Ginkgo biloba extract enhances noncontact erection in rats: the role of dopamine in the paraventricular nucleus and the mesolimbic system.

Penile erection is essential for successful copulation in males. Dopaminergic projections from the paraventricular nucleus (PVN) to the ventral tegmental area (VTA) and from the VTA to the nucleus accumbens (NAc) are thought to exert a facilitatory effect on penile erection. Our previous study showed that treatment with an extract of Ginkgo biloba leaves (EGb 761) enhances noncontact erection (NCE) in male rats. However, the relationship between NCE and dopaminergic activity in the PVN, VTA, and NAc remains unknown. The present study examined the relationship between NCE and central dopaminergic activity following EGb 761 treatment. We report here that, in comparison with the controls, there was a significant increase in the number of NCEs in rats after treatment with 50 mg/kg of EGb 761 for 14 days. EGb 761-treated rats also showed more NCEs than the same group before EGb 761 treatment. A significant increase in the expression of catecholaminergic neurons in the PVN and the VTA was seen by means of tyrosine hydroxylase immunohistochemistry, and tissue levels of dopamine and 3,4-dihydroxyphenylacetic acid in the NAc were also markedly increased in the EGb 761-treated animals. However, the norepinephrine tissue levels in the PVN and the NAc in the EGb 761-treated group were not significantly different from those in the controls. Together, these results suggest that administration of EGb 761 increases dopaminergic activity in the PVN and the mesolimbic system to facilitate NCE in male rats.

Differential involvement of medial prefrontal cortex and basolateral amygdala extracellular signal-regulated kinase in extinction of conditioned taste aversion is dependent on different intervals of extinction following conditioning.

Extinction reflects a decrease in the conditioned response (CR) following non-reinforcement of a conditioned stimulus. Behavioral evidence indicates that extinction involves an inhibitory learning mechanism in which the extinguished CR reappears with presentation of an unconditioned stimulus. However, recent studies on fear conditioning suggest that extinction erases the original conditioning if the time interval between fear acquisition and extinction is short. The present study examined the effects of different intervals between acquisition and extinction of the original memory in conditioned taste aversion (CTA). Male Long-Evans rats acquired CTA by associating a 0.2% sucrose solution with malaise induced by i.p. injection of 4 ml/kg 0.15 M LiCl. Two different time intervals, 5 and 24 h, between CTA acquisition and extinction were used. Five or 24 h after CTA acquisition, extinction trials were performed, in which a bottle containing 20 ml of a 0.2% sucrose solution was provided for 10 min without subsequent LiCl injection. If sucrose consumption during the extinction trials was greater than the average water consumption, then rats were considered to have reached CTA extinction. Rats subjected to extinction trials lasting 24 h, but not 5 h, after acquisition re-exhibited the extinguished CR following injection of 0.15 M LiCl alone 7 days after acquisition. Extracellular signal-regulated kinase (ERK) in the medial prefrontal cortex (mPFC) and basolateral nucleus of the amygdala (BLA) was examined by Western blot after the first extinction trial. ERK activation in the mPFC was induced after the extinction trial beginning 5 h after acquisition, whereas the extinction trial performed 24 h after acquisition induced ERK activation in the BLA. These data suggest that the original conditioning can be inhibited or retained by CTA extinction depending on the time interval between acquisition and extinction and that the ERK transduction pathway in the mPFC and BLA is differentially involved in these processes.

Sexual motivation is demasculinized, but not feminized, in prenatally stressed male rats.

Sexual motivation and copulation in male rats are associated with dopamine release in the nucleus accumbens. Demasculinized copulatory behavior has been demonstrated in prenatally stressed adult male rats. We have previously reported that approximately 80% of prenatally stressed male rats do not exhibit copulation and that no significant changes in nucleus accumbens dopamine release are seen during exposure to estrous females. In the present study, we investigated whether prenatal stress affects sexual motivation in these animals as adults. Pregnant Wistar rats were subjected to immobilization stress for two hours daily from day 15-19 of gestation. The prenatally stressed male offspring at the age of 3 months were allowed contact with receptive female rats for a 30 min period per week for 10 weeks; then, between the age of 5 and 6 months, their sexual motivation and copulatory activity were measured. Sexual motivation was measured in terms of sexual partner preference. The number of visits and the duration of each visit to an estrous female (stimulus female) or to a sexually active male rat (stimulus male) were recorded. Compared with control males, prenatally stressed male rats showed a significantly lower number of visits and a shorter duration of each visit to stimulus females. Prenatally stressed males showed no preference for male or female stimulus rats in terms of the number of visits and the duration of each visit, whereas control rats showed a significantly higher number of visits and duration of visits to female stimulus rats than male stimulus rats. A significant decrease in copulatory activity was observed in the prenatally stressed male offspring compared with control male rats, with most of the prenatally stressed males failing to show copulation. In vivo microdialysis experiments were performed on the nucleus accumbens with concurrent observation of sexual behavior. The prenatally stressed rats that did not exhibit copulation showed no significant changes in nucleus accumbens dopamine release during exposure to a stimulus male behind a wire-mesh barrier and the amount of dopamine release remained at the basal levels during actual physical contact. These results, combined with those of our previous report, indicate that sexual motivation in prenatally stressed male rats is demasculinized, but not feminized.

