Responses of adrenal sympathetic nerve activity and catecholamine secretion to cutaneous stimulation in anesthetized rats

Reflex effects of cutaneous mechanical stimulation on adrenal sympathetic efferent nerve activity and secretion rates of the adrenal medullary hormones (epinephrine and norepinephrine) were studied in anesthetized rats. Noxious pinching stimulation of the lower chest or hindpaw skin for 3 min produced proportional reflex increases in both the nerve activity and secretion rates of epinephrine and norepinephrine from the adrenal medulla in animals with an intact central nervous system. However, lower chest stimulation elicited a longer lasting response than hindpaw stimulation, 7-17 min vs 1 min after cessation of the stimulation, respectively. After spinal transection at the C1-2 level, only lower chest stimulation was capable of producing a reflex response, lasting 1 min after cessation of the stimulation. Contrary to the responses elicited by pinching, non-noxious brushing stimulation of the lower chest or hindlimb skin for 3 min in animals with an intact central nervous system produced proportional reflex decreases in nerve activity and epinephrine and norepinephrine secretion rates during the stimulation period only. Some slight increases in both nerve activity and secretion rates, lasting several minutes, followed cessation of the stimulation. However, in spinalized animals, non-noxious lower chest or hindlimb stimulation produced opposite effects, increasing both the nerve activity and secretion rates of epinephrine and norepinephrine. In spinalized animals lower chest brushing stimulation elicited a much stronger response than hindlimb brushing stimulation. It was concluded that; (1) the secretion of adrenal medullary hormones can be controlled reflexly by mechanical cutaneous stimulation through the central nervous system via adrenal sympathetic efferent nerves; (2) the excitatory effect of the cutaneo-adrenal medullary reflexes was independent of noxious or non-noxious stimulation at the spinal level, whereas in rats with an intact central nervous system the effect was either excitatory or inhibitory in response to noxious or non-noxious stimulation, respectively; (3) there is a marked segmental organization of this reflex at the spinal level which is modified into a generalized response through supraspinal central structures.     

Somatic afferent regulation of plasma corticosterone in anesthetized rats.

Effects of cutaneous stimulation on plasma corticosterone were examined in adult male Wistar rats anesthetized with pentobarbital. Under the resting condition, plasma corticosterone measured every 15 min between 1430 and 1630 h revealed no significant circadian fluctuations. Nociceptive mechanical stimulation of bilateral hindpaws by pinching for 10 min significantly increased plasma corticosterone for the following 1 h, whereas innocuous mechanical stimulation of bilateral hindlimbs by brushing for 10 min produced no significant change in plasma corticosterone. These results indicate that somatic sensory information from skin can influence secretion of corticosterone from the adrenal cortex after emotional factors are eliminated by anesthetizing the subjects.     

Somatosensory regulation of regional hippocampal blood flow in anesthetized rats.

The effect of noxious or non-noxious mechanical stimulation of various cutaneous areas on cerebral blood flow in hippocampus was examined with laser Doppler flowmetry in urethane-anesthetized artificially-ventilated rats. Noxious mechanical stimulation (pinching) of the skin on the face, forepaw, chest, or hindpaw for 20s increased regional hippocampal blood flow (Hpc-BF) and systemic blood pressure, but non-noxious mechanical stimulation (brushing) had no such effect. After the spinal cord was transected at T1 level a forepaw pinch caused no change in blood pressure but still increased Hpc-BF. This suggests that cutaneous noxious stimulation can induce pressor-independent increases in Hpc-BF. The increase in Hpc-BF induced by a forepaw pinch in T1-transected rats was partially reduced by intravenous administration of mecamylamine (2 mg/kg), a nicotinic cholinergic receptor antagonist. Atropine (0.5 mg/kg), a muscarinic cholinergic antagonist was ineffective. These data indicate that the cholinergic vasodilative system is involved in the somatically-induced increase in Hpc-BF via activation of the nicotinic cholinergic receptors.     

Somatic afferent regulation of plasma luteinizing hormone and testosterone in anesthetized rats.

