Intracerebroventricular LHRH relieves behavioral deficits due to vomeronasal organ removal

0361-9230/92 $5.00 + .OO Copyright 0 1992 Pergamon Press Ltd.

Bra/n Rewmch

Bulkfin, Vol. 29, pp. 75-79, 1992 Printed in the USA. All rights reserved.

RAPID COMMUNICATION

Intracerebroventricular LHRH Relieves Behavioral Deficits Due to Vomeronasal Organ Removal MICHAEL

Newoscience Program, Department

AND

MEREDITH’

qf Biological Science, Received

B-221,

30 December

GAY

HOWARD

Florida State University, Tallahassee, FL 32306 I99 1

M. AND G. HOWARD. Inlru~c~rc~brovenlri~~rlur LHRH rc~licves hehuviorul deficirs due to vomeronasal orgun BRAIN RES BULL 29(I) 75-79, 1992.-Contact between the sexes in many species is known to produce hormonal changes in the male [increases in serum luteinizing hormone (LH) and/or testosterone] that can be interpreted as due to an intracerebral release of LH-releasing hormone (LHRH). In some circumstances. these hormonal changes appear to depend on an intact vomeronasal sensory system. Exogenous LHRH is also known to facilitate mating behavior in several species. We show here that LHRH delivered into the cerebral ventricles can restore some mating behavior lost when the vomeronasal organs are removed from sexually inexperienced male hamsters. The results are consistent with our working hypothesis that intracerebral LHRH release is an intermediate in the facilitation of mating behavior by vomeronasal sensory input. MEREDITH.

removal.

LHRH

lntracerebroventricular

Mating behavior

Vomeronasal organ

THE vomeronasal organ (VNO), located at the base ofthe nasal septum, contains the receptor neurons of the accessory olfactory system. The system projects rather directly to areas of the forebrain concerned with reproductive physiology and behavior, including the corticomedial amygdala and preoptic and hypothalamic regions. Damage to the VNO or vomeronasal nerves, designed to interrupt vomeronasal sensory input, results in deficits in some reproductive functions in both sexes and several species (especially rodents), presumably because the reception of pheromone signals via the VNO is interrupted [see reviews: (7.10,22,24)]. In male hamsters, mating behavior is seriously impaired by removal of the VNO if animals are sexually inexperienced (I I). Sexual experience apparently allows animals to learn other cues (primarily olfactory) that permit them to maintain mating behavior when VNOs are subsequently removed. VNO damage also impairs hormonal responses to chemosensory cues (in mice). The luteinizing hormone (LH) release that follows exposure of males to female urine odors does not occur after vomeronasal lesions (2). The testosterone increase that follows exposure to females is also lost after VNO lesions (23). Both these hormonal responses are presumably preceded by a release of LH-releasing hormone (LHRH) in the brain. LHRH is known to be facilitatory to mating behavior in both male and female rodents (3,16,17).

If vomeronasal sensory input normally facilitates mating behavior via an intracerebral release of LHRH, it seemed possible that exogenous LHRH might restore mating behavior impaired as a result of VNO removal. We show here that intracerebroventricular (ICV) injection of LHRH significantly relieves deficits in mating behavior in animals with vomeronasal organs removed (VNX) when compared with sham-operated (SHAM) animals. We used intracerebral injection because in previous tests (Meredith. unpublished) subcutaneous LHRH (500 ng) produced no change in mating behavior in either intact or VNX animals. A preliminary account of this study has appeared in abstract/summary form ( I2,13). METHOD

Sexually inexperienced, male golden hamsters (Hsd/SYR, Harlan-Sprague-Dawley) bred in the lab and approximately 3 months old were assigned randomly (within litters where possible) to two groups. Ten animals had their VNOs removed (VNX) surgically through the palate (1 I) and 10 animals underwent sham surgery, where the palate was opened, to expose but not damage the vomeronasal capsule, and then sutured (SHAM). After at least 3 days’ recovery, each animal was im-

’ To whom requests for reprints should be addressed.