Striatal glutamate release during novelty exposure-induced hyperactivity in olfactory bulbectomized rats.

Striatal glutamate release during novelty exposure-induced hyperactivity was studied by microdialysis in freely-moving olfactory bulbectomized (OBX) rats. After collecting three 10 min basal striatal dialysate samples, the animals were transferred to an open-field apparatus (novelty) and locomotor activity recorded for 60 min. OBX rats showed significantly more locomotor activity (1210+/-270 cm) than sham-operated rats (420+/-70 cm), but only in the first 10 min after exposure to the novel environment. During the same period, striatal glutamate levels increased to 163+/-21% of the basal value in OBX rats, while no changes were seen in the striatum of sham-operated controls. These findings suggest that olfactory bulbectomy results in an increased response of the striatal glutamatergic system to novelty stress, and may consequently cause hyperactivity.

Male sexual behavior is associated with LHRH neuron number in middle-aged rats.

LHRH administration is reported to facilitate male sexual behavior. The aim of the present study was to investigate whether male sexual behavior is associated with the number of LHRH neurons in the forebrain in middle-aged rats. Male Long-Evans rats (18-19 months) were assigned to three groups on the basis of sexual performance: (1) group MEI consisted of rats showing complete copulatory patterns, including mounts, intromissions and ejaculations, (2) group MI was composed of rats showing mounts and intromissions, but no ejaculation and (3) group NC were non-copulators, i.e. they did not show any copulatory behavior. Young adult rats (4-5 months), displaying sexual behavior, were used as controls. Following the sexual behavior tests, the number of LHRH neurons in the medial septum (MS), organum vasculosum of the lamina terminalis (OVLT), preoptic area (POA) and anterior hypothalamus (AH) was determined by immunocytochemistry. No difference was seen in the total number of LHRH neurons in these combined brain areas between group MIE and young controls. In the three middle-aged groups, the total number of LHRH neurons was greatest in group MIE, less in group MI, and lowest in group NC. In general, a similar trend was seen separately in the MS, OVLT and POA. These results suggest that changes in the number of LHRH neurons in the forebrain, in most cases, are age-related, at least in the middle-aged rats, but they also seem to be associated with male sexual performance.

Dopamine release in the nucleus accumbens during sexual behavior in prenatally stressed adult male rats.

In vivo microdialysis experiments were performed on the nucleus accumbens (NAc) during observation of sexual behavior (including motivation and copulation) to determine if there were any changes in NAc dopamine (DA) transmission in prenatally stressed (PS) adult male rats. Approximate 37% of control males and 83% of PS males did not exhibit copulation during the sexual behavior tests and no significant changes in NAc DA release were seen during exposure to estrous females. In contrast, both control and PS males that displayed copulatory behavior showed a marked increase in NAc DA release when presented with a sexually receptive female behind a screen and this increased further during actual copulation. The increase in DA release in copulatory PS males was not significantly different from that in sexually active control males. In addition, a similar extent in DA release induced by high potassium perfusate was observed in all rats. These results suggest that prenatal stress may result in a deficit in DA neurotransmission in the NAc and this deficit may possibly cause impaired male sexual behavior in rats.

Effects of age on dopamine release in the nucleus accumbens and amphetamine-induced locomotor activity in rats.

The effects of age on dopamine (DA) release in the nucleus accumbens (NAc) and on amphetamine (AMPH)-induced locomotor activity were studied by microdialysis in freely-moving young (5 month) and old (24 month) rats. Both basal extracellular DA and 3,4-dihydroxyphenylacetic acid (DOPAC) release and that following intra-accumbens perfusion of AMPH (1-10 microM) were significantly lower in old rats. After intraperitoneal injection of AMPH (1.5 mg/kg), no age-related change in DA release was seen in the NAc, but locomotor activity was found to increase much more in young rats than in old ones. These results indicate that (1) old rats show decreased extracellular DA and DOPAC release, both in the basal state and following intra-accumbens infusion of AMPH, and (2) the age-related locomotor activity induced by systemic injection of AMPH is not paralleled by changes in DA release in the NAc.