The effects of cutaneous stimulation on plasma luteinizing hormone (LH) and testosterone were examined in adult male Wistar rats anesthetized with pentobarbital. Under the resting condition without somatic sensory stimulation, plasma LH and testosterone measured every 30 min between 1400 and 1730 h revealed no significant fluctuations. Nociceptive mechanical stimulation of bilateral hindpaws by pinching for 10 min significantly increased plasma LH and testosterone. Plasma LH was increased 30 and 60 min after stimulation, while plasma testosterone was increased 60-150 min after stimulation. However, innocuous mechanical stimulation of bilateral hindlimbs by brushing for 10 min did not significantly change either plasma LH or testosterone. These results indicate that, when emotional factors were eliminated by anesthetization, cutaneous nociceptive information led to the increased secretion of LH from the anterior pituitary, resulting in an increase in testosterone secretion from the testes into the plasma.     

Tactile skin stimulation increases dopamine release in the nucleus accumbens in rats

We investigated the effect of mild (non-noxious) tactile stimulation (stroking) of skin on dopamine (DA) release in the nucleus accumbens (NAc) of rats. A coaxial microdialysis probe was stereotaxically implanted in the NAc and perfused with modified Ringer’s solution. Dialysate output from consecutive 5-min periods was injected into a high-performance liquid chromatograph and DA was measured using an electrochemical detector. Bilateral tactile stimulation of the back for 5 min significantly increased DA release in conscious and anesthetized animals. Increased DA release was observed by stimulation of the contralateral, but not ipsilateral, back. DA secretion was also increased with stimulation of the forelimb, hindlimb, and abdomen. These effects were abolished after lesioning the ventral tegmental area (VTA). In contrast, noxious stimulation (pinching) of these areas had no effect on DA secretion. In conclusion, innocuous mechanical stimulation of the skin increases DA release in the contralateral NAc via the VTA.     

Luteinizing hormone releasing factor in rat hypophysial portal blood collected during electrical stimulation of the hypothalamus

1. Ovulation was induced in Nembutal-blocked pro-oestrous rats by electrical stimulation of the hypothalamus.2. The same type of electrical stimulation was applied during the collection of hypophysial portal blood.3. Pooled hypophysial portal plasma from donors in pro-oestrus, oestrus and met-oestrus was assayed for ovarian ascorbic acid depleting (OAAD) activity.4. Electrical stimulation of the hypothalamus increased the OAAD activity, believed to be due to luteinizing hormone releasing factor (LRF), in pro-oestrus and met-oestrus, but not in oestrus.5. It is concluded that the hypothalamic nerve fibres responsible for releasing LRF into the hypophysial portal vessels are depleted of their store of this releasing factor, or are refractory to electrical stimulation, during oestrus.     

Convergence of noxious and non-noxious cutaneous afferents and baroreceptor afferents onto single adrenal sympathetic neurons in anesthetized rats

Single units of the adrenal sympathetic nerve (n = 46) were dissected and characterized with respect to tonic discharge and response to cutaneous and baroreceptor stimulation. The frequency of tonic discharge averaged 1.6 Hz and cardiovascular rhythmic modulation was observed in 53% of the units. The stimuli employed in the present study included phenylephrine-induced increases in blood pressure and pinching or brushing of lower chest skin. Mean unit activity increased 27% on lower chest pinching stimulation, decreased 12% on lower chest brushing stimulation and decreased 62% on phenylephrine-induced baroreceptor stimulation. Although there was a tendency for units with higher tonic firing frequency to have a greater response to stimulation, this relationship was not significant for pinching or brushing of lower chest skin. The close correlation between tonic activity and response to phenylephrine was explicable on the basis of a near total depression of many units, which resulted in a larger decrease in firing frequency for units with initially high spontaneous discharge rates. As might be expected, units with cardiovascular rhythmicity manifested greater responses to baroreceptor activation. This correlation was independent of tonic rate of discharge since rhythmic and non-rhythmic units did not significantly differ in tonic activity. While a majority of units responded in a typical fashion to all three stimuli (i.e. with increases to pinching and decreases to brushing and phenylephrine administration), there was little correlation between the response magnitude of individual units to any two of the stimuli employed. We conclude, therefore, that most adrenal sympathetic units receive convergent reflex input from cutaneous noxious and non-noxious afferents as well as from baroreceptor afferents, although for any individual unit the quantitative significance of each input varies.     