75

MEREDITH planted stereotaxicaffy with a 26-ga guide tube into the left lateral ventricle. The animal’s head was positioned in the stereotaxic holder with lambda and bregma in the same horizontaf plane ~skulf-flat”). The guide tubes (Plastics One, Roanoke, VA) were cut to a length of 4-mm and implanted through a hole drilled in the skull at (cm) AP-0.03, ML 0.2277, DV -0.3400, with reference to bregma (coordinates previously calculated from several brains cut in the standard vertical plane and checked by test implantations in animals of the same age and weight). The exposed skull and su~ounding tissues were treated prophytacticaffy with antiseptic powder (Nitrofurazone, TechAmerica, Kansas City, MO) that was then scraped away from the skull around the guide tube placement. The guide tube was attached to the skull with dental acrylic and two sterilized O-80 screws fitted so that they did not protrude significantly through the interior surface of the skull. The guide tube was closed when not in use by a screw cap with an attached styfet, cut so that it extended fess than 111mm beyond the end of the tube. The skin incision was closed by bringing the cut edge up to the dental cement pedestal around the guide tube and lapping the dental cement over the edge of the skin. After surgery, animals were housed individually in plastic cages (44 X 21 X 18 cm), in a room with a partially reversed 14 L: IO D light cycle, which also housed cycling females. Food and water were available ad fib except during behaviorai tests and for 24 h before food preference tests (see below). Animals were inspected every day after guide tube implantation. Behavioral tests began no sooner than 3 days after surgery. After the completion of the experiments, all animals were deeply anesthetized with sodium pentobarbital and injected through the cannufa with a small volume of india ink. They were then perfused through the heart with saline followed by Bouin’s fixative. The skull was decalcified in RDO fluid (Apex Engineering, Plainfield, IL) and coronal sections were cut through the regions of the guide tube implant ( 10 Mm) and the VNO and olfactory epithefium (15 pm). Sections 150 pm apart were mounted and stained with haematoxylin and 1~x01fast blue to assess the effectiveness of surgical procedures and possible associated damage. Intermediate sections were retained in order and mounted to fill in the 150~pm gaps in the initially mounted series where necessary. Thus. serial sections were examined for some critical areas.

Animals were momentarily restrained while the cap and stylet were removed from the guide tube and a 33-ga cannula attached via a Tygon tube to a Picospritzer (General Valve Co., Fairfield, NJ) was inserted. The animal was then released into a clean plastic cage (44 X 21 X 18 cm) and the Picospritzer used to inject substances into the ventricles of the unrestrained animal (Fig. I). The cannula and Tygon tube were filled before insertion with peptide or saline solution and the Picospritzer operated to ensure that the cannufa was filled to the tip. A standard 2 ~1 volume of solution was injected by three pressure pulses spaced 30 s apart and followed by 90 s to allow solutions to diffuse away from the tip of the cannula. The cannula was then removed, the cap and stylet replaced, and the animal returned to its home cage. Before each test, cannuiae were individually calibrated by weighing saline ejected onto small filter paper discs. Behavioral tests were conducted 30 min after the start of intracerebral injection.

Thirty minutes after peptide or saline injection, the test animal was placed in a clean cage (44 X 2 I X 18 cm) and allowed

AND

HOWARD

Picospritzer i..“l

r\ \

injected

into the

cerebral

ventricles

/

\

n

VOMERONASAL

.

