Enhancement of the acoustic startle reflex by an alarm pheromone in male rats

Recently, we reported that an alarm pheromone released from the perianal region of male rats aggravated stress-induced hyperthermia and increased defensive and risk assessment behaviors in recipient male rats. Based on these results, we hypothesized that the primary effect of the alarm pheromone is to increase anxiety; however, there is still no clear evidence for this pheromone effect. Therefore, we examined this issue by assessing the effect of the alarm pheromone on the acoustic startle reflex (ASR), which is a useful index for studying negative emotions such as anxiety in rats. The alarm pheromone enhanced the ASR for 105-dB auditory stimuli, but not for those of 90 and 120 dB, when these three intensities of sound were used randomly. The same results were obtained when one of these three intensities was used repeatedly. In addition, pretreatment with diazepam (i.p.) at doses of 0.7 and 2.0 mg/kg suppressed the ASR of the pheromone recipients, whereas the lower dose (0.2 mg/kg) slightly attenuated the pheromone effect and the control injection (vehicle) had no effect. These results indicate that the alarm pheromone enhances the ASR by increasing anxiety in recipient rats, suggesting that the primary effect of the alarm pheromone is to increase the anxiety level.     

The volatility of an alarm pheromone in male rats

The volatility of an alarm pheromone in male rats. PHYSIOL BEHAV 00(0) 000-000, 2008. We previously reported that an alarm pheromone released from the perianal region of male rats is perceived by the vomeronasal organ and evokes stress-induced hyperthermia and defensive and risk assessment behavior. In addition, we recently reported that the alarm pheromone enhances the acoustic startle reflex (ASR). However, in contrast to our knowledge about such biological aspects of the pheromone, information concerning the physical character of the alarm pheromone is extremely limited. In this study, we investigated the volatility of the alarm pheromone using enhancement of the ASR as an index of the pheromone effect. The alarm pheromone enhanced the ASR when it was presented at a distance of 10 mm but not at 200 mm. In addition, the pheromone effect was observed even after the pheromone was trapped in the adsorbent (Tenax) and then extracted using purified water. These results suggest that the alarm pheromone is both volatile and water soluble.     

Alarm pheromone enhances stress-induced hyperthermia in rats

Behavioral and physiological effects of alarm pheromones emanating from stressed conspecific animals were investigated. Experimentally naive male Wistar rats were exposed to the test chambers containing alarm pheromones, which had been released by other rats receiving foot shocks in the same chamber beforehand. Along with behavioral analysis, the heart rate (HR) and core body temperature (cBT) were measured simultaneously using a biotelemetory system. Exposure to the alarm pheromones increased freezing, sniffing and walking and decreased resting as compared with rats exposed to control odors. In addition, these pheromone-exposed animals showed consistent increases in body temperature, i.e., stress-induced hyperthermia. After exposure to the alarm substances, immunoreactivity to nuclear Fos protein in the mitral cell layer in the accessory olfactory bulb (AOB) also increased compared with the reaction to control odors. These results suggest that an alarm pheromone enhances stress responses of conspecific animals both behaviorally and physiologically, and that these effects are mediated via activation of the AOB.     

Effects of anti-estrogens on testosterone stimulated male sexual behavior and peripheral target tissues in the castrate male rat.

In the first experiment castrated male rats were injected daily with either vehicle or 800 mug testosterone together with either 6 hr pretreatment or concurrent treatment with the anti-estrogens CI-628 (4 mg) or MER-25 (20 mg). The only treatment found to significantly reduce male copulatory behavior was concurrent treatment with CI-628. Anti-estrogen treatment was also found to slightly reduce body weights, increase seminal vesicle weights in response to testosterone and to have no significant effects on androgen stimulated increases in penis weight and length. In the second experiment castrated male rats were injected daily with either vehicle or 500 mug testosterone together with 2.5 mg injections of CI-628 given 6 hr before and concurrent with the androgen injections. The anti-estrogen treatment was found to markedly inhibit the desplay of male sexual behavior, to reduce body weights, and to stimulate seminal vesicle weights. Penile weights and lengths were again not effected by the anti-estrogen therapy. These results were interpreted as supporting the theory that testosterone stimulated male sexual behavior in the rat following its aromatization to estradiol in the brain.     

