Influence of age on nicotinic cholinergic regulation of blood flow in rat's olfactory bulb and neocortex.

The olfactory bulb receives cholinergic basal forebrain inputs as does the neocortex. With a focus on nicotinic acetylcholine receptors (nAChRs), this review article provides an overview and discussion of the following findings: (1) the nAChRs-mediated regulation of regional blood flow in the neocortex and olfactory bulb, (2) the nAChR subtypes that mediate their responses, and (3) their activity in old rats. The activation of the α4ß2-like subtype of nAChRs produces vasodilation in the neocortex, and potentiates olfactory bulb vasodilation induced by olfactory stimulation. The nAChR activity producing neocortical vasodilation was similarly maintained in 2-year-old rats as in adult rats, but was clearly reduced in 3-year-old rats. In contrast, nAChR activity in the olfactory bulb was reduced already in 2-year-old rats. Thus, age-related impairment of α4ß2-like nAChR function may occur earlier in the olfactory bulb than in the neocortex. Given the findings, the vasodilation induced by α4ß2-like nAChR activation may be beneficial for neuroprotection in the neocortex and the olfactory bulb.

Nicotinic cholinergic regulation of olfactory bulb blood flow response in aged rats.

In our previous research, we had demonstrated the crucial role of neuronal nicotinic acetylcholine receptors (nAChRs) in potentiation of the olfactory bulb blood flow response to olfactory stimulation in adult rats. The present study examined the effects of nAChR activation on the olfactory bulb blood flow response in rats aged 24-27 months. We found that, under urethane anesthesia, unilateral olfactory nerve stimulation (300 µA, 20 Hz, 5 s) increased blood flow within the ipsilateral olfactory bulb, without changes in the systemic arterial pressure. The increase in blood flow was dependent upon the current and frequency of the stimulus. Intravenous administration of nicotine (30 µg/kg) had little effect on the olfactory bulb blood flow response to nerve stimulation at either 2 Hz or 20 Hz. These results suggest a reduction in nAChR-mediated potentiation of the olfactory bulb blood flow response in aged rats.

Effects of nicotine on regional blood flow in the olfactory bulb in response to olfactory nerve stimulation.

This study examined the effect of olfactory nerve stimulation on regional cerebral blood flow and assessed the effect of intravenous nicotine administration on this response in anesthetized rats. Regional cerebral blood flow was measured with laser Doppler flowmetry or laser speckle contrast imaging. Unilateral olfactory nerve stimulation for 5 s produced current (≥ 100 µA) and frequency-dependent (≥ 5 Hz) increases in blood flow in the olfactory bulb ipsilateral to the stimulus. The increased olfactory bulb blood flow peaked at 30 ± 7% using stimulus parameters of 300 µA and 20 Hz. Nerve stimulation did not change frontal cortical blood flow or mean arterial pressure. The intravenous injection of nicotine (30 µg/kg) augmented the olfactory bulb blood flow response to nerve stimulation (20 Hz, 300 µA) by approximately 1.5-fold (60-s area after the stimulation). These results indicate that olfactory nerve stimulation increases olfactory bulb blood flow, and the response is potentiated by the activation of nicotinic cholinergic transmission.

Neural Mechanisms Involved in the Noxious Physical Stress-Induced Inhibition of Ovarian Estradiol Secretion.

Stress is known to change the secretion of ovarian steroid hormones via the hypothalamic-pituitary-ovarian (HPO) axis. Noxious physical stress can cause reflex responses in visceral function via autonomic nerves. This article reviews our recent animal studies on neural mechanisms involved in ovarian estradiol secretion induced by noxious physical stress stimulation. In anesthetized rats, noxious physical stress (pinching the hindpaw or electrical stimulation of the tibial nerve) decreased ovarian estradiol secretion. These noxious stress-induced ovarian hormonal responses were observed after decerebration but were abolished after spinal transection. Electrical stimulation of the ovarian sympathetic nerves (superior ovarian nerves: SON) decreased ovarian estradiol secretion. The reduced secretion of ovarian estradiol induced by hindpaw pinching was abolished by bilateral severance of the SON. Efferent activity of the SON was increased following hindpaw pinching. Thus, the inhibition of ovarian estradiol secretion during noxious physical stress was mainly integrated in the brainstem, and this inhibitory response was due to reflex activation of sympathetic nerves to the ovary. In rats, the sympathetic inhibitory regulation of ovarian estradiol secretion was pronounced when the HPO axis was inhibited by chronic estradiol treatment. Considering the female life cycle, extensive physical stress may inhibit ovarian function, especially before puberty and during old ages when the HPO axis is inactive. Anat Rec, 302:904-911, 2019. © 2019 Wiley Periodicals, Inc.

