Modulation of neuronal activity in dorsal column nuclei by upper cervical spinal cord stimulation in rats.

Clinical human and animal studies show that upper cervical spinal cord stimulation (cSCS) has beneficial effects in treatment of some cerebral disorders, including those due to deficient cerebral circulation. However, the underlying mechanisms and neural pathways activated by cSCS using clinical parameters remain unclear. We have shown that a cSCS-induced increase in cerebral blood flow is mediated via rostral spinal dorsal column fibers implying that the dorsal column nuclei (DCN) are involved. The aim of this study was to examine how cSCS modulated neuronal activity of DCN. A spring-loaded unipolar ball electrode was placed on the left dorsal column at cervical (C2) spinal cord in pentobarbital anesthetized, ventilated and paralyzed male rats. Stimulation with frequencies of 1, 10, 20, 50 Hz (0.2 ms, 10 s) and an intensity of 90% of motor threshold was applied. Extracellular potentials of single neurons in DCN were recorded and examined for effects of cSCS. In total, 109 neurons in DCN were isolated and tested for effects of cSCS. Out of these, 56 neurons were recorded from the cuneate nucleus and 53 from the gracile nucleus. Mechanical somatic stimuli altered activity of 87/109 (83.2%) examined neurons. Of the neurons receiving somatic input, 62 were classified as low-threshold and 25 as wide dynamic range. The cSCS at 1 Hz changed the activity of 96/109 (88.1%) of the neurons. Neuronal responses to cSCS exhibited multiple patterns of excitation and/or inhibition: excitation (E, n=21), inhibition (I, n=19), E-I (n=37), I-E (n=8) and E-I-E (n=11). Furthermore, cSCS with high-frequency (50 Hz) altered the activity of 92.7% (51/55) of tested neurons, including 30 E, 24 I, and 2 I-E responses to cSCS. These data suggested that cSCS significantly modulates neuronal activity in DCN. These nuclei might serve as a neural relay for cSCS-induced effects on cerebral dysfunction and diseases.

Roles of dorsal column pathway and transient receptor potential vanilloid type 1 in augmentation of cerebral blood flow by upper cervical spinal cord stimulation in rats.

Clinical and basic studies have indicated that upper cervical spinal cord stimulation (cSCS) significantly increases cerebral blood flow (CBF), but the mechanisms are incompletely understood. This investigation was conducted to differentiate between stimulation of dorsal column fibers and upper cervical spinal cord cell bodies in cSCS-induced increases in CBF and decreases in cerebrovascular resistance (CVR). cSCS (50 Hz, 0.2 ms, 1 min) was applied on the left C1-C2 dorsal column of pentobarbital anesthetized, ventilated and paralyzed male rats. Laser Doppler flowmetry probes were placed bilaterally over the parietal cortex, and arterial pressure was monitored. cSCS at 30%, 60%, and 90% of motor threshold (MT) produced vasodilation bilaterally in cerebral cortices. Subsequently, cSCS was applied at 90% MT, and ipsilateral responses were recorded. Ibotenic acid (0.3 mg/ml, 0.1 ml) placed on dorsal surface of C1-C2 (n=7) to suppress cell body activity, did not affect cSCS-induced %DeltaCBF (42.5+/-8.1% vs. 36.8+/-7.1%, P>0.05) and %DeltaCVR (-19.4+/-4.2% vs. -15.2+/-5.6%, P>0.05). However, bilateral transection of the dorsal column at rostral C1 (n=8) abolished cSCS-induced changes in CBF and CVR. Also, rostral C1 transection (n=7) abolished cSCS-induced changes in CBF and CVR. Resinferatoxin (RTX), an ultrapotent transient receptor potential vanilloid type 1 (TRPV1) agonist, was used to inactivate TRPV1 containing nerve fibers/cell bodies. RTX (2 microg/ml, 0.1 ml) placed on the C1-C2 spinal cord (n=7) did not affect cSCS-induced %DeltaCBF (60.2+/-8.1% vs. 46.3+/-7.7%, P>0.05) and %DeltaCVR (-25.5+/-3.5% vs. -21.4+/-8.9%, P>0.05). However, i.v. RTX (2 microg/kg, n=9) decreased cSCS-induced %DeltaCBF from 65.0+/-9.5% to 27.4+/-7.2% (P<0.05) and %DeltaCVR from -28.0+/-7.6% to -14.8+/-4.2% (P<0.05). These results indicated that cSCS-increases in CBF and decreases in CVR occurred via rostral spinal dorsal column fibers and did not depend upon C1-C2 cell bodies. Also, our results suggested that cerebral but not spinal TRPV1 was involved in cSCS-induced cerebral vasodilation.

