The effects of repetitive compression on nerve conduction and blood flow in the rabbit sciatic nerve.

The objective of this study was to clarify the effect of repetitive compression on nerve physiology in an experimental rabbit model. We defined 80 mmHg as a compression force which caused temporary disturbance of nerve conduction and blood flow with a brief compression. The following compressions were applied for 30 minutes to rabbit sciatic nerves: continuous compression, low frequency release compression (1 second of release time every 30 seconds) and high frequency release compression (1 second of release time every 10 seconds). Compound nerve action potentials and nerve blood flow were evaluated from the start of compression until 30 minutes after release. Endoneurial microvascular permeability was evaluated with Evans Blue albumin. The repetitive compression groups showed delay in recovery of compound nerve action potentials and blood flow after release, with endoneurial oedema. These findings suggest that repetitive compression may increase the risk of breakdown of the blood nerve barrier.

Leptin injection into white adipose tissue elevates renal sympathetic nerve activity dose-dependently through the afferent nerves pathway in rats.

Recent studies suggested that leptin in white adipose tissue (WAT) affected the sympathetic out flow to several tissues. We examined whether elevations of renal sympathetic nerve activity (RSNA) and blood pressure (BP) could be observed by leptin injection into WAT in rats. Injections of leptin (10 and 100 ng/ml per kg) into WAT evoked the activation of RSNA dose-dependently. Circulating sympathetic nerve activators, such as leptin, insulin, glucose and lactate, were unchanged by any doses of leptin. In addition, BP was not affected by leptin injections during a 90 min experimental period. These data suggested that leptin activated the afferent nerves through the sensors in WAT, resulting in elevation of RSNA.

Different cardiovascular neuron groups in the ventral reticular formation of the rostral medulla in rabbits: single neurone studies.

To examine whether the cardiovascular neurons of the ventral medulla consist of functionally different kinds of neurons, single neuronal activity of the ventral medulla, activity of the renal sympathetic nerves (RSNA), blood flow of the ear (EarBF) and arterial pressure (AP) were recorded in urethane-anesthetized, vagotomized and immobilized rabbits during electrical stimulation of the aortic nerve (AN, baroreceptor afferent fibers) and electrical stimulation of the dorsomedial hypothalamus (DMH) that reduced EarBF but less affected on AP and RSNA. The dorsolateral funiculus of the second cervical cord was stimulated to evoke antidromic spikes of medullary neurons. Two kinds of reticulo-spinal neurons were identified. Activities of one kind of neurons were facilitated by stimulation of DMH (latency 48.6+/-27.6 ms, n=11) but they did not respond to stimulation of the AN. Therefore, it was presumed that these neurons controlled vasomotion of the ear through the vasoconstrictor neurons in the spinal cord but did not participate in regulation of systemic AP. Activities of the other neurons were inhibited by stimulation of the AN (latency 47.8+/-8 4 ms, n=16) but they did not respond to the DMH stimulation. These neurons were identical to those reported previously as the RVLM neurons, and they contributed to regulate systemic AP but might not participate in control of cutaneous vascular movement. The former neurons were located medially to the latter in the reticular formation of the rostral ventral medulla. These results provided evidence at the single neuronal level that the cardiovascular neurons in the ventral medulla were consisted of functionally different sympatho-excitatory neurons and they were located at the different sites in the rostral ventral medulla.

Functional significance of the 10 Hz rhythmic discharges in sympathetic nerves.

1. By measuring renal vascular conductance in anaesthetized and immobilized rabbits, the functional significance of the 10 Hz rhythmic discharges in the sympathetic nerves was assessed by electrical stimulation of the renal nerve. This stimulation mimicked the intermittently occurring 10 Hz rhythmic discharges. 2. Comparison of high-frequency (10-20 Hz) intermittent electrical stimulation with continuous low-frequency (5 Hz) stimulation showed that the intermittent high-frequency stimulation evoked faster (smaller time constant) and larger responses of the renal vascular conductance if the total number of stimulus pulses was the same. 3. Therefore, the intermittently occurring 10 Hz rhythmic discharges in the sympathetic nerve produces faster and larger effects on peripheral organs than effects produced by continuous discharges if the number of spikes is equal.