Effects of nicotine on odor-induced increases in regional blood flow in the olfactory bulb in rats.

The present study examined the effects of nicotinic acetylcholine receptor activation on the odor-induced blood flow response in the olfactory bulb. In urethane-anesthetized rats, odor stimulation (5% amyl acetate, 30 s) produced an increase in olfactory bulb blood flow (reaching 107% ± 3% of the pre-stimulus basal values), without changes in frontal cortical blood flow or mean arterial pressure. Intravenous injection of nicotine (30 µg/kg), a nicotinic acetylcholine receptor agonist, significantly augmented the odor-induced increase response of olfactory bulb blood flow, without changes in the basal blood flow level. The nicotine-induced augmentation of the olfactory bulb blood flow response to odor was negated by dihydro-ß-erythroidine, an α4ß2-preferring nicotinic acetylcholine receptor antagonist. Our results suggest that the activation of α4ß2-like neuronal nicotinic acetylcholine receptors in the brain potentiates an odor-induced blood flow response in the olfactory bulb.

Mechanism of physical stress-induced inhibition of ovarian estradiol secretion in anesthetized rats.

This study examined the site of main integration center in the physical stress-induced inhibition of ovarian estradiol secretion because of ovarian sympathetic nerve (superior ovarian nerve: SON) activation in anesthetized rats. In central nervous system-intact rats, electrical stimulation of the tibial afferent nerve at 10V increased the efferent activity of the SON by 39±13% and reduced the ovarian secretion of estradiol by 34±7%. These responses were observed in decerebrate rats but were abolished in spinal rats. Thus, the main integration center for this ovarian hormonal response is located in the brain stem.

Sympathetic regulation of ovarian functions under chronic estradiol treatment in rats.

Activation of the sympathetic nerve to the ovary (superior ovarian nerve: SON) decreases ovarian blood flow and estradiol secretion in rats in the estrous phase. The present study examined the effects of long-term estradiol treatment on the sympathetic regulation of both ovarian blood flow and estradiol secretion. Non-pregnant Wistar rats received sustained subcutaneous estradiol (5µg/day) or saline for 4weeks. Chronic estradiol treatment did not affect ovarian blood flow at rest, while changed the basal ovarian estradiol secretion rate, i.e., narrow ranges (4-34pg/min) in estradiol-treated rats, versus wide ranges (3-192pg/min) in saline-treated rats of different estrous cycles. SON was electrically stimulated at different frequencies (2, 5 and 20Hz). Ovarian blood flow was decreased by SON stimulation in a stimulus frequency-dependent manner in both saline- and estradiol-treated rats, but the threshold was shifted from 2Hz to 5Hz after chronic estradiol treatment. Ovarian estradiol secretion rate was not significantly changed by SON stimulation at any frequency in saline-treated rats, while it was markedly decreased by SON stimulation at high frequencies (5 and 20Hz) in estradiol-treated rats. In conclusion, chronic estradiol treatment augments sympathetic inhibition of ovarian estradiol secretion perhaps by inhibiting the hypothalamic-pituitary-ovarian axis.

Afferent fibers involved in the bradykinin-induced cardiovascular reflexes from the ovary in rats.

Bleeding or rupture of the ovary often accompanies ovarian cysts and causes severe pain and autonomic responses such as hypotension. It would be expected that ovarian afferents contribute to cardiovascular responses induced by ovarian failure. The present study examined cardiovascular responses to noxious chemical stimulation of the ovary by bradykinin, an algesic substance released by tissue damage, and explored the role of ovarian afferents in the ovarian-cardiovascular responses in anesthetized rats. Non-pregnant adult rats were anesthetized with pentobarbital and artificially ventilated. The carotid artery was cannulated to monitor blood pressure and heart rate. Noxious chemical stimulation was achieved by applying a small piece of cotton soaked with bradykinin to the surface of the ovary for 30s. Application of bradykinin (10(-4) M) to the ovary decreased heart rate and blood pressure. These cardiovascular responses were not significantly influenced by severance of the vagal nerves or the superior ovarian nerve, but were abolished by severance of the ovarian nerve plexus (ONP). Application of bradykinin (10(-4) M) to the ovary evoked afferent activity of the ONP both in vivo and in vitro preparations. These results indicate that the decreases in heart rate and blood pressure following chemical noxious stimulation of the ovary with bradykinin are reflex responses, whose afferent nerve pathway is mainly through afferent fibers in the ONP.