Non-noxious skin stimulation activates the nucleus basalis of Meynert and promotes NGF secretion in the parietal cortex via nicotinic ACh receptors

The effects of non-noxious skin stimulation on nerve growth factor (NGF) secretion in the parietal cortex were examined in anesthetized rats. Innocuous skin stimulation was delivered to the left hindlimb with a soft-hair brush. Extracellular NGF in the right parietal cortex was collected by microdialysis methods using a protein-permeable probe and was measured using an enzyme-linked immune-sorbent assay. Brushing produced a significant increase in extracellular NGF levels. This NGF response was not observed in rats pretreated with a nicotinic ACh receptor (nAChR) antagonist mecamylamine. We further examined whether brushing could activate the basal forebrain nucleus (nucleus basalis of Meynert, NBM), which is the main source of cholinergic fibers in the cerebral cortex, by means of functional MRI. The blood oxygen level-dependent signal in the right NBM was significantly higher during brushing compared to baseline. The results suggest that non-noxious skin stimulation activates NBM and promotes NGF secretion in the parietal cortex via nAChRs.     

Reflex changes in heart rate after mechanical and thermal stimulation of the skin at various segmental levels in cats

Heart rate can be changed by cutaneous stimulation. In anesthetized cats with the central nervous system intact, a reflex increase in heart rate was elicited after natural stimuli such as pinching (noxious mechanical stimulation), rubbing (non-noxious mechanical stimulation), warming and cooling (thermal stimuli) were applied to the skin of the neck, chest, abdomen or perineum. It was shown that this cutaneo-cardiac acceleration reflex was produced mainly by a reflex increase in the discharges of the cardiac sympathetic efferent nerves and partially by a reflex decrease in the discharges of the cardiac vagal efferent nerves. On the other hand, in spinal cats, only stimulation of the chest and abdominal skin produced a reflex increase in heart rate. A possible explanation of this difference between central nervous system intact and spinal cats is that a spinal, segmentally organized component of the cutaneo-cardiac acceleration reflex is dominated by a supraspinal, diffusely distributed component in central nervous system intact cats.     

Reflex responses evoked in the adrenal sympathetic nerve to electrical stimulation of somatic afferent nerves in the rat

The present study was initiated to determine the role of somatic A (myelinated) and C (unmyelinated) afferent fibers in both responses of increases and decreases in adrenal sympathetic nerve activities during repetitive mechanical pinching and brushing stimulations of the skin in anesthetized rats with central nervous system (CNS) intact. Accordingly, changes in adrenal sympathetic nerve activity resulting from repetitive and single shock electrical stimulation of various spinal afferent nerves, especially the 13th thoracic (Th13) spinal nerve and the sural nerve, were examined in urethane/chloralose-anesthetized rats. Repetitive electrical stimulation of A afferent fibers in Th13 spinal or sural nerve decreased the adrenal nerve activity similarly as brushing stimulation of skin of the lower chest or hindlimb did, while repetitive stimulation of A plus C afferent fibers of those nerves increased the adrenal nerve activity as pinching stimulation of those skins did. Single shock stimulation of spinal afferent nerves evoked various reflex components in the adrenal nerve: an initial depression of spontaneous activity (the early depression); the following reflex discharge due to activation of A afferent fibers (the A-reflex); a subsequent reflex discharge due to activation of C afferent fibers (the C-reflex); and following post-excitatory depressions. These reflexes seem to be mediated mainly via supraspinal pathways since they were abolished by spinal transection at the C1-2 level. Although the supraspinal A- and C-reflexes could be elicited from stimulation of a wide variety of spinal segmental afferent levels, the early depression was more prominent when afferents at spinal segments closer to the level of adrenal nerve outflow were excited. It is suggested that the decreased responses of the adrenal nerve during repetitive electrical stimulation of A afferent nerve fibers are attributable to summation of both the early depression and post-excitatory depression evoked by single shock stimulation, while the increased responses during repetitive stimulation of A plus C afferent fibers are attributable to summation of the C-reflex after single shock stimulation. In spinalized rats, repetitive stimulation of Th13 always increased the adrenal nerve activities regardless of whether A fibers alone or A plus C fibers were stimulated, just as brushing and pinching of the lower chest skin always increased them. The increased responses in spinal animals seem to be related to the fact that single electrical stimuli of Th13 produced A- and C-reflexes of spinal origin without clear depressions.     

