Effects of ganglionic blocking agents on behavioral responses to centrally administered CRF

Brain Research, 478 (1989) 205-210 Elsevier

20~

BRE 14176

Research Reports

Effects of ganglionic blocking agents on behavioral responses to centrally administered CRF Donald R. Britton 2 and Elisabeth Indyk 1 t University of Health Sciences/Chicago Medical School, North Chicago, IL (U.S.A.) and 2NeuroscienceResearch Division, Pharmaceutical Discovery, A b bott Laboratories, Abbott Park, I L 60064 ( U.S.A.)

(Accepted 5 July 1988) Key words: Corticotropin-releasing factor; Novelty; Ganglionic blocker; Chlorisondamine; Hexamethonium; Locomotion; Grooming

The ganglionic blocking agents, chlorisondamine (CL) and hexamethonium (HEX) were used to examine the role of altered autonomic function in the behavioral resnense to i,c.v.-administered corticotropin-releasing factor (CRF). Animals were tested in either a novel modified open field or in their home cages. CRF-induced alterations in locomotion, grooming and eating were assessed in both environments in the presence or absence of CL or HEX. In the home cage the ability of CRF to increase grooming was attenuated by pretreatment with either CL or HEX. In the modified open field only HEX significantly suppressed grooming. In the familiar environment CRF-induced increases in locomotion were significantly inhibited by CL. However, in a novel environment, where CRF suppresses locomotion, CL was inactive. The competitive ganglionic nicotinic blocking agent, HEX, on the other hand, inhibited both the increased locomotion produced by CRF in the home cage and also the decreased locomotion induced by CRF in the modified open field. CRF suppression of food consumption was attenuated by CL. These results indicate that while centrally-mediated activation of the sympathetic nervous system cannot account for the full magnitude of the behavioral effects of i.c.v. CRF, such activation may play a part in both the locomotor activating components of the CRF response seen in the familiar environment as well as the suppressive eff'~ts seen in the novel environment. INTRODUCTION Previous work has demonstrated that centrally administered corticotropin-releasJng factor (CRF) produces a profile of effects charat,~eristic of a stress response as measured by sympathetic activation 9a°, pituitary-adrenal stimulation 7 and expression of behaviors often associated wi~h stressful stimuli. The behavioral profile of C R F includes decreased food consumption 4.5.7.16'17, increased grooming 4'5'7'17'2°, alterations in locomotor activity 4'5"7'~4'19'2°, disruption of sexual activity ~s and 'anxiogenic-like' effects in tests sensitive to anxiolytic drugs 5'8'12. There is now compelling evidence that the pituitary-adrenal response is neither sufficient nor necessary for the expression of the behavioral effects of centrally administered CRF7,15,17. Still an open question is the degree to which CRF-

mediated activation of the sympathetic nervous system may be responsible for or contribute to the expression of the behavioral responses to centrally administred CRF. Indeed, there is evidence from a variety of sources that activation of the sympathetic system can elicit a ~tress-like response, perhaps most obvious clinically in patients with pheochromocytoma 1. A plausible interpretation of much of the behavioral data thus far reported would be that the behavioral effects of centrally administered C R F arise from the animals' response to elevations in blood pressure, hyprglycemia or other discernable components of symapthetic activation. The present work was undertaken to study the behavioral effects of C R F in animals in which the potential for sympathetic activation was compromised by pretreatment with the ganglionic blocking agents, chlorisondamine (CL) and hexamethonium (HEX).

Correspondence: D.R. Britton, Neuroscience Research Division, Pharmaceutical Discovery, D-47H, AP10, Abbc'~: Laboratories, Abbott Park, IL 60064, U.S.A.