A study in male and female 5-HT transporter knockout rats: an animal model for anxiety and depression disorders

Human studies have shown that a reduction of 5-HT transporter (SERT) increases the vulnerability for anxiety and depression. Moreover, women are more vulnerable to develop depression and anxiety disorders than men. For that reason we hypothesized that homozygous 5-HT transporter knockout rat (SERT(-/-)) models, especially female, are valuable and reliable animal models for humans with an increased vulnerability for anxiety- and depression-related disorders. As rats are extensively used in neuroscience research, we used the unique 5-HT transporter knockout rat, that was recently generated using N-ethyl-N-nitrosurea (ENU) -driven mutagenesis, to test this hypothesis. Behavioral testing revealed that male and female SERT(-/-) rats spent less time in the center of the open field and spent less time on the open arm of the elevated plus maze compared with wild-type 5-HT transporter knockout rats (SERT(+/+)). In the novelty suppressed feeding test, only male SERT(-/-) rats showed a higher latency before starting to eat in a bright novel arena compared with SERT(+/+) controls. Both male and female SERT(-/-) rats showed a higher escape latency from their home cage than SERT(+/+) littermates. Moreover, SERT(-/-) rats were less mobile in the forced swim test, and sucrose consumption was reduced in SERT(-/-) rats relative to SERT(+/+) rats. Both effects were sex-independent. Neurochemically, basal extracellular 5-HT levels were elevated to a similar extent in male and female SERT(-/-) rats, which was not influenced by the selective 5-HT reuptake inhibitor citalopram. 5-HT immunostaining revealed no difference between SERT(+/+) and SERT(-/-) rats in the dorsal raphe nuclei, in both males and females. These findings demonstrate that SERT(-/-) rats show anxiety and depression-related behavior, independent of sex. Genetic inactivation of the SERT has apparently such a great impact on behavior, that hardly any differences are found between male and female rats. This knockout rat model may provide a valuable model to study anxiety- and depression-related disorders in male and female rats.     

The behavioral genetics of colony defense in honeybees: Genetic variability for guarding behavior

Guard honeybees stand at the entrance of colonies and facilitate the exclusion of nonnestmates from the colony. In this study, we examined the hypothesis that genetic variability among individuals in colonies might explain variability in guarding activity. To do this, we cross-fostered honey bees between colonies with high-defensive responses and colonies with low-defensive responses in alarm pheromone tests. Individuals from high-defensive colonies were more likely to guard in their own colonies (controls) than cross-fostered bees from low-defensive colonies. Cross-fostered high-defensive bees also were more likely to guard in low-defense colonies. These results support the hypothesis that interindividual differences in guarding behavior are at least partially under genetic control. A positive correlation between number of guards and response to alarm pheromone demonstrates a link between behaviorally separated components of the overall defensive response.This work was supported by NSF Grant BNS 8605604.     

Sexually dimorphic effects of forced exercise on food intake and body weight in the rat

The food intake (FI), body weight (BWt) and water intake (WI) of adult male and female rats were compared during a seven day period of forced exercise in a treadmill. Work loads for the exercised groups were gradually increased across the seven-day test period, whereas work loads for the sedentary controls were maintained at the same level used during a previous three-day training period. Relative to their respective control groups, male rats showed a decrease in FI and BWt in response to the exercise challenge, but female rats showed an increase in FI sufficient to maintain their BWt at control levels. Both male and female rats showed a reliable increase in WI during the period of forced exercise. These sexually dimorphic changes in FI and BWt in response to forced exercise indicate that female rats are capable of demonstrating a more vigorous defense of BWt than are male rats and suggest that there is a sex difference in the long-term control of feeding behavior in the rat.     

Housing influences exploration and social interaction of control and DSP-4-treated rats.