REMOVED IVNX) or sham

FJG. I. Cut-away diagram ofthe hamster head to expose the nasal septum and show the location of the vomeronasal organ and olfactory epithelium (stippled). Two microliters LHRH or saline was delivered by calibrated pressure pulses from a Picospritzer into the cerebral ventricles of inexperienced animals with vomeronasal organs removed (VNX) or with shamsurgery. The arrows indicate routes ofaccess to the olfactory region (open) and the vomeronasai duct (solid). I min to become accustomed to its new surroundings. A behaviorally receptive. naturally cycling female was then introduced and various aspects of mating behavior were recorded for 5 min or until the males achieved five intromissions (generalfy insufficient to allow ejaculation) by an observer who was aware of the treatment (peptide or saline) but was not aware of the group to which the test animal belonged (VNX or SHAM). Mounts, thrusts, intromissions, and investigation ofthe perineal and nonperineal areas, as well as other behaviors, were scored by holding down different keys of a hexadecimal keypad connected to a portable microcomputer. The number, duration, and timing of key presses was recorded by the computer and later uploaded into an IBM PC for further analysis. Tests were repeated six times, with at least 3 days between tests, and afternating between peptide and saline injection. Because test animals had no sexual experience at the start, some increase in performance was expected with repeated testing even though animals did not ejaculate. LHRH injection tests were therefore given before saline injection tests in each cycle because we expected LHRH to facilitate mating behavior and were anxious not to bias the results in favor of our hypothesis (see the Discussion Section). Tests were conducted during the first 4 h of the dark part of the 14 L: 10 D cycle. Performance in the mating tests was primarily measured as number of introm~ssions per minute. This measure allows data from tests where males achieved five intromissions before 5 min to be compared directly with data from tests that were terminated at 5 min, before the male had achieved five intromissions. Differences in the effects of LHRH on performance of VNX compared to SHAM animals were tested with the Mann-Whitney U-test (19). Changes in the performance of a group after LHRH injection, compared to the

LHRH

AND

VNO INFLUENCES

SEXUAL

BEHAVIOR

ON MATING

intromissions

77

BEHAVIOR

I Min

LHRH injection tests compared to saline injection tests (1. I intromissions/min. median over six tests; p < 0.05 Walsh test, two tailed).

The two groups did respond differently to LHRH injection when their performance was referred to the saline injection baseline. The difference in performance for each animal between the saline and LHRH injection tests in each repeated cycle of testing was calculated by subtracting the former from the latter to produce an “L - S” score. The L - S scores for each animal were averaged over the six repeated cycles of testing. Those for VNX animals were signi~cantly different from those for SHAM animals 0, < 0.05, Mann-Whitney P-test. two tailed).

SHAM

VNX

icv Saline

SHAM

VNX

icv LHRH

FIG. 2. Sexual (mating) behavior of VNX and SHAM animalsafter ICV injection of LHRH (hatched bars) or saline (open bars). Median intromissions/min for each animal and each test cycle were averaged over all six test cycles. VNX animals significantly increased performance after LHRH and SHAM animals significantly decreased performance compared to their respective performances after saline. The significant difference between VNX and SHAM animals (after saline) was eliminated by LHRH (see text).

performance of the same group after saline injection. were evaluated with the Walsh test (19). In three VNX and four Sham animals, the guide tubes became loose before the series of tests was half completed. Data from these animals was discarded, leaving data from seven VNX and six SHAM animals. Two additional SHAM animals lost their guide tubes between the fifth and sixth rounds of tests so the data for the last test is from seven VNX and four SHAM animals. The data for each group are averaged over all six tests so the missing data comprises 2/[(7 + 6) X 61 or 2.6% of the total data set. The possible consequences of the missing data are discussed Iater. RESULTS

As expected from previous results, inexperienced VNX animals had significant deficits in mating behavior compared with SHAM animals when both were injected with saline, as shown by the open bars in Fig. 2. VNX animals had 0.2 intromissions/ min compared with 1.6 intromissions/min for SHAM animals (medians in the six tests for each group; p < 0.0 1, Mann-Whitney -!&test, two tailed).