Functionally different neurons are organized topographically in the rostral ventrolateral medulla of rabbits.

To examine whether the sympatho-excitatory neurons in the rostral ventrolateral medulla (RVLM) were divided into subgroups, gamma-amino-n-butyric acid (GABA) was injected into multiple sites of the medulla while simultaneous recordings of blood flows were made from the renal artery with an ultrasonic pulsed Doppler flowmeter and from the ear skin and muscles of fore- and hind-limbs with laser Doppler flow meters in urethane-anesthetized, vagotomized and immobilized rabbits. The magnitude of the responses of mean systemic arterial pressure (MAP), heart rate (HR) and conductance of each vascular bed, calculated by its blood flow and MAP, were represented as a contour map of the ventral surface of the medulla. Microinjection of GABA (50 mM, 9-27 nl) into the RVLM produced a decrease in MAP (-27 +/- 10 mmHg) and HR (-14 +/- 7 beat min-1) and an increase in the vascular conductance of the ear skin (ESC; 33 +/- 25 microliters min-1 100 g-1 (mmHg)-1), the fore-limb muscle (FLMC; 93 +/- 84 microliters min-1 100 g-1 (mmHg)-1), the hind-limb muscle (HLMC; 18 +/- 7 microliters min-1 100 g-1 (mmHg)-1) and the kidney (KC; 49 +/- 25 microliters min-1 (mmHg)-1). Comparing the sites into which the injection of GABA evoked the maximal response of MAP (the 'center' of the RVLM), the maximal responses of HR, ESC and KC were obtained from caudal, caudo-medial and slightly rostral sites, respectively. In more than half of cases, the maximal responses of FLMC and HLMC were obtained from the 'center' of the RVLM. These results indicated that the functionally different sympatho-excitatory reticulospinal neurons are located at different sites in the RVLM, although they considerably intermingle with each other.

The spinally mediated 10-Hz rhythm in the sympathetic nerve activity of cats.

To examine the origin of the so-called '10-Hz rhythm' in the sympathetic nerves, the mass discharges of the white ramus of the third thoracic segment (T3WR) and the inferior cardiac nerve (ICN) and the activities of single postganglionic neurons in the stellate ganglion were recorded in spinal cats. During the chemical or electrical stimulation of the spinal cord, the time of peak of discharges in the sympathetic nerves was analyzed. Both intrathecal administration of N-methyl-D-aspartic acid (NMDA; 3-10 mM) and continuous high frequency (80-200 Hz) electrical stimulation of the dorsolateral funiculus at the second cervical level increased activity of the sympathetic nerves in a similar fashion. In these conditions, modes of the inter-peak interval histograms (IPIH) were about 100 ms (range; 90-130 ms), the inverse of about 10 Hz, but no correlation was observed in autocorrelograms of these peaks of discharges. Therefore, this 100-ms interval activity might have some significance for the 10-Hz rhythm. In order to make this point clear, we stimulated the dorsolateral funiculus with intermittent trains of electrical pulses (0.2-ms duration, 10-35 pulses of 80-200 Hz frequency, in every 300-800 ms). While intermittent trains of pulses were applied, multiple peaks of discharges were evoked in the sympathetic nerves. IPIHs of the nerves were multimodal. The first mode (shortest interval) was about 100 ms. The first mode depended on none of the stimulus parameters but the probability of the about 100-ms interval activity depended on the interval of trains of pulses and the stimulus intensity. With this intermittent stimulation, the autocorrelogram of the peaks revealed the 100-ms interval rhythm. To confirm that the peak of discharges in the ICN was composed of synchronized spikes of postganglionic fibers, single neuronal activities of postganglionic neurons were recorded during the intermittent stimulation. Inter-spike interval histograms showed almost same profile as the IPIHs of the ICN. These results can be explained if the following two assumptions are valid; (i) There are mechanisms that limit minimum firing interval of most preganglionic neurons to about 100 ms. (ii) Simultaneously a interneuron in the spinal cord resets the spike generation of multiple preganglionic neurons. Similarity of the spike activities of the sympatho-excitatory reticulospinal neurons to the intermittent stimulation can explain the 10-Hz rhythm in the peripheral sympathetic nerves in intact spinal cord animals. It is not necessary to postulate the specific 10-Hz rhythm generator in the brain stem for the sympathetic nervous system.