Exploratory behavior and social interaction were investigated in rats that were reared in different social environments following neonatal injection with either water vehicle or the norepinephrine neurotoxin, DSP-4. At weaning, they were placed in a familiar or novel bedding type and were housed in either vehicle control-only, DSP-4-only, or mixed vehicle control and DSP-4 groups for 10 days. They were then observed in three different situations: the home cage, the cage of an unfamiliar rat, and an open field. Compared to rats housed in vehicle control-only or DSP-4-only groups, rats housed in mixed DSP-4 and vehicle control groups showed elevated exploration behavior in the home cage. Also, rats housed in mixed groups in the familiar bedding, but not the novel one, showed abnormally low levels of rearing in an open field test and reduced social interaction with unfamiliar rats. The implications of these results for a new animal model of anxiety are discussed.     

Response to alarm substance in different rat strains.

Two studies were conducted. In the first study, four strains of male rats--Long-Evans (LE), Sprague-Dawley (SD), Wistar, Fisher 344--were immersed in fresh water or water soiled by a rat of the same strain which presumably contains an alarm substance. LE rats were less immobile than SD, Wistar, and Fisher rats in fresh water. All strains were significantly less immobile in soiled water; however, relative to immobility times in fresh water, the decrease in immobility in soiled water was greatest for SD rats and least for Wistar rats. A second study tested SD and Wistar rats in water soiled by animals of the same strain compared to water soiled by animals of the other strain. This study indicated that Wistar rats produce less alarm substance or less potent substance and are also less reactive to it than SD rats. The results of these two studies indicate that some strains are more affected by alarm substance than others and the differential response is due both to decreased production/potency and reaction to the substance.     

Level searching: a new assay of sexual motivation in the male rat

Mendelson and Gorzalka recently described a bilevel chamber for the evaluation of rodent sexual behavior. In initial studies it was observed that during 5 min adaptation periods prior to the introduction of a sexually receptive female rat, male rats with prior sexual experience in these chambers would move from level to level in apparent search for the female rat. In Experiment 1, we examined the acquisition of this level searching behavior in male rats. Sexually active male rats were given access to either sexually receptive or nonreceptive female rats following a 5 min period alone in the bilevel chamber. Only male rats that pursued and copulated to ejaculation with sexually receptive females in the bilevel chamber significantly increased the number of their level to level movements in subsequent tests during the 5 min periods prior to the introduction of the female rat. In Experiment 2, male rats that had acquired asymptotic rates of level searching showed a significant attenuation of this behavior when the presentation of a female rat into the chamber was discontinued. These findings lead us to conclude that the increase in level to level movement by the male rat represents a sexually motivated search for the female rat. We suggest that the analysis of the acquisition, maintenance, and extinction of level searching behavior might serve as a simple assay of sexual motivation in the male rat.     

Gradient of alarm substance in the forced swimming test

Three studies were conducted with rats to study the effects of a proposed alarm substance released during the forced swimming test. In the first study, rats were retested in water previously swum in but which had been subsequently diluted to varying degrees. When water was diluted by more than 25% the proposed alarm substance could no longer be detected. In the second study, rats were retested at various times after initial testing. This study showed that the alarm substance was still active by eight days after it had been initially released. The final study tested animals to see if the alarm substance could be depleted by repeated testing. Testing animals for more than an hour did not result in depletion of the alarm substance. These three studies suggest that the proposed alarm substance secreted by rats during the forced swimming test has considerable biological significance.     

Central tegmental field and sexual behavior in the male rat: effects of neurotoxic lesions

The medial preoptic area/anterior hypothalamus (MPOA/AH) is a key structure in the control of male sexual behavior. This area has reciprocal connections with mesencephalic and brainstem structures including the central tegmental field (CTF). It has been suggested that the CTF receives somatosensory information generated in the genitals promoting activation of the MPOA/AH. In the present study we evaluated the effects of bilateral neurotoxic lesions of the CTF upon male rat sexual behavior. We also explored the effects of these lesions on sociosexual behaviors, partner preference, sexual incentive motivation and motor execution. Tests were performed before and after bilateral quinolinic acid infusions. The lesion was evaluated by quantifying neuronal nuclei (Neu-N) and by the presence of glial fibrillary acidic protein (GFAP) immunohistochemistries. A significant reduction in the percentage of animals displaying mounts, intromissions, and ejaculations was observed in the bilateral and misplaced lesion groups 1 week after the lesion. In the second week post-lesion, only animals with bilateral damage of the CTF showed a significant reduction in sexual behavior. In the third post-lesion test, the percentage of animals displaying sexual behavior returned to control levels. The frequency of pursuit and self-grooming was reduced, and genital exploration was increased after the lesion. Partner preference and sexual incentive motivation were not affected by the lesion suggesting that the CTF is not involved in the appetitive aspects of sexual behavior. Mount, intromission, and ejaculation latency were increased in animals with damage of the CTF and in animals with lesions outside this region. Motor execution was also affected in both groups, suggesting that alterations in latencies could be associated with damage not specific to the CTF.     

