Effects of the opiate antagonist naloxone upon hypothalamically elicited affective defense behavior in the cat

Behavioural Brain Research, 33 (1989) 23-32 Elsevier

23

BBR00914

Effects of the opiate antagonist naloxone upon hypothalamically elicited affective defense behavior in the cat Martin

Brutus

and Allan

Siegel

New Jersey Medical School, Newark, NJ 07103 (U.S.A.)

(Received 28 March 1988) (Revised version received 16 September 1988) (Accepted 20 September 1988) Key words: Naloxone; Affcctive defense; Quiet biting attack behavior; Opiate peptide; Itypothalamus; Limbic system; Aggressive behavior

The opiate antagonist naloxone hydrochloride was employed in order to determine whether endogenous opioids play a role in the control of affective defense behavior elicited from the medial hypothalamus in the cat. The effects of naloxone upon quiet biting attack behavior elicited from the lateral hypothalamus were also assessed. A comparison of the differences in response latencies or thresholds before and after naloxone (i.p.) administration was made. Naloxone (1, 4 and 10 mg/kg) was found to significantly facilitate affective defense behavior in a dose- and time-dependent manner. The duration of facilitation ranged from 30 min after a I mg/kg injection to 180 min after a 10 mg/kg injection. The data also suggest that the effects of naloxone upon affective defense behavior are opposite to those seen x~ith quiet biting attack. In two animals, quiet biting attack behavior was suppressed for 30 min following a 10 mg/kg injection ofnaloxone. Naloxone was also administered to cats in which hypothalamic stimulation elicited predatory responses coupled with components of affective defense behavior. In these cases, naloxone was ineffective in altering latencies for this 'mixed' response. These findings suggest that the opiate peptide system selectively inhibit~ affective defense behavior elicited from the medial hypothalamus of the cat.

INTRODUCTION T h e limbic-hypothalamic-midbrain axis forms a reciprocally interconnected functional system which is involved in the expression and modulation o f aggressive reactions ~'8"9"1~-~s'2~ 58-62 and related processes 2'5'7'22'25"3~ In addition, these regions o f the brain are also k n o w n to contain high concentrations of b o t h enkephalin-containing perikarya and nerve terminals 57"65"66 and receptors6:34'47'48'52 which a p p e a r to functionally overlap with those sites that mediate affective defense behavior. Opiates are k n o w n to m o d u l a t e such behavioral responses as

intraeranial self-stimulation m2.42 and feeding behavior 3~ which can be elicited by electrical stimulation o f the hypothalamus. T h e s e peptides are also involved in other processes including h y p o t h a l a m i c regulation of b o d y t e m p e r a ture 38'39'45, stress 5'14"37"41, sexual behavior 22, and drinking 26.27. E n d o g e n o u s opioids a p p e a r t o m o d u l a t e aggression as well. D a t a obtained from experim e n t s utilizing rodents indicate that peripheral administration o f m o r p h i n e or fl-endorphin decreases aggressive reactions 24,63, while facilitation o f aggressive behavior has been found to o c c u r after n a l o x o n e administration '~176

Correspondence: M. Brutus, Department of Neurosciences, New Jersey Medical School, Medical Science Building, Room H517, 185 South Orange Avenue, Newark, NJ 07103, U.S.A.

0166-4328/89/$03.50 9 1989 Elsevier Science Publishers B.V. (Biomedical Division)

24 Despite these findings, the role that endogenous opioids have in aggression has been challenged23.33.56. Our laboratory has recently begun to explore the possible effects of endogenous opioids upon several forms of feline aggression elicited by brain stimulation. Two forms of aggressive behavior affective defense and quiet biting attack behavior -can be elicited by stimulation of the cat's 7,2~ medial and lateral hypothalamus, respectively. In one experiment, thresholds for affective defense behavior elicited from the midbrain periaqueductal gray were lowered following peripheral administration of naloxone 6~ In another study, we observed that when naloxone was microinjected into midbrain periaqueductal gray sites which suppressed hypothalamically elicited affective defense behavior, the suppressive effects of periaqueductal gray stimulation were blocked 49. The results of these preliminary investigations obtained from the cat, as well as those conducted in other species, have led us to test the hypothesis that endogenous opioid peptides serve to selectively suppress affective defense behavior elicited from the hypothalamus of the cat. MATERIALS AND METttODS