The 10-Hz rhythm in the sympathetic nerve activity of cats, rats and rabbits.

Since the 10-Hz rhythmic activity in the sympathetic nerves was reported only in cats, we examined whether the activity of the same frequency could be observed in rats and rabbits as well as in the cats. Histograms of inter-burst-peak intervals of discharges of the renal nerves revealed that the 100 ms interval activity, the reverse of the 10-Hz, was observed in all the three mammals, of which the baroreceptor afferents were intact or inactivated. Further, the same frequency activity could be evoked by intermittent electrical stimulation of the dorsolateral funiculus of the cervical cord in spinal animals. It was suggested that the 10-Hz rhythmic activity in the sympathetic nerves was a common phenomenon throughout mammals and this activity was produced in the spinal cord. The physiological significance of the 10-Hz activity in the sympathetic nerves was discussed.

Quantitative analysis of central terminal projections of visceral and somatic unmyelinated (C) primary afferent fibers in the guinea pig.

In guinea pigs, intracellular labeling of the dorsal root ganglion (DRG) cells with Phaseolus vulgaris-leucoagglutinin (PHA-L) was used to demonstrate the central projections of somatic and visceral afferent C-fibers. The terminations of the afferent fibers were analyzed qualitatively and quantitatively with the aid of camera lucida drawings. Terminal branches of C-fibers of both somatic and visceral origin were, in general, distributed in accord with the organization of the neuropil in lamina of the spinal cord. Terminal boutons arranged from longitudinally coursing fibers were distributed in lamina I, while boutons in lamina II were scattered in an apparent random fashion. The synaptic enlargements were counted in gray matter of the spinal dorsal horn and measured on each terminal branch of a fiber. All synaptic boutons (over one thousand) of somatic fibers were found in the superficial dorsal horn (laminae I and II). More than 60% of the synaptic enlargements of the visceral afferents also were localized superficially (lamina I and adjacent dorsal funiculus) while 10-20% of the visceral enlargements appeared in deeper layers of the spinal cord. Boutons of somatic C-fibers were larger than those of visceral origin. Quantitative data of the unmyelinated afferent fibers are discussed in the context of the sensory functions of myelinated afferent fibers.

Action of endothelin-1 on vasomotor neurons in rat rostral ventrolateral medulla.

Intracisternal administration of endothelin-1 (ET-1) elicits sympathetically mediated cardiovascular responses by acting on the ventral surface of the medulla oblongata (VSM) subjacent to the rostral ventrolateral medulla (RVLM). We examined, in urethane-anesthetized rats, whether intracisternal ET-1 affected activity of vasomotor neurons (VMNs) in the RVLM, by acting either directly on the VMNs or indirectly via the VSM. VMNs were identified electrophysiologically. Intracisternal administration of ET-1 altered activity of all the 13 VMNs tested. At a dose of 0.1 pmol, ET-1 invariably caused transient excitation in six VMNs examined, whereas at a dose of 1 pmol in separate experiments all the seven VMNs tested were inhibited with (n = 6) or without (n = 1) preceding excitation. Similarly, topical application of ET-1 (0.1-1 pmol) to the VSM caused inhibition with (n = 3) or without (n = 2) preceding excitation in all the five VMNs tested. Direct iontophoretic application of ET-1 to the VMNs caused excitation in four of seven VMNs examined but did not affect the other three neurons. These results support the view that intracisternally administered ET-1 alters activity of VMNs in the RVLM, by acting directly on neurons themselves and indirectly via the VSM.

Neurons in the caudal ventrolateral medulla mediate the somato-sympathetic inhibitory reflex response via GABA receptors in the rostral ventrolateral medulla.

