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.

Olfactory function and discrimination ability in the elderly: a pilot study.

We recently reported that subjects with a higher olfactory identification threshold for rose odor declined more in attentional ability in the elderly. This study focuses on discrimination ability and olfactory identification threshold in twelve elderly subjects living in a community (age: 80.9 ± 1.6). Olfactory function was assessed by the rose odor identification threshold. We assessed the discrimination ability by distinguishing 5 similar odor pairs. Our results showed that the subjects with a higher olfactory identification threshold (≥ 5) declined more in discrimination ability (14% ± 14%, p = 0.03) compared to those with a lower threshold (≤ 4) (averaged value set at 100%). As discrimination ability is related to the basal forebrain cholinergic system, our results suggest that olfactory impairment links to the decline in cognitive function relating the cholinergic system.

The relationship between olfaction and cognitive function in the elderly.

This study investigated the relationship between olfaction and cognitive function in 12 elderly people (age: 80.9 ± 1.6) living in the community. Olfactory function was assessed by the identification threshold for rose odor. Four cognitive measures consisting general cognitive ability assessed by Mini-Mental State Examination (MMSE), its sub-domains, and attentional ability assessed by drawing a line to connect the numbers consecutively (trail-making test part A; TMT-A), were assessed. Subjects with a higher olfactory threshold (≥ 5) declined more in the performance speed of TMT-A (73% ± 7%, p = 0.05) compared with those subjects with a lower threshold (≤ 4) (averaged value was set at 100%). Other cognitive statuses assessed by MMSE tended to decline in subjects with higher thresholds. Because attentional function relates to the basal forebrain cholinergic system, our results suggest that olfactory impairment links to the decline in cognitive function, particularly of attention-relating cholinergic function.

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.

Somatosensory regulation of resting muscle blood flow and physical therapy.

Somatosensory stimulation can affect skeletal muscle blood flow (MBF) at rest in anesthetized animals via pressor reflex response or antidromic and local vasodilation. Increase in MBF due to reflex pressor response occurs generally in the skeletal muscles of the entire body, while antidromic and local vasodilation are limited to the peripheral stimulation site. Since increased MBF improves several disorders (muscle stiffness, pain, etc.), it is reasonable to further explore the effective use of somatic stimulation in physical therapies, such as massage, acupuncture, anma, and shiatsu or acupressure, in treating skeletal muscle disorders.

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.

Blood pressure-independent increase in the cortical cerebral blood flow induced by manual acupuncture of the auricular region in rats.

We aimed to determine whether acupuncture to the auricular region increases cortical regional cerebral blood flow (rCBF). The rCBF was measured using laser speckle contrast imaging in urethane-anesthetized rats. Acupuncture stimulation was performed manually at the auricular concha or abdomen. The former's stimulation significantly increased the rCBF of the bilateral cerebral cortex in the frontal, parietal, and occipital lobes without altering the systemic arterial pressure. In contrast, abdominal stimulation affected neither rCBF nor systemic arterial pressure. The increase in the rCBF was completely abolished by the severance of the somatic nerves that innervated the auricular region, comprising the trigeminal nerve, facial nerve, auricular branch of the vagal nerve, glossopharyngeal nerve, and great auricular nerve. Thus, application of acupuncture to the auricular region increases the rCBF without increasing arterial pressure.

Effect of basal forebrain stimulation on extracellular acetylcholine release and blood flow in the olfactory bulb.

The olfactory bulb receives cholinergic basal forebrain input, as does the neocortex; however, the in vivo physiological functions regarding the release of extracellular acetylcholine and regulation of regional blood flow in the olfactory bulb are unclear. We used in vivo microdialysis to measure the extracellular acetylcholine levels in the olfactory bulb of urethane-anesthetized rats. Focal chemical stimulation by microinjection of L-glutamate into the horizontal limb of the diagonal band of Broca (HDB) in the basal forebrain, which is the main source of cholinergic input to the olfactory bulb, increased extracellular acetylcholine release in the ipsilateral olfactory bulb. When the regional cerebral blood flow was measured using laser speckle contrast imaging, the focal chemical stimulation of the HDB did not significantly alter the blood flow in the olfactory bulb, while increases were observed in the neocortex. Our results suggest a functional difference between the olfactory bulb and neocortex regarding cerebral blood flow regulation through the release of acetylcholine by cholinergic basal forebrain input.

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.

Somatoautonomic reflexes in acupuncture therapy: A review.

Oriental therapies such as acupuncture, moxibustion, or Anma, have been used to treat visceral disorders since ancient times. In each of these therapies, stimulation of the skin or underlying muscles leads to excitation of afferent nerves. The sensory information is carried to the central nervous system, where it is transferred to autonomic efferents, thus affecting visceral functions. This neuronal pathway, known as the "somatoautonomic reflex", has been systematically studied by Sato and his colleagues for over a half century. Nearly all their studies were conducted in anesthetized animals, whereas human patients are conscious. Responses in patients or the events following therapeutic somatic stimulation may differ from those observed in anesthetized animals. In fact, it is increasingly apparent that the responses in patients and animals are not always coincident, and the differences have been difficult for clinicians to reconcile. We review the mechanism of the "somatoautonomic reflex" as described in anesthetized animals and then discuss how it can be applied clinically.

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.

Effect of acupuncture-like stimulation on cortical cerebral blood flow in aged rats.

This study aimed to examine the effect of acupuncture-like stimulation on cortical cerebral blood flow (CBF) in aged rats and the contribution of the intracranial cholinergic vasodilatory system on its response. In urethane-anesthetized rats of 30-37 months of age, manual acupuncture-like stimulation of a forepaw produced an increase in the CBF, independent of systemic arterial pressure. The increase in the CBF induced by forepaw stimulation was abolished by intravenous administration of cholinergic receptor antagonists. Manual acupuncture-like stimulation of a forepaw increased extracellular acetylcholine release in the cerebral cortex. These results suggest that natural somatic afferent stimulation, such as acupuncture-like stimulation, activates the intracranial - most likely, basal forebrain - cholinergic vasodilatory system in the cerebral cortex, even in extremely aged rats.

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.

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.

Control of cerebral cortical blood flow by stimulation of basal forebrain cholinergic areas in mice.

We examined whether activity of the nucleus basalis of Meynert (NBM) regulates regional cerebral cortical blood flow (rCBF) in mice, using laser speckle and laser Doppler flowmetry. In anesthetized mice, unilateral focal stimulation, either electrical or chemical, of the NBM increased rCBF of the ipsilateral cerebral cortex in the frontal, parietal and occipital lobes, independent of changes in systemic blood pressure. Most of vasodilative responses to low intensity stimuli (2 times threshold intensity: 2T) were abolished by atropine (a muscarinic cholinergic blocker), whereas responses to higher intensity stimuli (3T) were abolished by atropine and mecamylamine (a nicotinic cholinergic blocker). Blood flow changes were largest when the tip of the electrode was located within the area containing cholinergic neurons shown by choline acetyltransferase-immunocytochemistry. These results suggest that cholinergic projections from basal forebrain neurons in mice cause vasodilation in the ipsilateral cerebral cortex by a combination of muscarinic and nicotinic mechanisms, as previously found in rats and cats.