Ten adult eats of either sex which did not spontaneously attack rats and whichweighed between 2.5 and 4.0 kg were employed in this study. They were maintained on an ad libitum feeding and drinking schedule throughout the duration of the experiments. The eats were anesthetized with sodium pentobarbital (45 mg/kg of body weight) prior to aseptic surgical procedures. A total of 6 stainless steel guide tubes (17-gauge) were then stereotaxically mounted on each side of the skull directly overlying the medial and lateral hypothalamus (AP: 8.5-12.5; L: 1.0-2.5) zg. This allowed us to explore a total of 3 medial and 3 lateral hypothalamic sites for elicitation of affective defense and quiet biting attack, respectively, on each side of the cat's brain. Moveable electrodes were subsequently lowered through these guide tubes into the hypothalamus. Details concerning the type of electrodes and surgical procedures used are described elsewhere8"9. All experi-

ments were conducted in a test chamber constructed of wood (61 cm x 61 cm x 61 cm) with a clear Plexiglas door. Electrical stimuli were generated by a Grass S-8 stimulator and channeled through a stimulus isolation unit (Grass SIU 4678) to the animal. Constant current conditions were approximated by a 40-kf~ resistor placed in series with the cat. Monopolar stimulation of the hypothalamus consisted of balanced biphasic pulses delivered at 62.5 Hz, with a pulse width of I ms per halfcycle duration. Peak-to-peak current was measured on a Tektronix 51i3 oscilloscope with differential inputs.

Elicitation of attack behavior The first phase of the research protocol required the identification of those hypothalamic sites from which an affective defense or quiet biting attack response could be elicited. One week after surgery, electrodes were passed through the guide tubes and the brain was stimulated at 0.5-ram steps as the electrode was lowered through the hypothalamus. When aggressive reactions were consistently elicited from a site, the electrode was cemented in place with dental acrylic. Of the 10 cats utilized in this study, affective defense was elicited from 12 medial hypothalamic sites in nine cats, while quiet biting attack was elicited from 5 lateral hypothalamic sites in two cats. A deeply anesthetized rat and food were always present within the observation chamber when tests were conducted. Measurements of response thresholds and latencies The next phase of the experimental paradigm involved the determination of baseline thresholds for each type of aggressive behavior. The baseline current threshold for each of these responses was determined by a modified Method of Limits, where ascending and descending trials were employed. In this test series, current intensity was raised or lowered in 0.05-mA steps in a counterbalanced a-b-b-a manner with intensity 'a' above threshold and intensity 'b' below. The response threshold for either affective defense or quiet biting attack behavior was defined as the lowest

25 current intensity at which 5 consecutive responses were elicited. After the threshold for a particular site was determined, the experimental paradigm was begun. Two types of dependent measures were recorded 5 min following drug administration. The response threshold, as described in ~the preceding paragraph, and response latency were the dependent variables measured both before and after drug and vehicle control injections. Latency measures were determined with a stop watch with an accuracy of 0.1 s. The latency for elicitation of affective defense behavior was defined as the duration of time required to elicit a hissing response following the onset of electrical stimulation. Current values delivered to the hypothalamus ranged from 0. I to 0.8 mA. The latency for elicitation of quiet biting attack behavior was defined as the time at which the cat's teeth made contact with the back of the neck of an anesthetized rat following stimulation onset. Latency for initial movement to attack was defined as the time from stimulation onset to the moment the cat moved its forepaws. Alterations in the attack latencies during stimulation of a particular brain site following drug treatment were always observed before changes occurred in threshold values.

Drug admblistration and paradigm for behavioral testing Drug and vehicle control injections were usually administered on the same day. Control injections of a volume of saline, ( 0 . 9 ~ ) equal to that employed for experimental conditions, preceded baseline threshold and latency determinations by 5 min. This procedure was then followed by i.p. administration of naloxone hydrochloride (Sigma). For studies involving affective defense behavior, each of 9 animals received naloxone injections at a dose of 10 mg/kg of body weight. Six of these animals also received doses of 1 and 4 mg/kg. Latency and threshold determinations were again taken 5 min postinjection. The sequence of delivery of drug doses were varied for different animals. A minimum interval of two days separated test sessions involving delivery of different doses of naloxone. With respect to quiet