In urethane-anesthetized rabbits, stimulation of the sural nerve, consisting of cutaneous afferents (A-fibers), evoked reflex responses consisting of an early small excitatory component followed by a prolonged inhibitory component in renal sympathetic nerve activity. Bilateral injections of GABA antagonist, bicuculline (4 nmol/site), into the rostral ventrolateral medulla (RVLM), where sympatho-excitatory reticulospinal neurons are located, attenuated the inhibitory component in a dose-dependent manner as well as the inhibition evoked by stimulation of the aortic nerve A-fibers (baroreceptor afferents). Bilateral injections of a neurotoxic agent, kainic acid (4 nmol/site, 3 sites/side), into the caudal ventrolateral medulla (CVLM), where sympatho-inhibitory neurons with axonal projection to the RVLM are located, diminished these sympatho-inhibitory responses. Therefore it is concluded that the sympatho-inhibition evoked by activation of somatic afferents was mediated by neurons in the CVLM and by GABA receptors in the RVLM, as was the sympatho-inhibition associated with the arterial baroreceptor reflex. Bilateral injections of kynurenic acid (4 nmol/site, 3 sites/side) into the CVLM did not affect the somato-sympathetic reflex response, but diminished the sympatho-inhibition produced by activation of the baroreceptor afferents. Sympatho-inhibitory neurons in the CVLM were activated by glutamate when baroreceptor afferents were activated, but another excitatory transmitter may participate in the somato-sympathetic reflex in the CVLM.

[Autonomic reflexes: focusing on the arterial baroreceptor-vasoconstrictor reflex].

The arterial baroreceptor-sympathetic vasoconstrictor reflex (baroreceptor reflex), among autonomic reflexes, is summarized. Arterial baroreceptor afferents terminate in the caudal part of the nucleus tractus solitarius. Excitation of the baroreceptor afferents activates neurons in the caudal ventrolateral medulla that send axonal projections to the rostral ventrolateral medulla. These neurons (CVLM neurons) then inhibit tonic activity of sympathoexcitatory reticulospinal neurons in the rostral ventrolateral medulla (RVLM neurons). Tonic activity of RVLM neurons are responsible for tonic activity of the peripheral vasoconstrictors. These two kinds of neurons are essential neuronal components for the baroreceptor reflex. Both the neurons may also participate in various vasoconstrictor reflexes, such as the somato-and viscero-sympathetic vasoconstrictor reflexes.

Endothelin-sensitive areas in the ventral surface of the rat medulla.

In urethane-anesthetized rats, subregions of the ventral surface of the medulla (VSM) in which endothelin (ET) caused cardiorespiratory effects were mapped by topically applying 1 pmol of ET-1. Two distinct subregions, termed the rostral and caudal ET-sensitive areas, were identified. The rostral area was also sensitive to L-glutamate and glycine. It extended between the caudal end of the trapezoid body and the rootlet of the XIIth nerve partly overlying the pyramidal tract. In this position ET-1 caused the type I response consisting of an initial increase (excitatory component) in arterial pressure (AP), renal sympathetic nerve activity (RSNA), heart rate (HR), phrenic nerve activity (PNA) and the number of bursts of PNA (burst rate) followed by a sustained decrease (inhibitory component) in them. The caudal ET-sensitive area was located near the rootlet of the XIIth nerve. In this position ET-1 caused the type II response consisting of a decrease in PNA and an increase in burst rate. Part of this area responded to nicotine but not to glutamate or glycine. ET-3 (10 pmol) applied to the two ET-sensitive areas produced responses similar to those elicited by ET-1. The dose-response relationship was investigated by delivering ETs to the rostral area. The excitatory component of most of the variables was elicited at a dose of 1 fmol of ET-1 or 1 pmol of ET-3, whereas the inhibitory component was produced at 10 fmol of ET-1 or 10 pmol of ET-3. These results suggest that subregions of the rat's VSM may participate in the central cardiorespiratory control by ET.

Neurons in the caudal ventrolateral medulla mediate the arterial baroreceptor reflex by inhibiting barosensitive reticulospinal neurons in the rostral ventrolateral medulla in rabbits.