Effects of fetal brain grafting on adult behavioral masculinization and defeminization in neonatally androgenized female rats.

Treatment of neonatal female rats with androgen results not only in decreased female sexual behavior but also in enhanced male sexual behavior examined in adulthood. The effects of grafting fetal preoptic area (POA) neurons into the POA, and fetal hypothalamic (HPT) neurons into the ventromedial hypothalamus (VMH), were tested in neonatally androgen-sterilized rats (ASR). The rats were injected subcutaneously with 80 micrograms testosterone propionate within the 24 hours after birth to see if sexual behavior could be normalized by fetal brain grafts. In repeated tests on ASR grafted with fetal HPT into the VMH, the lordotic response was seen to increase to the level seen in non-ASR controls, while the increase in mounting behavior in ASR was suppressed following grafting of fetal POA or cerebral cortex into the POA. These results suggest that there are dysfunctions of POA and VMH in ASR, and that the dysfunctions revealed by sexual behavior can be overcome by fetal POA or HPT grafting.

Dissociation of the restoration of copulatory behavior and the testicular morphology in old male rats with fetal brain grafting.

Improvement of copulatory activity has been reported in aged male rats bearing the fetal preoptic area (POA), however, whether the fetal POA graft has any effect on the gonad of aged male rats has not been established. In the present study, the testicular histology and the sexual behavior were studied in 20 old (18-19 months) Long-Evans male rats after fetal brain transplantation. Animals were divided into three groups: (1) POA-grafted group (n = 6): fetal POA neurons were grafted into the POA, (2) COR-grafted group (n = 7): fetal cerebral cortex tissue was grafted into the POA and (3) SHAM-grafted group (n = 7): the POA of rats received glucose-saline injection. Thirty days after the grafting, only those bearing POA graft showed an increase in mount frequency and intromission frequency. Sixty days after fetal brain grafting, testicular morphology was examined. Testicular volume was measured and paraffin sections (8 microns) of the testis were stained with Hematoxylin-Eosin. Seminiferous tubular diameter and epithelial height were analyzed quantitatively. No significant changes in testicular volume, seminiferous tubular diameter and epithelial height were observed in all three groups. The shrunk testicular morphology in the aged male rats cannot be restored by fetal POA grafting. These results suggest that copulatory behavior and testicular morphology may be dissociated in old male rats.

Age-related changes in the pattern of luteinizing hormone release induced in female rats by male rat urine.

The release of luteinizing hormone (LH) in young (3-4 months) and aged (24-25 months) ovariectomized female Sprague-Dawley rats, s.c. implanted with a 17 beta-estradiol benzoate silastic capsule, was studied in the presence and absence of stimulation by exposure to male rat urine. After taking an initial blood sample at 12:00 (reference sample), either urine, collected from young adult male rats, or distilled water was poured into the female's cage. Blood samples were then collected hourly up to 18:00 via a catheterized jugular cannula. The concentration of LH in the plasma was measured by RIA. The basal plasma LH level in young control rats was found to increase significantly at 16:00 compared with the 12:00 reference sample while no statistically significant change in plasma LH concentration occurred in old controls over the same period. Male rat urine caused a significantly earlier (at 15:00) and prolonged (from 15:00 to 18:00) elevation of plasma LH in young rats compared with young controls. In contrast, exposure of old female rats to male rat urine resulted in no marked change in plasma LH levels. These results suggest that both basal LH release and the response to pheromonal stimulation by male rat urine may be modified with increasing age in female rats.

Comparison of brain dopamine depletion induced by low-dose 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in young and aged rats.

Effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on dopamine levels in the striatum (ST), substantia nigra (SN), olfactory tubercle (OT), ventral tegmental area (VTA) and nucleus accumbens (NA) were studied in young (1 month) and old (21 months) male Long-Evans rats. Following repeated treatment with MPTP at low dose (3 mg/kg i.p. daily for 8 days) both young and old rats showed a marked depletion of dopamine (DA) concentrations in the ST compared to those of age-matched saline-treated controls. However, a significant reduction in DA levels was observed in the OT and NA of old but not young rats treated with MPTP. In contrast, no changes in 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) levels in these three regions were seen in either young or old MPTP-treated rats. However, the ratio of DOPAC/DA and HVA/DA in the ST and NA were significantly higher in old MPTP-treated rats than those in old controls. The concentrations of DA, DOPAC and HVA remained unchanged in the SN and VTA of both young and old rats after MPTP treatment. These results indicate that multiple treatment of low-dose MPTP selectively causes more widespread damage to dopaminergic neurons in old rats than in young ones, and further support the view that effects of MPTP on the dopaminergic system in rats may depend on age.