biting attack behavior, testing was limited to a drug dose of 10 mg/kg. At the 10-mg dose level, 12 sites in the medial hypothalamus of 9 cats were tested for the effects of naloxone upon affective defense, 5-30 min postinjection. In 8 of these cats, one or more replications were made. In 6 cats, latency and threshold determinations were recorded at 30-min intervals, starting at 5 rain and extending to 300min postinjection. Each 30-min period contained between 10 and 14 trials of hypothalamic stimulation, utilizing an interstimulus interval of 2 min. At the completion of all experiments, the animals were deeply anesthetized and small lesions were placed at hypothalamic sites at which attack responses were elicited, in order to facilitate identification of electrode tips. The animals were then perfused with 0 . 9 ~ saline and 10~ formalin. The brains were removed, blocked, sectioned at 40 iLm and stained with Cresyl violet for localization of electrode tips. A 4 x 8 repeated measures Analysis of Variance (ANOVA) was performed to determine the effects of different doses of naloxone upon affcctive defense behavior at each time period postinjection. One-way repeated measures ANOVA were then performed across each of the treatments at each time period, followed by a Duncan's Multiple Range Test with the alpha level set at P = 0.05 for each comparison. In addition, for all sites at which a 10 mg dose of naloxone was utilized, t-tests were employed to determine the significance of changes in response latencies 5 - 3 0 m i n following naloxone administration, relative to vehicle control as well as preinjection baseline values. RESULTS

Affective defense behavior was elicited by electrical stimulation of brain sites distributed within the medial hypothalamus, extending from the preoptic region to the posterior hypothalamus. Quiet biting attack was obtained from sites located in the perifornical lateral hypothalamus. Affective defense behavior was typically characterized by marked autonomic activation and

26 TABLE I

Effects of peripheral administration of naloxone upon hypothalamically elicited affective defense and predatory attack behavior AD, affective defense; QBA, quiet biting attack; QBA/AD, quiet biting attack with components of affective defense; F, response facilitation at each of the doses tested (P < 0.05); S, response suppression at each of the doses tested (P < 0.05); n.s., not significant; X, dose of naloxone administered; M t t , medial hypothalamus; LIt, lateral hypothalamus. Numbers next to attack site loci indicate site numbers referred to in the text and depicted in the illustrations. Note that, in 4 animals, several different attack sites were tested from the same animal. All effects shown in this table were based upon changes in response latencies over the first 30-min period following naloxone administration.

Animal

1 1 2 2 2 3 4 5 6 7 8 9 3 10 10 3 3

Attack site locus

Dose of naloxone (mg[kg)

Response

Effect

X X X X X X X X X X X X

AD AD AD AD AD AD AD AD AD AD AD AD

F F F F F F F F F F F F

X X X X X

QBA QBA QBA/AD QBA/AD QBA/AD

S S n.s. n.s. n.s.

1.0

4.0

10.0

MHI MH2 MII3 MtI4 MH5 MH6 MH7 MII8 MH9 MHI0 Mill I MHI2

X X X X X X X X X

X X X X X X X X X

LI I 1 LIt2 LH3 LH4 LIt5

-

-

included such components as pupillary dilatation, piloerection, arching of the back, retraction of the ears, growling and hissing. Defensive attack may be classified as either intraspecific or interspecific, since stimulation can elicit vocalization and striking directed at a variety of targets such as another cat or at other species 6~. In contrast, quiet biting attack behavior is a form of interspecific aggression. It consists of ar~ initial stalking of a rat followed byvigorous biting of its neck. Aside from the presence of pupillary dilatation, other overt signs of autonomic activation are not apparent.

Naloxone modulation of affective defense behavior The principal finding of this study - that i.p. injections ofthe opiate antagonist naloxone facilitate affective defense behavior elicited from 12 sites in the medial hypothalamus - is summarized in Fig. 1. At all sites examined, injections of

naloxone (10mg/kg) resulted in significant ( P < 0.05) reductions in hissing latencies 5-30 min postinjection, relative to vehicle control values. A more detailed analysis of this effect, shown in Fig. 2, demonstrates a facilitation of affective defense behavior in a dose- and timedependent manner following naloxone administration. A two-way repeated measures ANOVA indicated highly significant drug treatment effects ( F = 7.40, d f = 3,18, P < 0 . 0 0 2 ) . One-way ANOVAs indicated highly significant differences as well in the magnitude of the naloxone effect at each of the different dose levels for the following post-injection time periods: 5-30 min (F = 11.13, dr-- 3,17, P < 0.003); 30-60 min ( F = 5.08, df = 3,18, P < 0.01); and 60-90 min (F = 5.56, df = 3,17, P < 0.007). At both 4 and 10 mg/kg doses, affective defense thresholds were signifi-

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Fig. 1. Naloxone facilitates hypothalamically elicited affective defense behavior. Naloxone (10 mg/kg, i.p.) significantly reduced latencies for affective defense 5-30 min postinjection. For each pair of histograms, the first (open) histogram corresponds to the vehicle control; the second (hatched) histogram represents injections ofnaloxone (10 mg/kg, i.p.); numbers below bar graphs indicate site numbers (attack loci in Table I). Subscripts next to site numbers indicate the number ofreplications at a particular brain site. ""P < 0.001; "'P < 0.01; "P < 0.05.