Participation of the caudal ventrolateral medulla in the arterial baroreceptor reflex was examined in urethane-anesthetized, vagotomized and immobilized rabbits whose aortic nerve was cut bilaterally. The extent of the caudal ventrolateral medulla was mapped by decreases in the renal sympathetic nerve activity and arterial pressure following a local microinjection of a neuroexcitatory amino acid, sodium glutamate (0.075-1.5 nmol). It extended between the levels 1.3 mm rostral and 3.0 mm caudal to the obex. An injection of sodium glutamate into the caudal ventrolateral medulla also diminished spontaneous activity of barosensitive reticulospinal neurons in the rostral ventrolateral medulla. In the 'split medulla preparation' in which the medulla was split along the midsagittal plane to disrupt fiber connections associating both sides, a neurotoxic agent, kainic acid, was injected unilaterally into the rostral ventrolateral medulla. This treatment markedly attenuated responses of renal sympathetic nerve activity and arterial pressure induced by a sodium glutamate injection into the ipsilateral caudal ventrolateral medulla, whereas responses to an injection into the contralateral caudal ventrolateral medulla were totally preserved. In four separate experiments, three to five injections of kainic acid were made unilaterally to cover the whole extent of the caudal ventrolateral medulla. The sympathoinhibitory and depressor responses to stimulation of the ipsilateral aortic nerve were then totally abolished. Simultaneously, the cardiac cycle-related rhythmic fluctuation of renal sympathetic nerve activity, which represented activity of the carotid sinus baroreceptor reflex, was attenuated to the noise level. These results, together with our previous electrophysiological demonstration of barosensitive caudal ventrolateral medulla neurons with axonal projections to the rostral ventrolateral medulla, strongly support the hypothesis that neurons in the caudal ventrolateral medulla mediate the arterial baroreceptor-vasomotor reflex through inhibition of barosensitive reticulospinal neurons in the rostral ventrolateral medulla.

Cardiorespiratory effects of topical application of endothelin-1 to the ventral surface of the rat medulla.

In urethane-anesthetized and vagotomized rats, we examined the cardiorespiratory effects of a topical application of endothelin-1 (ET-1) to the ventral surface of the medulla (VSM) and surveyed subregions of the VSM influenced by these effects. ET-1 (0.1 fmol) delivered to the S area of the VSM via a needle (i.d. of approximately 100 microns) gently pressed on the VSM had no effect on mean arterial pressure (MAP), heart rate (HR), renal sympathetic nerve activity (RSNA), phrenic nerve activity (PNA), or the burst rate of PNA. However, a dose of 1 fmol of ET-1 induced transient but significant increases in MAP, HR, RSNA, and burst rate while at a dose of 10 fmol or more, PNA also increased and simultaneously longer-lasting decreases in MAP, RSNA, and PNA followed the initial increase. The subregion of the VSM in which ET-1 most prominently elicited these effects was the S and caudal part of the M area, where topical application of 50 nmol of L-glutamate caused cardiorespiratory changes. Additionally, there was a restricted region within the caudal VSM in which ET-1 caused a decrease in PNA with an increase in burst rate. These results support our hypothesis that the VSM is crucially involved in the cardiorespiratory changes induced by centrally administered ET-1.

Endothelin-1 modulates cardiorespiratory control by the central nervous system.

In urethane-anesthetized, vagotomized and immobilized rats under artificial ventilation, an intracisternal injection of 0.1 pmol of endothelin-1 resulted in immediate increases, lasting for 3-15 min, in arterial pressure, heart rate and renal sympathetic nerve activity. Phrenic nerve activity and the rate of its burst activity (burst rate) also increased initially but subsequently decreased for 5-20 min. At doses of 1 or 10 pmol, the initial increases (phase I) were followed by a period of decreases in all variables, that lasted for 20-80 min, below the pre-injection level (phase II). Phrenic nerve activity often disappeared completely. All the variables usually returned to, or often exceeded, pre-injection levels (phase III). However, arterial pressure sometimes remained below control for at least 2 h. Topical application of endothelin-1 to the ventral surface of the medulla produced the same pattern of changes as with intracisternal injection. This particular response pattern was not generated by local administration to any other brain sites examined. In conclusion, intracisternally administered endothelin-1 modulates cardiorespiratory control by the central nervous system. The effect on the central respiratory control was especially powerful. The ventral surface of the medulla appears to play a crucial role in this modulation.