The demonstration of luteinizing hormone releasing factor in hypophysial portal blood of pro-oestrous and hypophysectomized rats

1. A method for the routine collection of hypophysial portal blood from rats under direct vision has been described.2. This method has been used to obtain portal blood from adult female rats in pro-oestrus and adult female rats which had been hypophysectomized at least 14 days before collection.3. The pooled plasma from this blood was assayed for activity, resembling that of luteinizing hormone releasing factor (LRF), by the ovarian ascorbic acid depletion method.4. The portal plasma from both groups of donor animals exhibited significant activity when compared to peripheral plasma controls.5. It is concluded that the ovarian ascorbic acid depleting activity of portal plasma was due to LRF.6. The possibility that some of the activity exhibited by portal plasma from pro-oestrous rats was due to contamination of the samples by LH containing backflow blood from pituitary sinusoids is discussed.7. The possibility that the ovarian ascorbic acid depleting activity of the hypophysial portal plasma was due to either vasopressin or a nonspecific factor has been excluded.     

Responses of dorsal spinal cord blood flow to noxious mechanical stimulation of the skin in anesthetized rats

In urethane-anesthetized, artificially ventilated rats, alterations in dorsal spinal cord blood flow (SCBF) at the L4-6 level were measured with laser Doppler flowmetry in response to noxious mechanical cutaneous stimulation (pinching) of either a forepaw or a hindpaw. The stimulation was delivered ipsilaterally or contralaterally to the site of blood flow measurement. Pinching of the forepaw or the hindpaw on either side increased mean arterial pressure (MAP) to the same degree. However, the SCBF response to pinching of the ipsilateral hindpaw was significantly greater than that to other stimulations. These responses were not influenced by denervation of the baroreceptors. The responses of SCBF to pinching of the ipsilateral hindpaw persisted both after treatment with phenoxybenzamine and after spinalization at the C1-2 level, whereas the responses to pinching at other sites disappeared. The responses of MAP to stimulation at all four sites became negligible after treatment with phenoxybenzamine and after spinalization at the C1-2 level. These results indicate that noxious mechanical stimulation of the skin produces increases in SCBF via two mechanisms: one is via an elevation of systemic arterial pressure; the other is via a localized spinal mechanism evoked by ipsilateral, segmental inputs.     

The luteinizing hormone releasing activity of extracts of blood from the hypophysial portal vessels of rats

1. A method of acid ethanol extraction and gel filtration was used to obtain a `luteinizing hormone (LH)-free' fraction of blood collected from the cut pituitary stalk of rats (termed hypophysial portal blood).

2. The `LH-free' fraction of hypophysial portal plasma from hypophysectomized and from ovariectomized rats caused a greater depletion of ovarian ascorbic acid in immature rats, pretreated with gonadotrophins, than a similar fraction of systemic plasma obtained from the same donor animals.

3. The `LH-free' fraction of hypophysial portal plasma from ovariectomized rats evoked a rise in the level of LH in the systemic plasma of ovariectomized, oestrogen and progesterone treated, rats. This fraction also caused ovulation in rabbits when infused directly into the anterior pituitary gland of these animals. The activity of the `LH-free' fraction of systemic plasma was considerably less than that of portal plasma in either of these assay systems.

4. The results of these experiments suggest the presence of a factor in the `LH-free' fraction of hypophysial portal plasma which is capable of causing the release of luteinizing hormone from the anterior pituitary gland. The molecular weight of this factor, as assessed by its behaviour on filtration through `Sephadex G-25', is probably less than 5000.

5. The LRF activity of the `LH-free' fraction of hypophysial portal plasma obtained from rats at various phases of the oestrous cycle was measured by the ovarian ascorbic acid depletion method. There appears to be a decrease in the level of activity at oestrus. However, a rise in LRF activity, which was expected to occur at the `critical period' of prooestrus, was not evident. The significance of these findings is discussed.

     

A quantitative study of the insulin release induced by vagal stimulation in anesthetized cats.