Effects of age on open-field behavior of male rats.

As age advances, the performance of animal behavior declines gradually. The purpose of this study was to investigate the onset of age-related changes in open-field behavior of rats from a wide age spectrum. Male Long-Evans rats, ranging from 2 to 22 months, were placed in an open-field apparatus during the dark phase for a cumulative period of 28 min. Data were collected for the following parameters of open-field behavior: horizontal activity (HA), total distance (TD), stereotypy count (SC), vertical activity (VA), movement time (MT) and margin time (MGT). The highest value of HA, TD, SC and MT was found in rats at the age of 3 months. After 3 months, the values for these parameters gradually declined, subsequently reaching statistical significance by 6 months (HA, TD and SC) or 8.5 months (MT) of age. Maximum activity of VA occurred at 8.5 months of age and thereafter gradually declined with increasing age, however, no significantly statistical difference was reached as compared to the highest value. In contrast, the values for MGT gradually declined after 2 months of age, thereby reaching statistical significance by 11 months of age. These results indicate that the onset of age-related decline in open-field behavioral parameters can differ and the age-related changes in open-field behavior depends on observational parameters.

Age-related changes in locomotor behavior induced by MPTP in rats.

Effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on gait was studied in young (4 weeks) and old (21 months) male rats. No significant alteration in stride length (SL) or stride width (SW) was observed 30 min after single dose MPTP treatment (3 mg/kg, i.p.) in both young and old rats. Multidose treatment with MPTP (3 mg/kg, i.p. daily for 8 days) resulted in a marked decrease in SL in old rats compared to that of old control group, while an increase in SL was found in both MPTP-treated and control young rats. In contrast, multidose MPTP administration caused no changes in SW in either young or old rats. The present result of behavioral analysis suggests that multidose MPTP neurotoxic effect on locomotor activity may be age dependent in rats and that the old rat is an appropriate model for MPTP studies.

Norepinephrine levels in the vomeronasal system and their responses to male urine in young and aged female rats.

Norepinephrine (NE) levels in brain areas of the vomeronasal system in young (4-5 months) and aged (25-26 months) ovariectomized Sprague-Dawley rats, which were implanted with a 17 beta-estradiol silastic capsule and then exposed to male rat urine, were investigated. The unilateral vomeronasal organ was removed in all rats one week before exposure to urine stimulation. NE levels in the medial nucleus of the amygdala (MA), medial preoptic area (MPOA), ventromedial nucleus of hypothalamus (VMH) and bed nucleus of stria terminalis (BST) were measured. NE concentrations in these brain areas of the surgical side served as the control. Urine collected from young adult male rats was poured into the female's cage at 12:00h and the animals were sacrificed before and 1, 2, or 3 hours after the male urine was given. The NE basal levels in the MA and MPOA of young rats decreased significantly from 13:00h to 15:00h, and those in young rat VMH declined markedly from 13:00h to 14:00h compared to those at 12:00h. No marked alterations in NE basal levels in young rat BST were found. In contrast, no obvious changes in the NE concentrations were observed in these brain areas of old rats. Continuous exposure to male urine did not affect the NE levels in any of these brain areas of young and aged rats. We concluded that (1) the time-dependent fluctuation of the NE basal levels in some brain areas of the vomeronasal system in female rats is age-related, and (2) the NE in all these nuclei of the vomeronasal system is not involved in pheromone-induced effects.

Effects of long-term dietary restriction on body temperature are modified with increasing age.

Effects of long-term dietary restriction on body temperature and its circadian changes were investigated in 3.5- and 14.5-month-old male Long-Evans rats. Animals were either fed and libitum or kept on a restricted diet for 8 weeks. Purina Lab Chow was constantly available to the ad libitum-fed groups, while half portions of their daily food consumption were given to age-matched diet-restricted groups every day. A highly significant lowering of body temperature in middle-aged diet-restricted (MR) rats was not observed until their food intake had been restricted for 5 weeks compared with that of the middle-aged ad libitum-fed (MA) group as well as that of the young diet-restricted (YR) rats. Eight weeks after diet restriction, both the circadian pattern of body temperature and its diurnal peak-trough difference remained almost unchanged in all four groups, while the average body temperature of MR rats was greatly lower than that of the YR group and that of MA animals. No significant difference in average body temperature was found between the young ad libitum-fed (YA) rats and the MA group. These data suggest that the average body temperature and its circadian changes in ad libitum-fed rats, at least before the age of 14.5 months, is not age-related, while the effect of dietary restriction on body temperature may be modified with increasing age.