0006-8993/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

206 If.

MATERIALS AND METHODS

Animals Male Sprague-Dawley albino rats weighing 250300 g at the time of surgery were anesthetized with sodium pentobarbital (64.8 mg/kg) and surgically prepared with 23-gauge stainless steel guide cannulae stereotaxically positioned to project to just above the lateral ventricle. The guide cannulae were fixed to the skull with dental acrylic surrounding the cannula and two stainless steel screws which were placed in the skull just posterior to the cannula on either side of midline. Internal stylets were then placed in the guide cannulae until the time of i.c.v, injections. Injection cannula consisted of stainless steel tubing of a length which would project 0.5 mm past the tip of the guide cannula. After the last test animals were injected with 2.0pl of Toluidine blue dye i.c.v, and sacrificed 15 min later to verify correct cannula placement by dye distribution. Following 5-9 days of postsurgical recovery, animals were fasted for 24 h at the end of which time they were injected with either saline, CL (3.0 mg/kg, i.p.) or HEX (Sigma Chemical Co., St. Louis) (1.0, 3.0 or 10.0 mg/kg, i.p.). After 90 min, the rats received an i.c.v, injection of 2/~! consisting of either saline, or rat/human CRF (rCRF) in doses of 30, 60 or 120 pmol (corresponding to 200, 400 or 800 ng of rCRF). Rat/human CRF was generously provided by Dr. J. Rivier of the Salk Institute. Behavioral observations Immediately following the i.c.v, injections, animals were returned to their home cages. Thirty min later behavioral observations were b e ~ n with the animals either in their home cages or in a mt dified open field6. The home cage contained preweighed food in the food trough on the cage top. "(he modified open field contained a preweighed food pellet secured to a pedestal in the center of the otc,en field. In the home cage test, animals were observed at 2min intervals for 60 min. Behavioral activity was recorded and the number of observations of each class of activity was calculated to provide scores for rearing, horizontal locomotion (rearing + horizontal locomotion = total movement), grooming and eating. Testing in the modified open field was as previously described 6, except that time sampling techniques

were used to provide a better measure of the percentage of time in each class of behavior and also to allow a more direct comparison with the values obtained in the home cage observations. In tests of various doses of HEX, animals were observed first in their home cages for 15 min in the absence of food then placed in the open field for an additional 15 m]n observation. In other tests some animals were tested in both the modified open field and in the home cage with an intervening period of at least 7 days between tests. These animals were randomly assign,~d to a drug group for each test and therefore provided a heterogenous sample with respect to previous drug history. No animals were tested more than once in either setting. Data were compared by Newman-Keuls test. Statistical significance was accepted at the 0.05 level. RESULTS The results of the CRF treatment were very similar to those previously reported 5'7. In both novel and familiar emironments food consumption was decreased aad grooming was increased. Locomotion (expressed as combined rearing plus floor locomotion) was increased in the familiar home cage test environment but suppressed in the novel modified open field.

Effects on locomotion CRF-induced suppression of locomotion in the open field was not altered by CL treatment (Fig. 1). CL had only a slight and statistically non-significant suppressive effect by itself. However, the locomotor activating properties of CRF in the home cage were significantly but not completely suppressed by CL (Fig. 1). HEX, on the other hand, attenuated the activating properties of rCRF in the home cage environment in the dose-response study (see Fig. 3) and also reversed the locomotor suppressant effects observed following CRF in the open field (Figs. 2 and 3). As with CL, HEX alone had no effect on locomotor activity. Effects on grooming CL significantly attenuated CRF-induced grooming in the home cage at the 60 pmol CRF dose but not at the 120 pmol dose (Fig. 1). In the open field, CL-

207

HOME CAGE

MODIFIED OPEN FIELD 4

O SAIJNE, IP m~i CHLORISON~AMINE'IP

°z °~

4

q

T

SAUNE, iP CHLORI-~ONDAMINE,IP

12. 10.

/

+

+'~/~

6. 4..

2,

2,

0

O, 10,

°~

12

*

8

*

;

*

•

•

*+

4 0 0

60 pmol CRF"(icy)

120

0

30 60 prnol CRF"(icy)

120

Fig. 1. Effects of chlorisondamine (3.0 mg/kg, i.p.) on CRF.induced changes in locomotor activity and grooming in the home cage and in the modified open field. Value.~ represent the means + S.E.M. * Significantly different from saline-treated controls (P < 0.05), + significantly different from group receiving saline and equivalent dose of CRF (P < 0.05). The number of animals per group is as indicated in Table I.

MODIFIEDOPENFIELD

HOME CAGE 16 14, 12,

SALINE. IP iota HEX., IP

10 8 6

4

4,

•

•

2 0

2'I

t

20

12

/

•

6

-

4-

4

0

0

60 pmol CRF (icy)

120

0

60 pmol CRF"(icy)

120

Fig. 2. Effectsof hexamethonium(lO.Omg/kg, i.p.) on CRF-inducedchangesin locomotionand groomingin the homecageand in the modified open field. Values represent the means _+ S.E.M. * Significantly different from saline-treated controls; + significantly different from group receiving CRF alone (P < 0.05). The number of animals per group is as indicated in Table II.