Sympathetic regulation of estradiol secretion from the ovary.

It is well known that hormone secretion from endocrine glands is regulated by hierarchical feedback mechanisms. However, although Cannon revealed in the 1920s that sympathoadrenal medullary function increased during emergency situations, no studies on the autonomic nervous regulation of hormone secretion have been undertaken for many years. In the past 40 years, the autonomic nervous regulation of insulin secretion from the pancreas, gastrin secretion from the stomach, glucocorticoid secretion from the adrenal cortex, etc., has been demonstrated. Estradiol secretion from the ovary is strongly controlled by the hypothalamic-pituitary-ovarian axis, and its possible regulation by autonomic nerves has been largely unnoticed. Some histological studies have revealed rich adrenergic sympathetic innervation in the ovary. Recently, it has been demonstrated that the activation of the sympathetic nerves to the ovary directly reduces estradiol secretion from the ovary. This article reviews physiological and morphological studies, primarily in rats, on the sympathetic regulation of estradiol secretion from the ovary.

Autonomic nervous regulation of ovarian function by noxious somatic afferent stimulation.

It is well known that ovarian function is regulated by hypothalamic-pituitary-ovarian hormones. However, although several histological studies have described the autonomic innervation of the ovary, the involvement of these autonomic nerves in ovarian function is unclear. Recently, it has been shown that both the superior ovarian nerve (SON) and the ovarian nerve plexus (ONP) induce vasoconstrictor activity by activation of alpha 1-adrenoceptors, whereas the SON, but not the ONP, inhibits ovarian estradiol secretion by activation of alpha 2-adrenoceptors. Furthermore, reflex activation of these ovarian nerves by noxious cutaneous stimulation of the rat hindpaw results in ovarian vasoconstriction and inhibition of estradiol secretion. Thus, in addition to long-term regulation of ovarian function by hormones, ovarian autonomic innervation may be involved in rapid regulation of ovarian function by responding to either internal or external environmental changes.

Effects of electrical stimulation of autonomic nerves to the ovary on the ovarian testosterone secretion rate in rats.

Previously, we demonstrated that electrical stimulation of the superior ovarian nerve (SON), but not the ovarian nerve plexus (ONP), reduces the secretion rate of estradiol from the ovary via activation of alpha 2-adrenoceptors in rats. The inhibitory effect of SON on estradiol secretion may be due to reduced production of testosterone, a direct precursor of estradiol. Here, we examined the effects of electrical stimulation of the SON and the ONP on ovarian testosterone secretion in rats. On the day of estrous, ovarian venous blood samples were collected intermittently from the ovarian vein. The secretion rate of testosterone from the ovary was calculated from the difference in the testosterone concentration between ovarian venous plasma and systemic arterial blood plasma, and the rate of ovarian venous plasma flow. Stimulation of either the SON or ONP reduced the secretion rate of testosterone from the ovary. The reduction of the testosterone secretion rate by SON stimulation was not influenced by an alpha 2-adrenoceptor antagonist (yohimbine), but it was abolished by an alpha 1-adrenoceptor antagonist (prazosin). Our results show that ovarian nerves have an inhibitory role in ovarian testosterone secretion, via activation of alpha 1-adrenoceptors, but not alpha 2-adrenoceptors. This, therefore, indicates that the reduction of estradiol secretion by SON stimulation is independent of the reduction of testosterone secretion.

The missing link between long-term stimulation of nicotinic receptors and the increases of acetylcholine release and vasodilation in the cerebral cortex of aged rats.

In adult rats (4-9 months), chronic nicotine infusion increases the basal level of acetylcholine (ACh) release in the cerebral cortex and enhances responses of cortical ACh release and cortical vasodilation elicited by nucleus basalis of Meynert (NBM) stimulation. In the present study, we examined whether these effects of nicotine are detected in aged rats. Aged rats (27-30 months) received sustained subcutaneous nicotine (100 µg/kg/h) or saline for 14 days. Under urethane anesthesia, ACh release and regional blood flow in the parietal cortex were measured. The basal level of ACh release in the cerebral cortex was not changed by chronic nicotine. In addition, the magnitudes of ACh release and vasodilation by NBM stimulation were similar between the saline-treated and nicotine-treated groups. The lack of an effect of chronic nicotine in aged rats may be due to a decrease in nicotinic receptors in the cerebral cortex during aging (Nordberg et al., J Neurosci Res 31:103-111, 1992).

Reflex modulation of ovarian estradiol secretion by noxious mechanical stimulation of a hindpaw in anesthetized rats.