Influence of long-term continuous illumination on the nuclear volume of hypothalamic neurons in female rats.

A light microscopic quantitative analysis was performed for studying the effects of long-term exposure to continuous illumination on hypothalamic morphology in female rats. Nuclear volumes of neurons in the suprachiasmatic nucleus, paraventricular nucleus, ventromedial nucleus and preoptic area were measured in 16-18 month-old Long-Evans female rats which had been exposed to continuous light for 11 months. Significant nuclear volume shrinkage could be observed in all investigated regions except for the preoptic area in these animals when compared to that of control rats kept under a photoperiod of 14h light and 10h dark. The relationship between morphological changes of hypothalamic neurons and the marked alteration of melatonin and estrogen levels induced by long-term exposure to continuous light is discussed.

Morphological changes in the hypothalamic neurons of female rats exposed to continuous illumination.

The morphological changes of neurons in the paraventricular nucleus (PVN), suprachiasmatic nucleus (SCN), ventromedial nucleus (VMN), preoptic area (POA), supraoptic nucleus (SON) and arcuate nucleus (ARN) were studied in 16-18 month-old Long-Evans female rats which had been exposed to continuous illumination (LL) for 11 months. Most neurons in those hypothalamic areas showed compact cytoplasm with abundant Nissl substances and evenly distributed chromatin. These morphological features were not found in the same hypothalamic regions of rats placed under a regular alternative light-dark regimen (14h light and 10h dark). These findings suggest that decreased melatonin and elevated estrogen levels induced by long-term exposure to LL might be involved in the morphological changes in hypothalamic neurons of female rats.

Inhibition of platelet aggregation and antagonism of vasopressin-induced ECG changes in primates by a carboprostacyclin analogue, ZK 36374.

A chemically stable carboprostacyclin analogue, ZK 36374 has been compared with two other prostacyclin derivatives with respect to ADP-induced in vitro aggregation of baboon and human platelets and ex vivo platelet aggregation in the baboon. ZK 36374 was also tested on the systemic arterial blood pressure of the baboon and against vasopressin-induced ECG changes in primates. Compared to the other two compounds, ZK 36374 displayed enhanced anti-platelet aggregating activity; there was dissociation between this property and its hypotensive potency. ZK 36374 antagonized the vasopressin-induced ECG changes. These results indicate that ZK 36374 possesses therapeutic potential in vascular disease including that affecting the coronary vessels.

Inhibition of platelet aggregation with intravenous and oral administration of a carboprostacyclin analogue, 15-cyclopentyl-omega-pentanor-5(E)-carbacyclin (ONO 41483) in man.

Intravenous and oral administration of a chemically stable carboprostacyclin analogue, 15-cyclopentyl-omega-pentanor-5(E)-carbacyclin (ONO 41483), resulted in ex-vivo inhibition of ADP-induced platelet aggregation in man. The maximum tolerated intravenous dose was 2.5 ng/kg/min for 1 hour and this produced a mean of 27.1% inhibition in 3 volunteers. For oral administration the tolerated single dose was 200 microgram. At this dose, there was 56.3% inhibition of aggregation (mean of 3 results). High oral (400 microgram) and intravenous doses (5 and 10 ng/kg/min for 1 hour) of ONO 41483, which caused marked inhibition of aggregation (ranging 39-100%), was accompanied by flushing of face and extremities, headache and phlebitis. However, none of the doses tested produced significant changes in arterial blood pressure or heart rate.

Inhibition of platelet aggregation and reversal of vasopressin-induced ECG changes by a carboprostacyclin analogue, ONO 41483, in primates.

15-cyclopentyl-omega-pentanor-5(E)-carbacyclin (ONO 41483), a chemically stable carboprostacyclin analogue, was 3.3 times less active than prostacyclin but was 2.6 times more active than carboprostacyclin in inhibiting aggregation of ADP-induced baboon platelet in vitro. On human platelets in vitro, ONO 41483 was 9.4 times less active than prostacyclin and 12.7 times more active than carboprostacyclin. ONO 41483 was 3.7 times less active than prostacyclin but was 2.2 times more active than carboprostacyclin in producing a fall in arterial blood pressure in anaesthetised baboons. Intravenous and oral administration of ONO 41483 in baboons produced ex vivo inhibition of ADP-induced platelet aggregation at doses that did not affect blood pressure or heart rate. In addition, bolus intravenous doses (3 to 10 micrograms/kg) of ONO 41483 reversed vasopressin-induced ECG changes in the monkey, suggesting an ability of the compound to relieve coronary spasm.