Paced mating behavior persists in rats with vaginocervical Lidocaine

The present study tested whether the topical application of a local anesthetic (Lidocaine) to the vaginocervical region altered the pattern of paced mating behavior displayed by gonadectomized, hormone-primed female rats. Both rats receiving Lidocaine and rats receiving vehicle exhibited the expected lengthening of contact-return latency as the intensity of the mating stimuli increased (mount<intromission<ejaculation). Although rats given Lidocaine versus vehicle received a greater number of intromissions, no other group differences were observed. The present study found no evidence for a change in behavioral responsiveness following the vaginocervical application of Lidocaine.     

Increases in extracellular zinc in the amygdala in acquisition and recall of fear experience and their roles in response to fear

The amygdala is enriched with histochemically reactive zinc, which is dynamically coupled with neuronal activity and co-released with glutamate. The dynamics of the zinc in the amygdala was analyzed in rats, which were subjected to inescapable stress, to understand the role of the zinc in emotional behavior. In the communication box, two rats were subjected to foot shock stress and anxiety stress experiencing emotional responses of foot-shocked rat under amygdalar perfusion. Extracellular zinc was increased by foot shock stress, while decreased by anxiety stress, suggesting that the differential changes in extracellular zinc are associated with emotional behavior. In rats conditioned with foot shock, furthermore, extracellular zinc was increased again in the recall of fear (foot shock) in the same box without foot shock. When this recall was performed under perfusion with CaEDTA, a membrane-impermeable zinc chelator, to examine the role of the increase in extracellular zinc, the time of freezing behavior was more increased, suggesting that zinc released in the lateral amygdala during the recall of fear participates in freezing behavior. To examine the role of the increase in extracellular zinc during fear conditioning, fear conditioning was also performed under perfusion with CaEDTA. The time of freezing behavior was more increased in the contextual recall, suggesting that zinc released in the lateral nucleus during fear conditioning also participates in freezing behavior in the recall. In brain slice experiment, CaEDTA enhanced presynaptic activity (exocytosis) in the lateral nucleus after activation of the entorhinal cortex. The present paper demonstrates that zinc released in the lateral amygdala may participate in emotional behavior in response to fear.     

Aphrodisiac effect of Hunteria umbellata seed extract: Modulation of nitric oxide level and arginase activity in vivo

Hunteria umbellata has been reported useful in managing the male sexual related disorder, but there is a dearth of information on its possible mechanism of action. Hence, this study was designed to investigate the effect of H. umbellata on some sexual behavior parameters in male rats, in order to ascertain its acclaimed aphrodisiac property. Twenty-four male rats were divided into four groups (n?=?6). The extract (50 and 100?mg/kg body weight/day) and sildenafil citrate (5?mg/kg body weight) were administered orally by gavages for 28 days. Some sexual behavior parameters (mounting number, mounting latency, intromission number, and intromission latency), and some anxiety behavioral tests (dark-light box test and open field test) were monitored. In addition, the effect of the extract on arginase activity, nitric oxide and antioxidant level in male rats was assessed. Results revealed that administration of H. umbellata extract increases the mounting number, intromission number, time spent at the light compartment, nitric oxide and antioxidant level when compared with animals in the control group. Conversely, administration of H. umbellata caused a significant decrease in mounting latency, intromission latency, and arginase activity. Findings in this study revealed that H. umbellata improves sexual behavior parameters in male rats, and the results indicate that aphrodisiac effect of H. umbellata, identified by its effect on nitric oxide level and arginase activity, may play an important role in initiating and maintaining an erection.     