Previously, we demonstrated that electrical stimulation of the superior ovarian nerve in rats reduces the ovarian estradiol secretion rate. In the present study, we examined the effect of noxious mechanical afferent stimulation (pinching) of a hindpaw on the ovarian estradiol secretion rate in rats. The rats were anesthetized on the day of estrus, and the ovarian venous blood was collected intermittently. The secretion rate of estradiol from the ovary was calculated from differences in the estradiol concentration between ovarian venous plasma and systemic arterial blood plasma, and from the flow rate of ovarian venous plasma. Pinching stimulation of a hindpaw for 5 min decreased the estradiol secretion rate from the ovary. A significant reduction of the estradiol secretion rate began at 5 min after the end of the stimulation and lasted for 20 min. The minimum decrease in estradiol secretion rate was 71.1 ± 14.0% of the prestimulus basal values at 15 min after the stimulation ended. The decrease responses of the ovarian estradiol secretion rate were abolished by bilateral severance of the superior ovarian nerves. The efferent activity of the superior ovarian nerves was increased following hindpaw pinching. After spinal transection at the second cervical level, the increased response of the superior ovarian nerve activity by hindpaw pinching was abolished. These results indicate that noxious mechanical stimulation of a hindpaw decreases the estradiol secretion rate from the ovary, and that the response is due to reflex activation of ovarian sympathetic nerves, mediated by supraspinal structures.

The role of alpha adrenoceptors in the vascular and estradiol secretory responses to stimulation of the superior ovarian nerve.

Electrical stimulation of the superior ovarian nerve in rats reduces both the plasma flow rate of ovarian venous blood (ovarian blood flow) and the ovarian estradiol secretion rate. Here, we examined the possible roles of alpha-adrenoceptors in these processes. The reduction of the plasma flow rate was blocked by an alpha 1- (prazosin), but not by an alpha 2- (yohimbine) adrenoceptor blocker. In contrast, the reduction of the estradiol secretion rate was blocked by yohimbine but not by prazosin. We conclude that ovarian vascular and estradiol secretory responses to superior ovarian nerve activation are mediated by alpha 1- and alpha 2-adrenoceptors, respectively.

Sustained subcutaneous infusion of nicotine enhances cholinergic vasodilation in the cerebral cortex induced by stimulation of the nucleus basalis of Meynert in rats.

The present study examined the effects of sustained nicotine exposure on the cholinergic vasodilative system originating in the nucleus basalis of Meynert (NBM) and projecting to the cerebral cortex in rats. Rats received sustained subcutaneous nicotine (100µg/kg/h) for 14 days. Under urethane anesthesia, the vasodilation response and acetylcholine release in the parietal cortex induced by electrical stimulation of the NBM (10-200µA) were measured. The basal level of acetylcholine release was significantly higher in nicotine-treated rats than in saline-treated control rats. In the control rats, both the acetylcholine release and blood flow were increased by NBM stimulation in a stimulus intensity-dependent manner, and a threshold of 50µA. In nicotine-treated rats, the threshold intensity of NBM stimulation producing increases in acetylcholine release and blood flow was reduced to 20µA. The stimulus intensity-dependent acetylcholine release and vasodilation by NBM stimulation were significantly larger in nicotine-treated rats than in control rats. We conclude that sustained subcutaneous infusion of nicotine enhances cholinergic vasodilative system in the cerebral cortex originating in the NBM.

Long-term nicotine treatment reduces cerebral cortical vasodilation mediated by alpha4beta2-like nicotinic acetylcholine receptors in rats.

Regional cortical cerebral blood flow is increased via activation of brain nicotinic acetylcholine receptors. Acute intravenous injection of nicotine increases cortical blood flow, without changing systemic blood pressure in anesthetized rats. Here, we examined whether the nicotine-induced cerebral cortical vasodilation is affected by chronic nicotine treatment. Rats received chronic subcutaneous nicotine (at a low or a high-dose) short-term (1 h) or long-term (14 days). Under urethane anesthesia, blood flow in the frontal cortex, before and after bolus injection of nicotine (0.3-30 microg/kg, i.v.) was measured by laser Doppler flowmetry. The threshold dose of nicotine (3 microg/kg, i.v.) producing vasodilation was not affected by chronic nicotine treatment. However, the vasodilation induced by nicotine at 30 microg/kg was reduced after long-term nicotine treatment (but not after short-term exposure). The degree of reduction was marked and was statistically significant with high-dose (100 microg/kg/h) nicotine; low-dose (33 microg/kg/h) nicotine had a small effect that was not statistically significant. In contrast, the vasodilation in the cortical vessels obtained by hypercapnia (inhalation of 10% CO2) was not changed by chronic nicotine treatment. The nicotine-induced cortical vasodilation was not influenced by methyllycaconitine, an alpha7-selective nicotinic antagonist, while it was completely abolished by dihydro-beta-erythroidine, an alpha4beta2-preferring nicotinic antagonist. We conclude that long-term nicotine treatment reduces the functional activity of alpha4beta2-like nicotinic receptors that mediate cortical vasodilation.