Insulin was released by vagal stimulation in anesthetized and eviscerated cats. The plasma insulin concentration and blood flow in the portal vein were determined concomitantly and the insulin output was calculated. Stimulation of either the right or the left cervical vagus released the same amount of insulin, whereas bilateral stimulation released twice as much. Following a stimulation that depleted the "vagally-releasable pool", a recovery period of 15--20 min was needed before the same maximal output could be obtained again. With shorter interstimulatory periods the amounts of insulin released were reduced. When less than 2--3 00 impulses were applied during a stimulation period, the amount of insulin released per impulse was constant. Atropine (0.2--2 mg/kg) did not reduce the vagally-induced insulin release.     

A quantitative study of the insulin release induced by vagal stimulation in anesthetized cats

Insulin was released by vagal stimulation in anesthetized and eviscerated cats. The plasma insulin concentration and blood flow in the portal vein were determined concomitantly and the insulin output was calculated. Stimulation of either the right or the left cervical vagus released the same amount of insulin, whereas bilateral stimulation released twice as much. Following a stimulation that depleted the “vagally-releasable pool”, a recovery period of 15–20 min was needed before the same maximal output could be obtained again. With shorter interstimulatory periods the amounts of insulin released were reduced. When less than 2–3 000 impulses were applied during a stimulation period, the amount of insulin released per impulse was constant. Atropine (0.2–2 mg/kg) did not reduce the vagally-induced insulin release.     

The effects of SDZ NKT 343, a potent NK1 receptor antagonist, on cutaneous responses of primate spinothalamic tract neurones sensitized by intradermal capsaicin injection

Substance P, acting through neurokinin I receptors, is involved in the processing of nociceptive information in the spinal cord. Sensitization of spinothalamic tract neurons occurs to low-intensity stimuli following capsaicin injection. The current study tested the effects of the novel neurokinin 1 receptor antagonist, SDZ NKT 343, on the sensitization of spinothalamic tract cells by capsaicin in monkeys. Spinothalamic tract cells from the lumbar enlargement with receptive fields in the hindpaw were isolated and recorded before and after intradermal injection of capsaicin. The background activity and responses to brushing, pressing and pinching the skin were assessed. Thirty minutes after capsaicin injection there was an increase in background activity and responses to brush and pressure applied to the receptive field. Infusion of SDZ NKT 343 (for 30–45 min) significantly reversed the increased response to brushing without affecting the increased background activity or the increased response to pressure. Thus, blockade of neurokinin 1 receptors reduces the sensitized responses to innocuous mechanical stimuli but not to noxious mechanical stimuli. Received: 2 March 1998 / Accepted: 24 April 1998     

The effects of hypophysial portal plasma on the content of immunoreactive growth hormone in the anterior pituitary

1. Blood plasma obtained from the hypophysial portal vessels of rats has been tested in assay animals for effects on the content of growth hormone in the anterior pituitary.2. Growth hormone was estimated by radioimmuno-assay at 15 min, 30 min, 1 hr and 2 hr after intracarotid or intravenous infusion of portal plasma. There was a significant increase in growth hormone content at 15 min after infusion, but not at 30 min, 1 hr and 2 hr.3. Insulin, cold stress, haemorrhage and treatment with growth hormone had no effect on growth hormone content of the pituitary under the conditions of application of these stimuli.4. Growth hormone was measured in plasma obtained from the pituitary of control and insulin-treated animals. In control animals the amount of growth hormone in this plasma was higher than in base line samples and in the insulin-treated animals it was lower than in base line samples. A calculation based on the growth hormone content of blood obtained directly from the pituitary indicates a daily rate of synthesis and release of about 10 mug.5. The hypothesis that portal plasma stimulated the synthesis of immuno-assayable growth hormone by the anterior pituitary is considered.6. Discrepancies between our results using immuno-assay and those of others using bio-assay are discussed.     

