James A. Cherry

Olfactory sex discrimination persists, whereas the preference for urinary odorants from estrous females disappears in male mice after vomeronasal organ removal.

Based on observed changes in the social context for the display of ultrasonic vocalizations, scent marking, aggression, and mounting behavior by male mice with a null mutation of the transient receptor potential 2 ion channel, it was proposed recently that a primary function of the mouse vomeronasal organ (VNO)/accessory olfactory system is sex discrimination. We tested this hypothesis directly by studying the ability of male mice to discriminate between urinary odors of conspecifics of the two sexes and in different endocrine states using habituation-dishabituation tests. Male mice from which the VNO had been surgically removed (VNOx) resembled sham-operated controls (VNOi) in their ability to discriminate between volatile urinary odors from estrous females versus gonadally intact males, as well as between urinary odors from estrous versus ovariectomized females and from gonadally intact versus castrated males. When physical access to stimuli was permitted, VNOi control males strongly preferred to investigate volatile and nonvolatile urinary odorants from estrous females as opposed to intact males, whereas VNOx males showed no such preference. Mating performance in tests with estrous females was equivalent in VNOi and VNOx subjects. Both groups of males preferred to mount an estrous female instead of a castrated male. Our results suggest that the VNO is not required for sex discrimination but instead detects the nonvolatile components of opposite-sex urine that may be used to help prolong contact with individuals that produce these chemosignals.

Cyclic AMP phosphodiesterases are localized in regions of the mouse brain associated with reinforcement, movement, and affect

Four cyclic AMP‐specific, rolipram‐inhibited phosphodiesterases (PDE4s) have been identified in mammals; all four are homologs of dunce, a gene required for learning and memory in Drosophila. To determine the distribution of PDE4s in the mammalian brain, specific antibodies were generated against the proteins encoded by each of three dunce homologs PDE4A, PDE4B, and PDE4D in the mouse. On Western blots, these antibodies recognized multiple protein species in all brain regions studied. Immunohistochemical studies showed that both cell bodies and neuropil were well labeled in selected regions throughout the brain. Immunoreactivity for PDE4A was found predominantly in the anterior olfactory nucleus, subiculum, layer V pyramidal neurons from the cerebral cortex, and corticospinal tracts. By contrast, anti‐PDE4B–labeled neurons were observed in the inferior olive, the paraventricular and supraoptic nuclei of the hypothalamus, and in the ventral striatum. Regions of neuropil containing high levels of PDE4B immunoreactivity included the cerebellar molecular layer, globus pallidus, nucleus accumbens, and substantia nigra. Anti‐PDE4D antibody distinctly labeled cerebellar Purkinje cells as well as neurons in the medial habenula and thalamic nuclei. Fibers in the fasciculus retroflexus, interpeduncular nuclei, and periaqueductal gray were also stained with this antibody. These findings indicate that the distribution of PDE4s in the brain is remarkably segregated, and suggest that each of these enzymes has a unique functional role. Furthermore, the data support the notion that rolipram, the PDE4‐specific inhibitor that acts as an antidepressant in humans, may mediate its behavioral effects through PDE4B, which is highly localized to neural pathways known to underlie reward and affect in mammals. J. Comp. Neurol. 407:287–301, 1999.

A direct main olfactory bulb projection to the 'vomeronasal' amygdala in female mice selectively responds to volatile pheromones from males.

The main olfactory system, like the accessory olfactory system, responds to pheromones involved in social communication. Whereas pheromones detected by the accessory system are transmitted to the hypothalamus via the medial (‘vomeronasal’) amygdala, the pathway by which pheromones are detected and transmitted by the main system is not well understood. We examined in female mice whether a direct projection from mitral/tufted (M/T) cells in the main olfactory bulb (MOB) to the medial amygdala exists, and whether medial amygdala‐projecting M/T cells are activated by volatile urinary odors from conspecifics or a predator (cat). Simultaneous anterograde tracing using Phaseolus vulgaris leucoagglutinin and Fluoro‐Ruby placed in the MOB and accessory olfactory bulb (AOB), respectively, revealed that axons of MOB M/T cells projected to superficial laminae of layer Ia in anterior and posterodorsal subdivisions of the medial amygdala, whereas projection neurons from the AOB sent axons to non‐overlapping, deeper layer Ia laminae of the same subdivisions. Placement of the retrograde tracer cholera toxin B into the medial amygdala labeled M/T cells that were concentrated in the ventral MOB. Urinary volatiles from male mice, but not from female conspecifics or cat, induced Fos in medial amygdala‐projecting MOB M/T cells of female subjects, suggesting that information about male odors is transmitted directly from the MOB to the ‘vomeronasal’ amygdala. The presence of a direct MOB‐to‐medial amygdala pathway in mice and other mammals could enable volatile, opposite‐sex pheromones to gain privileged access to diencephalic structures that control mate recognition and reproduction.

Vomeronasal neuroepithelium and forebrain Fos responses to male pheromones in male and female mice.

