimulation was delivered during the first 3 days. Beginning on the fourth day the animals received a stimulation whose intensity was progressively increased at each session: 2, 4, 6, 8, 10, 15, 20, 25, 30, 35, 40 and 50 /xA. At each current intensity, the total amount of time that stimulation was ON or OFF during the 10 min session was divided by the number of ON or OFF responses; mean values of ON and OFF duration were thus determined. EXPERIMENT 2: SELF-STIMULATIONBY LEVER PRESSING The animals were rested for 10 days after the first experiment and were then placed in an ICSS cage which has been described elsewhere [8]. By pressing a lever, the mice obtained 200 msec (100 Hz sin•wave current) central stimulation. The experiment lasted for one week. No stimulation (0 /xA) was applied the first day; the current was fixed at 5/xA on the second day and was then increased daily from 10 to 50 tzA in 10/zA steps. Each ICSS session lasted 30 rain. EXPERIMENT 3: SWITCHON--SWITCHOFF UNDER INTERMITTENTSTIMULATION The animals were again rested for 10 days after the above experiment and were then placed in the shuttle-box used in Experiment 1. The sinusoidal stimulation was fractionated in order to reproduce the mean lever pressing rhythm observed during ICSS behavior in Experiment 2. Sessions lasted 10 min, and the same program of current increments was used as in Experiment 2.

Histological Control At the end of the third experiment, the animals were sacrificed and perfused with saline followed by 10% Formalin solution. Brains were sectioned at 40 ~m on a freezing microtome; sections were stained with 1%0 thionin solution.

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RESULTS

Switch ON-Switch OFF" Under Continuous Stimulation (Fig. I) The behavior exhibited by the two strains was quite similar during the three stimulation-less sessions. Moreover, the animals' activity remained relatively stable during these three sessions; we have only reported on Fig. 1 the time spent in front of each photoelectric cell as averaged from all the values observed during these three sessions. As soon as an actual central stimulation was begun, it was immediately noted that the mean ON duration value was higher in DBA/2 mice than in BALB/c animals, especially for intensities in the 2-20 /~A range. The observed difference was confirmed by the trend analysis [23], F(1,19)=7.39; 0.02>p>0.01. The mean OFF duration value was also higher in DBA/2 than in BALB/c mice for the quasi-totality of currents applied, F(1,19)=26.86; p<0.001. Moreover it was observed

REWARD AND AVERSION IN TWO STRAINS OF MICE

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INTENSITY (JJA) FIG. 2. Self-stimulation performance expressed in terms of mean number of lever presses in 30 min for the BALB/c and DBA/2 strains. Abscissa: applied intensities (~A). Ordinate: mean number of lever presses and S.E. **p<0.02; ***p<0.01. (Student t-test). that increasing the current intensity led to a progressively more rapid interruption of the stimulation by both strains (comparison between ON duration values at 2 and 50/zA, respectively, by Student t-test: BALB/c, p=0.02; DBA/2, 0.01>p>0.001). Stimulation onset was also triggered more rapidly (comparison between O F F duration values at 2 and 50/zA, respectively: 0.01>p>0.001 for both lines).

Self-Stimulation by Lever Pressing (Fig. 2) Following the acquisition of operant behavior in the ICSS cage during the first session (0 /zA), the mean number of responses given by the two strains was seen to become progressively different. The response level of BALB/c mice became much greater than that of DBA/2 animals beginning at 30/xA (Student t-test: 30/xA: p <0.01 ; 40/xA: p <0.02; 50/zA: p<0.01). Two DBA/2 mice underwent convulsive seizures during the 40 and 50/~A sessions, while no comparable behavior was observed in the BALB/c group.

Switch ON-Switch OFF Under Intermittent Stimulation (Fig. 3) Experiment 1 demonstrated the apparent aversive power of a continuous train of stimulation. It was thus important to determine the effects of a discontinous stimulation that would reproduce the rhythm of ICSS lever presses observed in the second experiment. During Experiment 1, it was the BALB/c mice that exhibited the more intense escape reactions (ON duration gen-

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erally shorter), and so the stimulation sequence was determined from their ICSS performance which reached 2.500 responses in 30 min at 50/xA (Fig. 2). The performance level of BALB/c mice implanted in the LH generally stabilized at around 50-60/xA [7]. This response frequency corresponds to a discontinous stimulation : "200 msec ON/720 msec OFF". Under these conditions, it was seen that the mean value of ON duration was much higher in BALB/c than in DBA/2 mice at the highest intensities tested (40 and 50 g.A). The mean O F F duration value however was lower in BALB/c than in DBA/2 mice, as in the first experiment. The OFF duration progressively decreases in both lines with increasing current (comparison between values at 5 and 50/.LA by Student t-test : 0.01>p>0.001 in both lines). Convulsions were noted in three DBA/2 mice at 30 and 40/~A and in one BALB/c mouse at 50/xA.