208 EFFECTS OF HEXAMETHONIUM ON CRF-INDUCED

TABLE II

CHANGES IN LOCOMOTOR ACTIVffY

Effects of hexamethonium (10.0 mg/kg, i.p.) on CRF-induced suppression of eating in a novel and a familiar environment

25. hi

.~ o

20.

I ,4-

o z

15-

o

10.

o u

5-

_z

g

g

0

9

ul E z b.I ft.

30.. 25-.

o cz o =Z

20.

o

10,.

0

5,.

z

q0

,4-

0

HEX

(mg/Kg. IP)

pmol CRF (i.c.v.)

mglkg HEY (i.p.)

Home cage

Modified open .field

Saline 60 pmol 120 pmol Saline 60 pmol 120 pmol

Saline Saline Saline 10.0mg/kg 10.0mg/kg 10.0 mg/kg

2.59+0.75 (14) 2.33+0.30 0.88+0.61" (8) 0.01+0.01"* 0.38+0.22** (6) 0.00+0.00"* 3.89+1.00 (11)1.72+0.26 1.78+0.69 (7) 0.13+0.05"* 0.79+_0.25** (10) 0.20+0.07**

(10) (7) (5) (15) (5) (10)

* Significant effect of CRF compared to saline/saline or saline/ hexamethonium group in same environment, P < 0.05. ** Significantly different from saline/saline or saline/hexamethonium, P < 0.01.

15.

pmol CRF. ICV

Values are expressed as mean +- S.E.M. for g eaten• n (number of animals per group) is given in parentheses•

0

60

60

60

60

0

0

1.0

3.0

10.0

Fig. 3. Effects of various doses of hexamethonium on CRF-induced changes in locomotor activity in the home cage (upper graph) and in the modified open field (lower graph). Values represent the means + S.E.M. * Significantly different from saline-treated controls (P < 0.05); + significantly different from group receiving CRF alone (P < 0.05). The numbe~of animals per group is as indicated in Table III.

treated animals t e n d e d to show less grooming, but there were no significant differences b e t w e e n the CL- and the saline-treated groups in response to C R F

(Fig. 2). Hexamethonium suppressed the increased grooming induced by both 60 and 120 pmol rCRF in the modified open field (Fig. 1). In the home cage test, HEX significantly suppressed grooming only at the higher (120 pmol) dose of CRF (Fig. 1). Effects on f o o d consumption In the home cage test, C L significantly reversed the appetite-suppressive effects of 60 pmoi of C R F but not the effect of 120 pmol. In the novel o p e n field, C L was without effect on food consumption (Table I). H E X failed to significantly alter the suppressive effects of C R F on food consumption by aniTABLE III

TABLE I

Effects of various doses of hexamethonium on CRF-induced grooming in a novel and a familiar environment

Effects of chlorisondamine (CL) on CRF-induced suppression of food consumption in a novel and a familiar environment

Values represent mean + S.E.M.

n (number of animals per group) is given in parentheses.

pmol CRF (i.c.v.)

mg/kg CL (i.p.)

Home c a g e

Modified open field

Saline 60pmol 120 pmol Saline 60 pmol 120pmol

Saline Saline Saline 3.0mg/kg 3.0 mg/kg 3.0mg/kg

3.4 +- 0.4 (16) 1.3 _+0.3* (7) 2.3 _+0.6 (18) 4.1 +-0.5 (15) 3.7 __ 0.4 + (13) 1.9 +- 0.4 (19)

0.60 +- 0.10 (10) 0.02 +- 0.01" (8) 0.00 __ 0.00" (7) 0.90+-0.10 (9) 0.05 _+0.03* (8) 0.10 + 0.05* (8)

* Significant effect of rCRF compared to saline/saline or saline/ chlorisondamine group in same environment. + Significantly different from group receiving equivalent dose of CRF and saline (P < 0.05).

pmol CRF (i.c.v.)

mg/kg HEX (i.p.)