Our simple working hypothesis predicted that VNX animals would increase their performance when injected with LHRH, partially or fully compensating for the deficits attributable to removal of vomeronasal input. VNX animals did increase their performance-to 0.7 intromissions/min, which was significantly greater than when saline was injected (medians in the six tests; 11< 0.05, Walsh test, one tailed)-and there was no longer any significant difference in the performance of VNX and SHAM groups, as shown by the hatched bars in Fig. 2. However, SHAM animals unexpectedly decreased their performance during

The amount of time males spent investigating the perineal and surrounding area of the female, compared to time spent investigating the rest of the body. was calculated from the raw key-press data. These “percent time at rear” (%R) scores for LHRH and saline injection tests were almost identical for both VNX and SHAM animals. VNX animals did have somewhat lower scores than SHAM animals but the difference was not significant @ < 0.05. .Mann-Whitney Cl-test). The data are shown in Table 1. Histology,

Five of the seven VNX animals and four of the six SHAM animals had india ink throughout the ventricies, indicating a free flow of solution from the cannula. The other two VNX animals had no obvious ink in the third ventricle but ink was seen near the site of injection in the lateral ventricle. Data from these animals was included in the analysis because they did show evidence of intracerebral delivery. The two SHAM animals that lost their cannulae were not injected with ink. There was no intrusion into the cranial cavity by the skull screws used to hold the implant. In the nose, the VNOs of the VNX animals appeared to have been substantially removed, together with the intracapsular parts ofthe vomeronasal nerves. In some animals. a vestige of the anterior end of the organ remained but appeared not to have bundles of nerves passing back toward the accessory olfactorv bulbs. In one VNX animal, a short length of the palate in&ion (five sections, approx. 75 pm) had healed without completely fusing across the midline but there was no sign of inflammation or accumulation of food particles in the nasal cavity. In all animals, the nasopalatine ducts appeared patent and, in sample sections through the posterior nasal cavity, the olfactory epithelium appeared normal in thickness and extent. Olfactory function was also confirmed in each animal in a simple food odor test (15).

TABLE 1 PERCENT TIME INVESTIGATING HINDQUARTERS (%R) (MEAN SECONDS IN ALL TESTS k SE) Saline

LHRH

SHAM

59.8 t

5.4

61.2 t

VNX

53.9 f

3.9

55.7 rk 2.3

5.4

78

MEREDITH DISCUSSION

Working Hypothesis Supported We have shown that 50 ng LHRH, injected ICV, significantly facilitates mating behavior at 30 min in VNX animals but not in SHAM-operated control animals. The effect of LHRH on the VNX and SHAM groups was significantly different. This result is consistent with the working hypothesis that LHRH release is an intermediate in the facilitatory influence of vomeronasal sensory input on mating behavior. Of course, this result does not prove the hypothesis by itself. It leaves two obvious questions that need to be addressed. First, LHRH might facilitate mating behavior independently of vomeronasal sensory input by increasing arousal. Arousal. The question of arousal is addressed by the results of the analysis of investigation times. The %R ratio (see the Results Section and Table 1) has been used in previous reports as a measure of the male’s interest in the female (8,9,11). This ratio was not significantly different here between LHRH- and saline-injected animals (Table 1), suggesting that LHRH does not facilitate mating behavior in VNX animals solely by increasing arousal. Changes in Controls. The second important question concerns the reduction in behavior seen in control animals. Our prediction that VNX animals would significantly increase their behavior after LHRH injection was correct and this result is independent of the performance of controls; but, our other prediction, that there would be no difference between VNX and SHAM animals after LHRH injection, only occurred because SHAM animals decreased their performance. We have no convincing explanation for this effect. One possibility is that the dose of LHRH may be too large in the presence of endogenously t&eased peptide, that is, in intact (SHAM) animals, but not in animals where release triggered by vomeronasal sensory input is absent, that is, in VNX animals. This possibility will be investigated in further experiments using different doses. There is some evidence for a lower effectiveness of higher doses, at least in females (17). Order of Testing In the behavioral tests, we did not counterbalance the order of presentation of LHRH and saline across each group of animals. Both VNX and SHAM animals received LHRH first in each of the six cycles of testing. We knew from previous work (11) that performance would increase on average with repeated testing, even among VNX animals. We preferred to bias the results against our expectation. The significant decrease in performance of SHAM animals treated with LHRH compared to their performance after saline may owe something to this bias, but the significance and opposite direction of the change in behavior is clear evidence for a real effect of LHRH on behavior.