Cerebral cortical vasodilatation mediated by nicotinic cholinergic receptors: effects of old age and of chronic nicotine exposure.

Nicotinic acetylcholine receptors in the brain play a crucial role in the vasodilation in the cerebral cortex induced by basal forebrain cholinergic activation. Nicotinic receptors can be up-regulated or down-regulated in various conditions, e.g., up-regulation (increase in number) is found in smokers and in rodents exposed to chronic nicotine, while down-regulation (decrease in number) is found in elderly humans and in aged rats. We found in anesthetized rats that the increase in cortical cerebral blood flow induced by bolus intravenous nicotine injection was due to a vasodilation mediated by nitric oxide following activation of nicotinic receptors, probably of alpha4beta2-like subtype, both in the basal forebrain nuclei (Meynert nucleus) and in the cortex. This vasodilative response was reduced by long-term nicotine treatment and also in aged rats. Our results show that functional changes in nicotinic receptors are not always in the same direction as the changes in number; they highlight the importance of investigating not only the changes in receptor numbers but also those in their functional activity.

Effects of electrical stimulation of the superior ovarian nerve and the ovarian plexus nerve on the ovarian estradiol secretion rate in rats.

The present experiments examined the effects of electrical stimulation of the superior ovarian nerve (SON) and the ovarian plexus nerve (OPN) on the ovarian estradiol secretion in rats. The rats were anesthetized on the day of estrus, and the ovarian venous blood was collected intermittently. The secretion rate of estradiol from the ovary was calculated from differences in the estradiol concentration between ovarian venous plasma and systemic arterial blood plasma, and from the flow rate of ovarian venous plasma. Either an SON or OPN, ipsilateral to the ovary from which ovarian venous blood was collected, was electrically stimulated at a supramaximal intensity for C-fibers. The secretion rate of estradiol was significantly decreased by 47 +/- 6% during SON stimulation, but it was not significantly changed during OPN stimulation. These results suggest that autonomic nerves, which reach the ovary via the SON, have an inhibitory role in ovarian estradiol secretion.

Effects of nicotine on regional blood flow in the olfactory bulb in rats.

Effects of nicotine on blood flow in the olfactory bulb were examined in anesthetized rats. Nicotine administered intravenously at 100 microg/kg increased regional blood flow in the olfactory bulb, irrespective of changes in systemic arterial pressure. Nicotine administered locally into the internal carotid artery at 10 microg increased blood flow, without changing arterial pressure; this response was abolished by hexamethonium. These results indicate that nicotine produces vasodilatation in the olfactory bulb via activation of nicotinic receptors located close to the olfactory bulb. Nicotine may be of therapeutic value in improving blood flow in the olfactory bulb.

Nicotine-induced NO-mediated increase in cortical cerebral blood flow is blocked by beta2-adrenoceptor antagonists in the anesthetized rats.

Involvement of nitric oxide (NO) and beta-adrenoceptors in an increase in the cortical cerebral blood flow (CBF) following an intravenous (i.v.) injection of a small dose of nicotine which did not affect the systemic blood pressure in the rats was investigated. I.v. injection of nicotine (30 microg/kg) for 1 min produced a significant increase in CBF lasting for more than 20 min without a significant effect on the systemic blood pressure. I.v. injection of L-N(G)-nitroarginine methylester (30 mg/kg) significantly attenuated nicotine-induced increase in the cortical CBF. The attenuation was reversed by i.v. injection of L-arginine (300 mg/kg), suggesting an intimate role of nitric oxide (NO) in nicotine-induced increase in the cortical CBF. The nicotine-induced increase in the cortical CBF was significantly attenuated by propranolol (10 mg/kg, i.v.) and ICI 118,551 (a beta2-adrenoceptor antagonist, 10 mg/kg, i.v.) but not by metoprolol (a beta1-adrenoceptor antagonist, 10 mg/kg, i.v.). Beta2-adrenoceptors on presynaptic nitrergic nerves may be involved in nicotine-induced NO-mediated increase in the cortical CBF.