Saline

Saline

60 pmol 60 pmol 60pmol 60 pmol

Saline 1.0 mg/kg 3.0mg/kg 10.0 mg/kg

Grooming Home cage 1.1 + 0.5 1.1"

9.2 + 7.5 + 4.5 + 11.3 +

Modified open field 0.1 + 0.1 17.6 + 1.5"*

1.0" 11.5+ 1.7 *+ 1.6 *+ 7.2 + 2.8 *++ 0.5* 12.5 + 1.7 **+

* Significant effect of CRF compared to saline/saline or saline/ hexamethonium group in same environment. + Significantly different from group receiving equivalent dose of CRF and saline (P < 0.05). ** or ++P < 0.01. Number of animals per group: sal/sal, 8; CRF/sal, 5; CRF/ HEX-1, 4; CRF/HEX-3, 4; CRF/HEX-10, 4.

209 mals in the home cage or in the open field. The apparent increased food intake by animals treated with CRF + HEX compared to CRF alone was not significant (Table II).

Dose-response effects of hexamethonium Some animals were pretreated with varying doses of HEX as described in Materials and Methods and then administered CRF (60 pmol, i.c.v.) and tested 45 min later. These animals were first observed in the home cage for 15 rain and then introduced into the novel open field for a second 15 min observation. In these animals, HEX also reversed both the locomotor-activating effects of CRF in the home cage and the locomotor suppressant effects observed in the modified open field. The former effect was significant only at the higher dose (10 mg/kg) while the suppressant effects of CRF were significantly reversed by all 3 doses of HEX (Fig. 4). CRF-induced grooming was significantly suppressed in both environments by all doses of HEX. These data are presented in Table III. DISCUSSION The hypothesis behind these experiments was that the demonstrated ability of i.c.v.-administered CRF to activate the sympathetic nervous system and alter other aspects of autonomic activity could be a contributing factor in the observed behavioral effects of CRF. We have used two ganglionic blocking agents to determine whether such blockade and the subsequent suppression of any CRF-mediated activation of autonomic activity dependent upon ganglionic transmission could depress or eliminate the behavioral response to centrally administered rCRF. The dose of CL used was chosen on the basis of published data, indicating that it eliminated the elevations in plasma catecholamines otherwise seen following i.c.v, administration of CRF l°. HEX was used in 3 separate doses with qualitatively similar results. Since entry of HEX into the CNS is very limited due to its quaternary nitrogens 21, it seems unlikely that it is altering the CRF response through central nicotinic receptor blockade. The results of this study are consistent with the possibility that ganglionic-transmitted neuronal activity contributes to the behavioral effects of rCRF.

A prime candidate for such neuronal activity would be sympathetic activation. CRF has been shown to produce such activation and the data are compatible with a prior report that adrenal demedulation blunted the suppressive effects of i.c.v. CRF on appetite ~3. However, given the multitude of metabolic and hormonal responses arising from sympathetic activation and the fact that there is evidence for the ability of i.c.v. CRF to alter parasympathetic activity as well, we cannot at present identify the precise component(s) of ganglionic activity which contribute(s) to the behavioral results. Overall, the results with CL are consistent with the possibility that the relative contribution of ganglionmediated events to the behavioral response to CRF may be more prevalent in the lower doses of CRF and in the less threatening, familiar environment. Higher doses of CRF or increased environmental stimuli such as the novel test setting appear to overcome the partial blocking effects of CL. HEX reversed not only the locomotor-activating effects of CRF in the home cage but also the locomotor-suppressive effects of CRF in the modified open field. This latter finding is consistent with a true antagonism rather than merely a non-specific reduction of high levels of activity as might be interpreted for the suppression of the locomotor-activating properties observed with CL. The present findings are conceptually consistent with other literature on the physiology of CRF. That is, CRF has a wide range of actions associated with elements of the stress response. We suggest that the present data support the additional view that multiple CRF-sensitive responses can also serve as stimuli which have behavioral significance. The activation of the autonomic nervous system appears to contribute such stimuli and thereby enhance the behavioral response to CRF. At the same time, it is clear that even at doses of CL which have been shown to completely reverse the effects of CRF on sympathetic activation, there is a large residual behavioral effect of CRF which is insensitive to ganglionic blockade. In recent years there have been several advances in understanding the details of the regulatory mechanisms of specific components of an animal's response to 'emergency' or "stressful' situations. The multiple factors contributing to neuroendocrine regulation serve as a prime example 2. There has been a long his-

210 tory of appreciation of the relationship of a u t o n o m i c to psychological events since the pioneering work of Bard, C a n n o n and others 381. The elucidation of the regulatory mechanisms of these systems allows increasingly detailed investigations of how a u t o n o m i c , endocrine and behavioral components may be inte-

ACKNOWLEDGEMENTS This research was supported in part by a grant from the N I M H , MH42480. We express our appreciation to Dr. J. Rivier of the Salk Institute for providing rCRF.

grated.