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CAZALA AND GARRIGUES

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FIG. 4. Histological control. The placements of the electrode tips are plotted on frontal section diagrams. The values in micrometers (~m) indicate the distance of the section from the interaural line. Abbreviations: CI, capsula interna; DMH, nucleus dorsomedialis hypothalami; FX, fornix; LM, lemniscus medialis; TMT, fasciculus mamillothalamicus; TO, tractus opticus; PH, nucleus posterior hypothalami; PM, nucleus premamillaris; SN, substantia nigra; SUT, nucleus subthalamicus; V III, ventriculus tertius.

Histological Control (Fi~,. 4) The histological control has confirmed the correct situation of the electrode tips in the two strains. These were implanted in the ventral part of the LH in a frontal plane corresponding to the posterior third of the ventro medialis nucleus of the hypothalamus. The scattering was similar in the two strains. Microscopical examinations of individual slices showed no tissue damage caused by the long duration of stimulation. DISCUSSION These results led to the following conclusions. " A v e r s i o n " circuits appeared to be activated in the LH during the first experiment. When sinusoidal stimulation was applied continuously, both strains of mice spontaneously interrupted it after having triggered it; this interruption was observed sooner in BALB/c than in DBA/2 mice. In addition, increasing the stimulation current led to a progressive reduction of the ON duration in both strains. Bower and Miller [5] and Roberts [18] reported that the onset of stimulation engendered only an appetitive effect and that the aversive component was noted only when stimulation was continued. In the framework of this hypothesis it appears perfectly conceivable that BALB/c mice, whose positive reinforcement systems have a lower reactivity threshold than that of the DBA/2 strain [9,10] would trigger stimulation more rapidly. The O F F duration, or approach latency, could in this given case be the index of the rewarding power of central stimulation 11]. We have previously shown I6] that this is not always the case. F o r the same reasons it is conceivable that during Experiment 2, BALB/c perform the greatest number of ICSS lever presses when stimulation is limited to 200 msec. The results of Experiment 3 reinforce these two points. Repeated "200 msec ON/720 msec O F F " stimulations produced effects comparable to those of Experiment 2, since

BALB/c mice tolerated a greater number of stimulations than did their DBA/2 counterparts. In addition, as in Experiment 1, they triggered stimulation more rapidly than did DBA/2. As we have already pointed out [7] convulsions occurred more frequently in DBA/2 than in BALB/c mice. Moreover seizures were only observed when the stimulus was limited to 200 msec (Experiments 2 and 3) which confirms that these accidents are not related to the aversive component of electrical stimulation [6]. The second point is that a parallel evolution was seen between the intensity of approach reactions and that of escape reactions induced by hypothalamic stimulation in a given strain. According to a number of workers, positive reinforcement and negative reinforcement result from the activation of different nervous elements in the LH. Thus, Schmitt et al. [20] reported that the fibers subtending the escape component of LH stimulation had a refractory period generally included between 0.4 and 0.6 msec, whereas the comparable figure for the approach component was included between 0.4 and 1.2 msec. Moreover it appears possible to modify one or the other of these two components independently either by pharmacological means [3, 13, 21] or by homeostatic imbalance [2], In our study BALB/c mice exhibit the more intense approach or escape reactions according to the length of the applied stimulation; and so in the context of the above-mentioned hypothesis it might be admitted that the respective nervous elements involved in the genesis of these two behavioral responses have a greater density and/or reactivity in this strain. Stimulation would thus activate two systems of nerve fibers either simultaneously or successively, and the system responsible for the aversive effect would progressively predominate compared to the one mediating the appetitive effect. It should not be forgotten that under our experimental conditions, characteristic escape responses appeared when the animals received a continuous train of stimulation. In this case, it is thus not impossible that the prolonged activation of the fibers involved in positive reinforcement led to the deterioration of certain of their functional properties. We recalled in the introduction that the hypothesis of a single anatomical substrate governing switch ON and switch O F F phenomena had already been advanced by other workers. Thus, it was reported that the positive reinforcement system progressively adapted itself to stimulation [11, 12, 14], leading to the disappearance of the appetitive effect. The animal would interrupt the stimulation in order to again benefit from its rewarding properties following the next triggering. The results obtained in our interstrain BALB/c-DBA/2 comparison are not really sufficient to enable us to choose between the two available hypotheses: " a v e r s i o n " or " a d aptation". We believe however that data obtained with the F1 generation and recombinant inbred strains BALB/c×DBA/2 could enable us to confirm if there is a genetic relationship between the relative intensities of approach and escape responses. Such a relationship, found in the present study, has been previously observed in the Rat by Lipp [15]. Taken together, such data would enable us to see whether approach and escape responses are mediated by one and the same set of nerve fibers or whether there are two anatomically and functionally distinct systems whose proportional densities would determine their respective effects.

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ACKNOWLEDGEMENTS We wish to thank Mrs. A. M. Perret, A. Zielinski and J. Ducout for technical assistance and Dr. S. P. Jarman (University of London) who was kind enough to translate this article into English. This investigation was supported by the C.N.R.S. (ERA n ° 416) and by Grant n ° 80.79.112 (Professor B. Cardo) of Institut National de la Sant~ et de la Recherche Mrdicale.

REFERENCES

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