Maudsley reactive and non-reactive rat strains: differential performance in a conflict task

Based on open field defecation rates, distinct strains of Maudsley rats have been identified. The Maudsley Reactive strains exhibit high levels of open field defecation, while the Maudsley Non-Reactive strains exhibit low levels of open field defecation. This difference in open field behavior has been interpreted as an indicator of "emotionality." The present studies were designed to compare the performance of two Maudsley strains, MR/Har and MNRA/Har, in the conditioned suppression of drinking (CSD) paradigm, another "model" behavior for the study of emotionality and/or anxiety in animals. In daily 10-minute sessions, water-deprived rats were trained to drink from a tube that was occasionally electrified (0.5 mA), electrification being signalled by a tone. In this procedure, MNRA/Har rats accepted significantly more shocks than did MR/Har rats. This difference between MR/Har and MNRA/Har strains, initially somewhat slight, became quite impressive over weeks of repeated testing. Although present in both male and female subjects, this strain difference was more prominent in the males. Thus, the MR/Har and MNRA/Har rat strains, bred originally for their differences in open field behavior, also differ markedly in their performance in the CSD paradigm. These findings suggest that the Maudsley rat strains may represent a useful genetically-based "animal model" for the study of emotionality and/or anxiety.     

Testosterone induced mounting behavior in adult female rats born in litters of different female to male ratios

Female rats can show mounting behavior towards isosexual or heterosexual conspecifics. The present experiments were designed to study whether the prenatal presence of male fetuses would affect display of this mounting behavior in adulthood. Therefore mounting behavior, after gonadectomy and during continuous treatment with testosterone propionate (TP) was studied in female rats which were born in litters without male siblings (“all female” litters) and in litters with a variable number of male siblings. Litter composition at birth did not affect display of adult mounting behavior during protracted tests (a total of 6 tests during 8 weeks of treatment with TP). The data indicate that TP induced mounting behavior in adulthood occurs independent of the prenatal presence of male fetuses. If mounting behavior in adulthood has to be “organized” by prenatally present androgen (the current way of thinking), then the present data indicate that female rat fetuses provide themselves with these hormones. It would then seem inappropriate to judge adult mounting behavior as a sign of “masculinization” of the rat brain.     

Brain serotonergic neurons: Their role in a form of dominance-subordination behavior in rats

The present study evaluated the possible role of brain serotonergic neurons in dominant-subordinate (D-S) behavior in Wistar male rats competing for water. Treatment of D rat with drugs that stimulate serotonergic neurons of receptors (tryptophan, 5-hydroxytryptophan, quipazine, femoxetine) resulted in D-S reversal. A similar effect was observed when the S animal was treated with drugs that blocked serotonin synthesis (p-chlorophenylalanine) or receptors (metergoline). The D-S relationship was unchanged when serotonergic drugs were given to the S subject (tryptophan or quipazine) or when D animal received p-chlorophenylalanine. None of the drugs tested influence the water intake and the general activity of rats. Rats with lesioned midbrain raphe nuclei were always dominant when paired with sham lesioned counterparts. Our results indicate that one form of dominance behavior can be inversely related to the activity of brain 5-HT system.     

Long-term kindling of the basolateral amygdala impairs copulatory behavior in male rats

This experiment examined the consequences of long-term kindling of the basolateral amygdala on male sexual behavior and the frequency of both spontaneous wet dog shakes (WDS) and those induced by the 5-HT_2A receptor agonist DOI. Results demonstrated that following 60 stimulations of the left basolateral amygdala over a 4-week period, male Long–Evans rats exhibited decrements in every aspect of sexual behavior. Specifically, latencies to mount, intromit and ejaculate were all prolonged following long-term kindling, and ejaculation frequencies were significantly reduced. Furthermore, spontaneous peri-copulatory WDS were increased in kindled rats, suggesting a possible role of the 5-HT_2A receptor. However, countering this suggestion, there were no differences between sham and kindled rats on WDS induced by the 5-HT_2A receptor agonist DOI. These results suggest that kindled rats may exhibit elevated levels of endogenous serotonin during exposure to a female rat, which would attenuate copulatory behavior, while concurrently increasing WDS expression.     

A developmental study of maternal responsiveness in the rat

Male and female rats will retrieve and group rat pups as early as 24 days of age. Injections of testosterone propionate failed to inhibit the onset of this behavior in the male.