Male urinary pheromones modulate behavioral and neuroendocrine function in mice after being detected by sensory neurons in the vomeronasal organ (VNO) neuroepithelium. We used nuclear Fos protein immunoreactivity (Fos-IR) as a marker of changes in neuronal activity to examine the processing of male pheromones throughout the VNO projection pathway to the hypothalamus. Sexually naive male and female Balb/c mice were gonadectomized and treated daily with estradiol benzoate (EB) or oil vehicle for 3 weeks. Subjects were then exposed to soiled bedding from gonadally intact Balb/c males or to clean bedding for 90 min prior to sacrifice and processing of their VNOs and forebrains for Fos-IR. Male pheromones induced similar numbers of Fos-IR cells in the VNO neuroepithelium of oil-treated male and female subjects; however, EB-treated females had significantly more Fos-IR neurons in the VNO than any other group. There was an equivalent neuronal Fos response to male odors in the mitral and granule cells of the anterior and posterior accessory olfactory bulb of males and females, regardless of hormone treatment. In central portions of the VNO projection pathway (i.e., bed nucleus of the stria terminalis, medial preoptic area) neuronal Fos responses to male pheromones were present in female but absent in male subjects, regardless of hormone treatment. In a separate experiment, mating induced neuronal Fos-IR in these brain regions at levels in gonadally intact male subjects which were equal to or greater than those seen in ovariectomized females primed with estrogen and progesterone. This suggests that neurons in the central portions of the males VNO pathway are capable of expressing Fos. Our results suggest that sexually dimorphic central responses to pheromones exist in mice that may begin in the VNO neuroepithelium.

Paced copulation in rats: Effects of intromission frequency and duration on luteal activation and estrous length

When estrous female rats regulate or pace (P) the timing of vaginal intromissions received from males during mating, the stimulation is more effective in inducing luteal function and abbreviating the period of receptivity than is nonpaced (NP) stimulation. The present studies examined whether the coital stimuli necessary for each of these functional consequences are similar. In Experiment 1, estrous females received either 5 or 10 intromissions from males in P or NP tests; control animals received mounts-without-intromission (MO). The duration of estrus was not affected by 5P, 5NP, or 10NP stimulation, but was significantly abbreviated in 10P animals. In contrast, activation of prolonged luteal function occurred in 70% of 5P females compared to only 10% of 5NP females; luteal activation was similar in 10P and 10NP females (74% for both groups combined). In Experiment 2, male copulatory behaviors were compared in tests with P and NP females. Males tested with P females exhibited significantly longer intromission durations (616 +/- 21 msec) than did males tested with NP females (527 +/- 30 msec). Other measures of male copulatory performance such as the number of intromissions to ejaculation and the ejaculation latency did not differ between groups. These studies demonstrate that luteal activation is more readily induced by paced coital stimulation than is abbreviation of estrus. In addition, they suggest that differences between P and NP females in the behavioral and neuroendocrine responses to coital stimulation may result from differences in intromission duration displayed by males under these test conditions.

Effects of lesions of a sexually dimorphic nucleus in the preoptic/anterior hypothalamic area on the expression of androgen- and estrogen-dependent sexual behaviors in male ferrets

The male nucleus of the preoptic/anterior hypothalamic area (MN-POA/AH) is a sexually dimorphic structure present in male, but not in female ferrets. Ovariectomized female ferrets given increasing dosages of estradiol benzoate (EB) normally run faster towards a stud male in an L-maze (i.e. become more proceptive). In two separate experiments, only gonadectomized males with bilateral damage to the MN-POA/AH following large or small electrolytic lesions approached stud males more quickly in response to EB. By contrast, males which received sham lesions, unilateral large POA/AH lesions, or bilateral lesions which missed the MN-POA/AH on at least one side failed to show EB-induced reductions in approach latencies in pre- or post-operative tests. Males with large POA/AH lesions also displayed significant post-operative decrements in masculine sexual behaviors during treatment with a high dose of testosterone propionate (TP). Less severe, but statistically significant deficits in masculine coital performance were also observed in males with small lesions which damaged the MN-POA/AH bilaterally; however, the ability of these males to achieve intromissions appeared normal. Together, these results suggest that the MN-POA/AH of the male ferret exerts an inhibitory influence on estrogen-dependent proceptive responsiveness, but play only a minor role in the control of masculine coital behavior.

Different profiles of main and accessory olfactory bulb mitral/tufted cell projections revealed in mice using an anterograde tracer and a whole-mount, flattened cortex preparation.

A whole-mount, flattened cortex preparation was developed to compare profiles of axonal projections from main olfactory bulb (MOB) and accessory olfactory bulb (AOB) mitral and tufted (M/T) cells. After injections of the anterograde tracer, Phaseolus vulgaris leucoagglutinin, mapping of labeled axons using a Neurolucida system showed that M/T cells in the AOB sent axons primarily to the medial and posterior lateral cortical amygdala, with minimal branching into the piriform cortex. By contrast, M/T cells in the MOB displayed a network of collaterals that branched off the primary axon at several levels of the lateral olfactory tract (LOT). Collaterals emerging from the LOT into the anterior piriform cortex were often observed crossing into the posterior piriform cortex. M/T cells in the dorsal MOB extended fewer collaterals from the primary axon in the rostral LOT than did M/T cells from the anterior or ventral MOB. MOB M/T cells that projected to the medial amygdala did not do so exclusively, also sending collaterals to the anterior cortical amygdala as well as to olfactory cortical regions. This arrangement may be related to the ability of social experience to modify the response of mice to volatile pheromones detected by the main olfactory system.