Activity of barosensitive neurons in the caudal ventrolateral medulla that send axonal projections to the rostral ventrolateral medulla in rabbits.

In urethane-anesthetized rabbits, we successfully recorded unit activity of four neurons in the caudal ventrolateral medulla (CVLM) that were excited by orthodromic stimulation of the aortic nerve and by antidromic stimulation of the rostral ventrolateral medulla (RVLM). The sum of mean onset latency of excitation to stimulation of the aortic nerve (37.5 ms) and mean conduction time of antidromic spikes (10.5 ms) was close to the mean onset latency of inhibition of reticulospinal neurons in the RVLM to stimulation of the aortic nerve (47.1 ms) as previously reported by us. Three of 4 neurons received excitatory input from carotid sinus baroreceptors as well. Our results provide strong evidence for the hypothesis that neurons in the CVLM subserve the arterial baroreceptor-sympathetic vasomotor reflex.

Modulatory effects of rat endothelin on central cardiovascular control in rats.

To characterize the modulatory action of rat endothelin (endothelin-3 or ET-3) on the cardiovascular control by the central nervous system (CNS), ET-3 was injected into the cisterna magna of urethane-anesthetized and immobilized rats. An injection of 100 pmol of ET-3 caused immediate rises in arterial pressure (AP), renal nerve activity (RNA), and heart rate (HR). These variables subsequently decreased and, in 5-20 min, fell below the pre-injection level. Simultaneously, the arterial baroreceptor reflex was almost totally suppressed. Although RNA and HR subsequently returned to, or often exceeded, pre-injection levels in 20 to 60 min and reflex activity recovered, AP sometimes remained below control for at least 2 h. A similar pattern of changes was elicited in unanesthetized precollicular decerebrated rats. The responses to ET-3 were abolished by hexamethonium chloride, but were not conspicuously altered by arginine vasopressin antagonist or angiotensin II antagonist. The CNS sites responsible for ET-induced changes were subsequently searched. Topical application of ET-3 to the ventral surface of the medulla (VSM) caused the pattern of changes in AP, RNA, and HR similar to that following intracisternal injection. Microinjection of ET-3 into the nucl. tractus solitarius (NTS) increased AP and RNA, whereas intrathecal administration of it decreased them. We conclude that intracisternally administered ET-3 centrally modulates both tonic and reflex control of AP by the sympathetic nervous system and that the VSM appears to be primarily responsible for the modulation, although NTS and spinal cord may also be involved in it.

Modulatory effects of endothelin-1 on central cardiovascular control in rats.

In urethane-anesthetized and immobilized rats, modulatory effects of endothelin-1 (ET-1) on central cardiovascular control were examined. An injection of 0.1 pmol of ET-1 into the cisterna magna caused immediate increases in arterial pressure (AP), renal sympathetic nerve activity (RSNA), and heart rate (HR) that lasted for 5-45 min. At doses of 1 and 10 pmol, intracisternal ET-1 elicited initial increases (phase I) followed by decreases in these variables below the pre-injection level (phase II). At the dose of 1 or 10 pmol, the arterial baroreceptor reflex was suppressed during the latter part of phase I and during phase II. The three variables subsequently returned to, or often exceeded, pre-injection levels in 30 to 60 min and reflex activity recovered (phase III). However, AP often remained below control throughout the 2-h observation period. Essentially identical responses to intracisternal ET-1 were observed in unanesthetized precollicular decerebrated or urethane-anesthetized rats. Application of a piece of filter paper soaked with 1 pmol of ET-1 to the ventral surface of the medulla (VSM) caused the pattern of changes similar to the following intracisternal injection. A microinjection of 4 pmol of ET-1 into the nucleus tractus solitarius (NTS) caused a moderate increase in RSNA with a minute fall in AP. Intrathecal administration of ET-1 resulted in moderate changes in AP and RSNA at the dose as high as 100 pmol. We conclude that intracisternally administered ET-1 modulates tonic and reflex control of AP and sympathetic vasomotor activity and that the VSM appears to be involved critically in this modulation.