cantly reduced (P < 0.05) relative to vehicle controis by as much as 25~o at 5-30 min following naloxone administration. At a dose of 10 mg/kg, significant ( P < 0 . 0 5 ) reductions in affective defense thresholds were still found at 180 min postinjection. At a dose level of 4 mg/kg of naloxone, significant ( P < 0.05) reductions in affective defense thresholds lasted only 60-90 min after the injections. Administration of a 1 mg/kg dose ofnaloxone produced a significant (P < 0.05) reduction in thresholds for affective defense only for 5-30 min postinjection. At this dose level, changes in affective defense latencies approached baseline levels ( P > 0 . 0 5 ) after 30 min postinjection.

Naloxone modulation of quiet bithlg attack behavior Alterations in quiet biting attack latencies following drug administration were examined for 5 sites (in two cats) in order'to assess the specificity of the effects of naloxone upon the affective defense behavior described above. This data, summarized in Fig. 3, indicates that, in the two cats, naloxone administration at a d o s e of 10 mg/kg resulted in a statistically significant ( P < 0.05) suppression of quiet biting attack behavior 5-30 min postinjection. In each of these cases, a statistically significant replication of such effects was also observed. In 3 additional animals, the effects ofnaloxone (I0 mg/kg) upon a form of

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TIME POST INJECTION (mln)

Fig. 2. Naloxone facilitates affective defense behavior in a dose- and time-dependent manner. Higher doses ofnaloxone result in progressively larger dose-dependent decreases in affective defense thresholds. These dose-dependent threshold decreases diminish in a time-dependent manner towards vehicle control levels. In addition, vehicle injections were not found to have any effects upon affective defense thresholds when compared to preinjection baseline values for the entire testing period. Each data point represents the grouped mean change in the affective defense threshold across stimulation trials for each 30-min period following naloxone administration. Error bars indicate the S.E.M. All naloxone percent changes were determined with respect to vehicle control values.

28 m

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Fig. 3. Peripheral injections of naloxone suppress quiet biting attack obtained from two cats in which 5 different attack sites were tested. Natoxone significantly increased latencies for the biting component of the quiet attack response 5-30 min postinjection (sites 1 and 2). At those sites where quiet attack also included affective components such as hissing, the suppressive effects ofnaloxone that were seen upon 'pure' forms of quiet attack were not observed (sites 3-5). Although not shown in this figure, vehicle control injections had no effects upon latencics for quiet attack.

quiet biting attack behavior, which included several components of affective defense such as piloerection, urination and some vocalization, were assessed. In all of these cases, the increases in attack latencies were observed to be statistically non-significant. In addition, the effects of naloxone administration upon the latencies for initial movement to quiet biting attack were examined. This measure was selected because changes in initial movement latencies can frequently reflect non-specific drug interference of the motor components of the quiet biting attack response 6'. The results of naloxone administration (10 mg/kg) upon the latencies for initial movement to attack are shown in Fig. 4. Here, naloxone was observed to have no significant effects upon latencies to initial movement for any of the 4 cases considered. In two of these animals, replication of this experiment also produced similar findings.

Fig. 4. Peripheral injections ofnaloxone had no effects upon latencies for initial movement for quiet biting attack 5-30 min postinjection for all sites examined. Initial movement represents a motor component of the attack response ofwhich biting is the final component. Since naloxone failed to significantly suppress the latencies to initial movement (sites 1-4), but did suppress the biting component of the quiet attack response in these animals, it would appear that the effects of naloxone were selective for the biting component.

DISCUSSION

The results of the present study indicate that naloxone powerfully facilitates affective defense behavior elicited from the medial hypothalamus ofthe cat. The specificity ofthe effects ofnaloxone upon affective defense behavior was revealed by the fact that quiet biting attack behavior was either suppressed by naloxone or unaffected by this drug. Naloxone was shown to have no effects upon quiet attack behavior that was mixed with components of the affective defense response. These results are quite consistent with several other experiments recently carded out in our laboratory. These include the demonstration that peripheral administration ofnaloxone lowered the threshold for affective defense behavior elicited from the midbrain periaqueductal gray matter 6~ and that suppression ofhypothalamically elicited affective defense behavior b y the midbrain periaqueductal gray could be prevented by microinjections of naloxone placed into these same central gray modulatory sites 49. The present results are also generally consistent with the findings obtained from other studies where opiate compounds were administered to rodents and another model of affective aggression