Effect of Vomeronasal Organ Removal From Male Mice on Their Preference for and Neural Fos Responses to Female Urinary Odors

Four experiments were conducted to determine whether vomeronasal organ (VNO) inputs in male mice mediate the rewarding properties of estrous female urinary odors. Sexually naive male mice with either an intact (VNOi) or lesioned (VNOx) VNO preferred to investigate female urine over water in Y-maze tests. Subsequently, VNOi males ran significantly more quickly and remained in nasal contact longer with estrous female urine than with male urine, whereas VNOx males investigated these odors equally. In home-cage habituation-dishabituation tests, VNOi males also investigated female urine significantly longer than did VNOx males, although both groups investigated female urine longer than other non-body odors. Finally, female urinary odors induced Fos in the nucleus accumbens core of VNOi males but not of VNOx males. Our results suggest that female urinary odors retain some incentive value in VNOx males. However, once direct nasal contact is made with female urine, VNO inputs further activate forebrain mechanisms that amplify the reward salience of this stimulus for the male mouse.

Sex and gonadal steroid modulation of pheromone receptor gene expression in the mouse vomeronasal organ.

Non-volatile chemosignals in rodents are detected by unique receptors in the vomeronasal organ of the accessory olfactory system. Although the vomeronasal organ has been implicated in the regulation of sexually dimorphic behavioral and neuroendocrine functions, the underlying cellular mechanisms are undetermined. In previous studies we showed that exposure to soiled male bedding augmented immediate early gene immunoreactivity in neurons of the basal zone of the vomeronasal organ, an effect that depended on gender and sex steroid expression. To determine whether this effect could be due to differences in vomeronasal organ receptor expression, we examined two representatives (VR1 and VR4) from different subfamilies of the V2R family of receptors that are expressed in the basal zone of the vomeronasal organ. Adult Swiss-Webster male and female mice were gonadectomized and implanted with capsules containing 17beta-estradiol, testosterone or neither steroid (control). Two weeks later vomeronasal organs were processed for in situ hybridization using probes from the N-terminal extracellular domains of VR1 and VR4. Expression of both VR1 and VR4 was significantly higher in males than in females. Estradiol, but not testosterone-treated, males had significantly lower levels of VR1 expression in the caudal vomeronasal organ compared with untreated gonadectomized males. In contrast, testosterone enhanced VR4 expression in males relative to similarly treated females. Despite these effects, we found no evidence that vomeronasal organ neurons express either androgen or estrogen receptors. These data show that expression of vomeronasal organ receptors in mice is sexually dimorphic and regulated by sex steroids. Thus, gonadal hormones may affect the response of vomeronasal organ neurons to chemosignals by altering levels of the receptors to which they bind.

Central forebrain Fos responses to familiar male odours are attenuated in recently mated female mice

Exposure of recently mated female mice to the urinary odours of an unfamiliar male blocks pregnancy (the Bruce effect). The absence of a pregnancy block in response to the stud males familiar odours depends on an olfactory memory that is formed in the accessory olfactory bulb (AOB) in response to vomeronasal organ (VNO) inputs during mating. Sexually naive Balb/c female mice in pro‐oestrus/oestrus were either placed onto soiled bedding (‘bedding‐only’ females) from, or allowed to mate with, a Balb/c male (‘recently mated’ females). After 42 h, females were placed for 90 min onto clean bedding (controls) or onto soiled bedding from either a C57BL/6 male (unfamiliar bedding) or a Balb/c male (familiar bedding). Significant increases in Fos‐immunoreactivity (Fos‐IR, a marker of neuronal activation) occurred in the medial amygdala and the medial preoptic area (MPA) of ‘bedding only’ females exposed to either unfamiliar or familiar bedding and in ‘recently mated’ females exposed to unfamiliar bedding but not to familiar bedding. This suggests that a mating‐induced memory prevents the later activation by the familiar stud males odours of neurons in forebrain regions that receive inputs from the VNO–AOB. ‘Bedding‐only’ females later exposed to either familiar or unfamiliar bedding had increased Fos‐IR in the Gαo protein‐expressing basal zone of the VNO whereas no such effect occurred in ‘recently mated’ females. Familiar, as well as unfamiliar, male odours augmented Fos‐IR in significantly more rostral than caudal AOB granule cells in all groups, with the effect being strongest in ‘recently mated’ females exposed to familiar male bedding. This outcome is consistent with the absence of odour‐induced Fos‐IR in forebrain regions of these females and, presumably, the absence of a pregnancy block.