AND HOWARD

from seven VNX and four SHAM animals. The remaining SHAM animals showed a slightly smaller difference between performance after LHRH and performance after saline in the last test cycle. Thus, the loss of data may have reduced the significance of the LHRH effect on SHAMS and reduced the difference between VNX and SHAM animals, but the conclusions would not have been changed. When the L - S value is assessed for each cycle, it is consistently positive for VNX animals and negative for SHAM animals for all cycles, including the sixth. Timing Another important consideration in the evaluation of our working hypothesis is timing. If endogenous LHRH, released in response to vomeronasal sensory input, is responsible for the facilitation of mating behavior in our 5-min tests, then its effect must be apparent within 5 min of release. Under our hypothesis, exogenous LHRH should be effective within 5 min of its arrival at the normal tatgets for endogenous release. Since we do not yet know where those targets are, we can only estimate the transit time necessary for ICV LHRH to reach them. Ben-Jonathan et al. (1) found that LHRH injected in the lateral ventricle appeared in pituitary portal blood within 10 min. If we estimate 10 min as a reasonable time for effective concentrations to reach the medial preoptic area and other likely sites of action, then 15 min would be an outside estimate of the overall latency for exogenous LHRH action under our hypothesis. So far, we have tested at 30 min after LHRH injection to increase the probability of detecting a response. We will use shorter delays in future experiments. Intracerebral Site of Action? In female rats, LHRH appears to be facilitatory to mating behavior via an extrapituitary action since it is effective in hypophysectomized animals (18). Also, an LHRH fragment, A?-“, which does not release pituitary LH, is still effective in facilitating mating behavior (4,5) as are some LHRH-like peptides that are antagonists for pituitary LH release (16). These findings suggest that mating behavior can be enhanced by LHRH action at an extrapituitary site and possibly by activation of a receptor different from that involved in pituitary LH release (16). Our preliminary evidence suggests that similar effects of LHRH-related peptides occur in male hamster mating behavior (6) supporting the idea that LHRH probably acts intmcerebmlly, not through the pituitary, in this situation too. Only about 50% of LHRH neurons in the forebrain project to the median eminence in rodents (2 1) so there is an extensive substrate for intracerebral release at the preoptic area, the medial amygdala, and in the midbrain, among many other areas (20). It remains to be proven that vomeronasal sensory input does in fact engage some part of these pathways and thereby facilitates behavior. The findings presented here and other work in progress suggests that it may do so.

Missing Data The animals whose data are presented here contributed to all six cycles of testing with the exception of two SHAM animals that lost their guide tubes before the final cycle (attachment is more secure in current experiments). The sixth test thus comprises data

ACKNOWLEDGEMENTS

We thank Mary Wisgirdafor help with these experiments and Charles Badland for the photography. This work was supported by NSF Grant BNS-85 15I59 and NIH Grant DC-00906.

REFERENCES I. Ben-Jonathan, N.; Mica], R. S.; Porter, J. C. Transport of LRF from CSF to hypophysial portal blood and the release of LH. Endocrinology 95: 18-25: 1974.

2. Coquelin. A.; Clancy, A. N.; Macrides,F.; Noble, E. P.; Go&i, R. A. Pheromonally induced releaseof luteinizing hormone in male mice: Involvement ofthe vomeronasaI system. J. Neumsci. 42230-2236; 1984.