REFERENCES 1 American Psychiatric Association, Diagnostic and Statistical Menual of Mental Disorders, 3rd edn., American Psychiatric Assoc., Washington, D.C., 1980. 2 Axelrod, J. and Reisine, T.D., Stress hormones: their interactions and regulation, Science, 224 (1984) 452-459. 3 Bard, P., A diencephalic mechanism for the expression of rage with special reference to the sympathetic nervous system, Am. J. Physiol., 84 (1928) 490-515. 4 Britton, D.R., Hoffman, D., Lederis, K. and Rivier, J., A comparison of the behavioral effects of CRF, sauvagine and urotensin I, Brain Research, 304 (1984) 201-205. 5 Bdtton, D.R., Koob, G.F., Rivier~J. and Vale, W., Intraventricular corticotropin-releasing factor enhances behavioral effects of novelty, Life Sci., 31 (1982) 363-367. 6 Britton, D.R. and Thatcher-Britton, K., A sensitive open field measure of anxioiytic drug activity. Pharmacol. Biochem. Behav., 15 (1981) 577-582. 7 Britton, D.R., Varela, M., Garcia, A. and Rosenthal, M., Dexamethasone suppresses pituitary-adrenal but not behavioral effects of centrally administered CRF, Life Sci., 38 (1986) 211-216. 8 Britton, K.T., Morgan, J., Rivier, J., Vale, W. and Koob, G., Chlordiazepoxide attenuates CRF-induced response suppression in the conflict test, Psychopharmacology, 86 (1985) 1070-1074. 9 Brown, M.R., Fisher, L., Rivier, J., Spiess, J., Rivier, C. and Vale, W., Corticotropin-releasingfactor: effects on the sympathetic nervous system and oxygen consumption, Life Sci., 30 (1982) 207-210. 10 Brown, M.R., Fisher, L.A., Spiess, J., Rivier, C., Rivier, .I. and Vale, W., Corticotropin-releasing factor: actions on the sympathetic nervous system and metabolism, Endocrinology, ii i (i982) 928-931.

11 Cannon, W.B. and Britton, S.W., Studies on the conditions of activity in endocrine glands. XV. Pseudoaffective medulliadrenal secretion, Am. J. Physiol., 72 (1925) 283-294. 12 Dunn, A. and File, S., Corticotropin-releasingfactor has an anxiogenic action in the social interaction test, Hormones Behav., 21 (1987) 193-202. 13 Gosnell, B.A., Morley, J.E. and Levine, A.S., Adrenal modulation of the inhibitory effect of corticotropin-releasing factor on feeding, Peptides, 4 (1983) 807-812. 14 Koob, G.F. and Bloom, F.E., Corticotropin-releasing factor and behavior, Fed. Proc., 44 (1985) 259-263. 13 Koob, G.F. and Thatcher-Britton, K., Stimulant and anxio~'.enic effects of corticc~tropin releasing factor, Prog. Clin. l~.iol. Res. (U.S.A.). 192 (1985)499-506. 16 Levine, A.S., Rogers, B., Kneip, J., Grace, M. and Morle,/, J.E., Effect of centrally administered corticotropin-rek':,~ing factor (CRF) on multiple feeding paradigms, Neuropharmacology, 22 (3A) (1983) 337-339. 17 Morley, J. and Levite, A.S., Corticotropin-releasing factor, grooming and ingestive behavior, Life Sci., 31 (1982) 1459-1464.

18 Rivier, C., Rivier, J. and Vale, W., Stress-induced inhibition of reproductive fm~ctions: role of endogenous corticotropin-releasing factor, Science, 231 (1986) 607-609. 19 Sherman, J.E. ano l~.al,~. N.H., l.c.v. CRH potently affects behavior without altering antinocioceptive responding, Life Sci., 39 (1985) ~33-441. 20 Sutton, R.E., Koob, G.F., LeMoal, M., Rivier, J. and Vale, W., Corticotropin-releasing factor produces behavioral activation in rats, Nature (Lond.), 297 (1982) 331-333. 21 Taylor, P., Ganglionic stimulating and blocking agents. In A.G. Goodman, L.S. Goodman and A. Gilman (Eds.), The Pharmacological Basis of Therapeutics, sixth edn., Macmillan, New York, 1980, pp. 211-220.