29 was employed 1~176 Although several authors have failed to observe any systematic relationship between manipulation of opiates and shock-induced fighting4~ the majority of studies utilizing this model of aggression have indicated that naloxone administration enhances shock-induced fighting19"43"5~ This suggests that an inhibitory relationship exists between endogenous opiate release and defensive behaviors 19,23,43, 50" The relationship between opioid peptides and aggressive behavior may also be understood in terms of the nature of the-response under consideration. With respect to affective defense behavior, it has previously been shown that a cat will learn a task to escape hypothalamic stimulation at sites where this behavior can also be elicited2. This observation would strongly imply that affective defense behavior has aversive properties. Accordingly, this response would likely share some of the features found in other aversive processes such as nociception and stress, which have also been shown to be suppressed by opioid peptides 3'31"44. Therefore, it is not surprising that the present results suggest that opioid peptides serve to inhibit affective defense behavior. In contrast, our preliminary data describing the suppressive effects of naloxone injections upon quiet biting attack behavior would suggest that, perhaps, a distinctly different mechanism governs opioid peptide control over this form of aggressive behavior. In fact, it should be pointed out that predatory aggression elicited from the perifornical lateral hypothalamus in the rat has been shown to have positive reinforcing properties 46,s3,64. It is likely that quiet biting attack in,the cat may have the same properties as well. Our findings that naloxone suppresses quiet attack are also consistent with the recent observations of Jenck et al.30 who showed that lateral hypothalamic stimulation-induced reward is facilitated by the action of mu and delta opioid receptor ~igonists. Endogenous opioids may then normally act to potentiate quiet attack by utilizing such a mechanism. Thus, it would appear that the role of endogenous opioids in regulating aggressive behavior may vary, depending upon the form of aggression considered.

While the present study has established the likelihood that the opioid peptide system can powerfully modulate affective defense behavior elicited from the hypothalamus, it has not addressed issues concerning the sites within the brain where these effects occur, nor the opioid receptor subtypes at these sites. Neurochemical and immunocytochemical studies have indicated that opioid peptides are present throughout the rostrocaudal extent of the medial hypothalamus and midbrain periaqueductal gray matter from which affective defense can be elicited 19'21"58,65,66. Opioid peptides are also extensively present in limbic system sites and related structures such as the amygdala, bed nucleus of the stria terminalis and hippocampal formation4:1'25's4,66. All of these regions have been shown to modulate affective defense behavior ~2.25.59.62.Thus, it is reasonable to conclude that the facilitation of affective defense behavior we have observed following naloxone administration in the present study may have been achieved by the antagonism of opioid receptors situated in some or all of these nuclei. Concerning the possible receptors involved in the regulation of affective defense behavior, naloxone is known to preferentially bind to mu receptors, although it has a much weaker affinity for delta and kappa receptors ~5-~7. If the effects of naloxone on defensive aggression were due primarily to delta and kappa receptor activity, then diprenorphine, which has a very high and equal affinity for these receptors ~5-~7, would be expected to have some effect upon shock-induced (irritable) aggression. While naloxone can facilitate this type of aggression, diprenorphine does not 55. Thus, it is plausible to suggest that the major component of the effects of naloxone upon affective defense in the cat are, at least, partially, mediated by the mu-opioid receptor. Although the present data are compatible with the hypothesis that naloxone facilitates affective defense behavior by blocking neural activity at the level of the endogenous opioid receptor, the known antagonism of GABA-mediated synaptic inhibition by naloxone ~8 provides an alternative explanation for these effects. Naloxone can antagonize GABA-induced inhibition of neuronal firing in rat brain, as well as potentiate the con-

30 vulsant activity ofbicuculline in mice ~8. In spite of the species, brain site and significant dose differences utilized in this study, we do not entirely rule out the possibility of GABAergic involvement in the facilitation of affective defense. However, our injections ofnaloxone had no apparent effects upon any ongoing natural behaviors in the cat, in contrast to the convulsions observed in mice in the experiments of Dingledine et al. ~8. Moreover, antagonism of GABA-receptor-mediated inhibition resulting in convulsions following large doses of naloxone also suggests a non-specific or generalized disinhibition which stands in marked contrast to the graded dose- and time-related effects we have found. Our laboratory is currently attempting to identify possible GABA mediation and specific opioid receptor subtypes and agonist actions at key synaptic regions within the limbic-hypothalamic-midbrain axis that also modulate hypothalamically elicited affective defense behavior. ACKNOWLEDGEMENTS

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