Responses of primate SI cortical neurons to noxious stimuli

Recordings were made from single SI cortical neurons in the anesthetized macaque monkey. Each isolated cortical neuron was tested for responses to a standard series of mechanical stimuli. The stimuli included brushing the skin, pressure, and pinch. The majority of cortical neurons responded with the greatest discharge frequency to brushing the receptive field, but neurons were found in areas 3b and 1 that responded maximally to pinching the receptive field. A total of 68 cortical nociceptive neurons were examined in 10 animals. Cortical neurons that responded maximally to pinching the skin were also tested for responses to graded noxious heat pulses (from 35 to 43, 45, 47, and 50 degrees C). If the neuron failed to respond or only responded to 50 degrees C, the receptive field was also heated to temperatures of 53 and 55 degrees C. Fifty-six of the total population of nociceptive neurons were tested for responses to the complete series of noxious heat pulses: 46 (80%) exhibited a progressive increase in the discharge frequency as a function of stimulus intensity, and the spontaneous activity of two (4%) was inhibited. One population of cortical nociceptive neurons possessed restricted, contralateral receptive fields. These cells encoded the intensity of noxious mechanical and thermal stimulation. Sensitization of primary afferent nociceptors was reflected in the responses of SI cortical nociceptive neurons when the ascending series of noxious thermal stimulation was repeated. The population of cortical nociceptive neurons with restricted receptive fields exhibited no adaptation in the response during noxious heat pulses of 47 and 50 degrees C. At higher temperatures the response often continued to increase during the stimulus. The other population of cortical nociceptive neurons was found to have restricted, low-threshold receptive fields on the contralateral hindlimb and, in addition, could be activated only by intense pinching or noxious thermal stimuli delivered on any portion of the body. The stimulus-response functions obtained from noxious thermal stimulation of the contralateral hindlimb were not different from cortical nociceptive neurons with small receptive fields. However, nociceptive neurons with large receptive fields exhibited a consistent adaptation during a noxious heat pulse of 47 and 50 degrees C. Based on the response characteristics of these two populations of cortical nociceptive neurons, we conclude that neurons with small receptive fields possess the ability to provide information about the localization, the intensity, and the temporal attributes of a noxious stimulus.4+.     

Modulation of noxious and non-noxious spinal mechanical transmission from the rostral medial medulla in the rat

Modulatory influences on spinal mechanical transmission from the rostral medial medulla (RMM) were studied. Noxious stimulation, produced by von Frey-like monofilaments, and non-noxious stimulation, produced by a soft brush, was applied to the glabrous skin of the hind foot. At 28 sites in RMM, electrical stimulation facilitated responses to noxious mechanical stimulation at low intensities (5-25 microA) and inhibited responses of the same neurons at greater intensities (50-100 microA) of stimulation. At 24 and 9 other sites in RMM, stimulation at all intensities only inhibited or only facilitated, respectively, responses to noxious mechanical stimulation of the hind foot. Stimulus-response functions to mechanical stimulation were shifted leftward by low intensities and decreased by high intensities of stimulation. Inhibitory influences were found to descend in the dorsolateral funiculi; facilitatory effects were contained in the ventral spinal cord. Descending modulation of non-noxious brush stimulation revealed biphasic facilitatory-inhibitory effects (9 sites in RMM), only inhibitory effects (14 sites) and only facilitatory effects (8 sites). The effects of electrical stimulation were replicated by intra-RMM administration of glutamate; a low concentration (0.25 nmol) facilitated and a greater concentration (2.5 nmol) inhibited spinal mechanical transmission, providing evidence that cells in RMM are sufficient to engage descending influences. Descending modulatory effects were specific for the site of stimulation, not for the spinal neuron, because modulation of the same neuron was different from different sites in RMM. These results show that spinal mechanical transmission, both noxious and non-noxious, is subject to descending influences, including facilitatory influences that may contribute to exaggerated responses to peripheral stimuli in some chronic pain states.     

The effect of clonidine on adrenal sympathetic nerve responses to mechanical, noxious and innocuous stimulation of the skin in rats

The effect of clonidine on the reflex responses of the adrenal sympathetic nerve to mechanical stimulation of the lower chest skin was studied in anesthetized CNS intact and spinalized rats. Clonidine (3-120 micrograms/kg i.v.) administration resulted in a dose-related decrease in spontaneous adrenal efferent nerve activity in both CNS intact and spinalized rats. In CNS intact rats, noxious pinching caused a reflex increase in adrenal nerve activity while innocuous brushing caused a reflex decrease. In both cases, clonidine reduced the reflex response in a dose-dependent manner. In spinalized rats, both noxious and innocuous stimulation caused reflex increases in adrenal nerve activity, and clonidine produced a similar dose-dependent decrease in these responses as seen in CNS intact animals. The similar dose-response relationships for adrenal nerve tone or reflex response in CNS intact and spinalized animals suggest, although not conclusively, that at least some of clonidine's effect in CNS intact animals may be mediated at the spinal level.