LHRH

AND

VNO

INFLUENCES

ON

MATING

BEHAVIOR

3. Dorsa, D. M.; Smith, E. R. Facilitation of mounting behavior in male rats by intracranial injections of luteinizing hormone-releasing hormone. Regul. Peptides I: 147- 155; 1980. 4. Dudley. C. A.: Moss, R. L. Facilitation of lordosis in female rats by CNS-site specific infusions of an LH-RH fragment, AC-LH-RH-(5 IO). Brainkes. 441:16l-167; 1988. 5. Dudley. C. A.: Vale, W.: Rivier, J.: Moss, R. L. Facilitation of sexual receptivity in the female rat by a fragment of the LHRH decapeptide. AC-LHRH(S-IO). Neuroendocrinology 36:486-488; 1983. 6. Fernandez. G.; Howard, A. G.; Meredith, M. Early vomeronasal lesions cause severe deficits in male hamster mating behavior; relieved in part by intracerebral LHRH peptides. Chem. Senses l6:5 19-520; 1991. and function of the vomeronasal sys7. Halpern. M. The organization tem. Annu. Rev. Neurosci. 10:325-362: 1987. 8. Macrides. F.; Johnson, P. A.: Schneider. S. P. Responses of the male golden hamster to vaginal secretion and dimethyl disulfide: Attraction versus sexual behavior. Behav. Biol. 20:377-386: 1977. 9. Meredith. M. The vomeronasal organ and accessory olfactory system in the hamster. In: Muller-Schwarze. D.: Silverstein. R. M.. eds. Chemical signals in vertebrates and aquatic animals. New York: Plenum Press: 1980:303-326. IO. Meredith. M. Sensory physiology of phermone communication. In: Vandenbergh. J. G.. ed. Pheromones and reproduction in mammals. New York: Academic Press: 1983: 199-252. I I. Meredith. M. Vomeronasal organ removal before sexual experience impairs male hamster mating behavior. Physiol. Behav. 36:737-743: 1986. 12. Meredith. M. Sensory processing in the main and accessory olfactory systems: Comparisons and contrasts. J. Steroid Biochem. Mol. Biol. 39:601-614: 1991. 13. Meredith. M.: Howard. G.: Wisgirda. M. LHRH injected intracerebrally relieves some behavioral deficits in male hamsters after vomeronasal organ lesions. Chem. Senses I5:6 19: 1990.

19

14. Meredith, M.: Marques, D. M.; O’Connell, R. J.: Stern, F. L. Vomeronasal pump: Significance for male hamster sexual behavior. Sci. 207:1224-1226; 1980. 15. Meredith, M.: O’Connell. R. J. HRP uptake by olfactory and vomeronasal receptor neurons: Use as an indicator of incomplete lesions and relevance for non-volatile chemoreception. Chem. Senses 13: 487-515: 1988. hormone 16. Moss. R. L.: Dudley. C. A. Luteinizing hormone-releasing (LHRH): Peptidergic signals in the neural integration of female reproductive behavior. Neurol. Neurobiol. 50:485-499: 1989. 17. Moss. R. L.; Dudley, C. A.; Foreman, M. M.: McCann, S. M. Synthetic LRF: A potentiator of sexual behavior in the rat. In: Motta. M.: Crosignani, P. G.: Martini, L., eds. Hypothalamic hormones. London: Academic Press; 1975:269-278. 18. Pfaff, D. W. Luteinizing hormone-releasing factor potentiates lordosis behavior in hypophysectomized ovariectomized female rats. Science 182:1148-l 149: 1973. 19. Siegel, S. Non-parametric statistics. New York: McGraw-Hill; 1956. 20. Silverman, A. J. The gonadotropin-releasing hormone (GnRH) neuronal systems: Immunocytochemistry. In: Knobil, E.; Neill, J., eds. The physiology of reproduction. New York: Raven Press; 1988: 1283-1303. 21. Silverman. A. J.: Jhamandas. J.: Renaud. L. P. Localization of luteinizing hormone-releasing hormone (LHRH) neurons that project to the median eminence. J. Neurosci. 7:23 12-23 19: 1987. 22. Wysocki. C. J. Vomeronasal chemoreception-its role in reproductive fitness and physiology. Neurol. Neurobiol. 50:545-566; 1989. 23. Wysocki. C. J.; Katz. Y.: Bernhard, R. Male vomeronasal organ mediates female induced testosterone surges in mice. Biol. Reprod. 28:9 17-922: 1983. 24. Wysocki. C. J.: Meredith, M. The vomeronasal system. In: Finger, T. E.: Silver. W. L.. eds. Neurobiology oftaste and smell. New York: Wiley; 1987:125-150.