Chemical activation of C(1)-C(2) spinal neurons modulates activity of thoracic respiratory interneurons in rats.

Discharge patterns of thoracic dorsal horn neurons are influenced by chemical activation of cell bodies in cervical spinal segments C(1)-C(2). The present aim was to examine whether such activation would specifically affect thoracic respiratory interneurons (TRINs) of the deep dorsal horn and intermediate zone in pentobarbital sodium-anesthetized, paralyzed, artificially ventilated rats. We also characterized discharge patterns and pathways of TRIN activation in rats. A total of 77 cells were classified as TRINs by location, continued burst activity related to phrenic discharge when the respirator was stopped, and lack of antidromic response from selected pathways. A variety of respiration-phased discharge patterns was documented whose pathways were interrupted by ipsilateral C(1) transection. Glutamate pledgets (1 M, 1 min) on the dorsal surface of the spinal cord inhibited 22/49, excited 15/49, or excited/inhibited 3/49 tested cells. Incidence of responses did not depend on whether the phase of TRIN discharge was inspiratory, expiratory, or biphasic. Phrenic nerve activity was unaffected by chemical activation of C(1)-C(2) in this preparation. Besides supraspinal input, TRIN activity may be influenced by upper cervical modulatory pathways.

Afferent pathways for cardiac-somatic motor reflexes in rats.

The present study used a rat model in which algogenic chemicals were infused into the pericardial sac to evoke spasmlike contractions in paraspinal muscles. The following techniques were used to study the roles of sympathetic (SCA) and vagal cardiac afferents (VCA) in electromyographic (EMG) responses to pericardial algogenic chemicals: chemical stimulation, electrical stimulation, and nerve transection. Activation with bradykinin (n = 46) produced a significantly higher peak response than infusion of an algogenic mixture (n = 53) containing chemicals that also activate VCA. Electrical stimulation of SCA produced bilateral EMG activities (7 of 7). Electrical stimulation of VCA did not evoke EMG activity but inhibited the chemically evoked EMG response (12 of 12). The chemically evoked response was decreased after transection of the left sympathetic chain (n = 22) and was increased after bilateral vagotomy (n = 19). These results suggest an excitatory and inhibitory role for SCA and VCA, respectively. Therefore, in addition to spinothalamic convergence of somatic and visceral afferents, activation of SCA to generate spasmlike muscle contractions could account in part for anginal pain, and VCA activation could attenuate this effect.

Effect of recurrent laryngeal nerve paralysis on superior laryngeal nerve afferents during evoked vocalization.

We tested the hypothesis that vocal fold paresis leads to a substantial reduction in activity from the internal branch of the superior laryngeal nerve (iSLN) during respiration and evoked vocalization. The iSLN afferent activity was measured before and after recurrent laryngeal nerve paresis by cold block in decerebrate cats during spontaneous respiration and electrically evoked vocalization. Response rate patterns of 33 iSLN single units from 11 cats were categorized into 5 groups based on responses to vocalization. Only 24% of single units during spontaneous respiration and 18% during evoked phonation displayed activity pattern changes as a result of paresis. Those fibers affected were heterogeneous in discharge pattern, but none of the units that followed voice frequency lost this characteristic when the motor nerve was cooled. The relative insensitivity of iSLN activity to motor paralysis suggests that the receptors studied are coupled to tissue such that passive interaction rather than active muscular contraction is the major stimulus.

Internal superior laryngeal nerve afferent activity during respiration and evoked vocalization in cats.

The purpose of this project was to identify and categorize patterns of activity of the internal branch of the superior laryngeal nerve during vocalization evoked by midbrain stimulation in cats anesthetized with alpha-chloralose. Unit activity was isolated from the cut distal end of the internal branch of the superior laryngeal nerve by means of floating bipolar electrodes that retained their contact with nerve fibers despite movement due to vocalization. The phases of respiration and vocalization were determined with a diaphragm electromyogram, a photoglottogram, and a microphone recording. Five groups of discrete afferent activities were defined according to relationships between the spike activity and the phases of vocalization. Group 1 cell activity peaked just before phonation, during expiratory airflow (n = 26). Group 2 cells (n = 19) followed a vocal fold vibratory pattern during phonation. Group 3 cell activity (n = 6) peaked during phonation, but did not follow vocal fold vibration. Group 4 cells (n = 3) were active during inspiration between phonations. Group 5 cells (n = 4) showed both inspiratory and expiratory modulation. These results indicate that laryngeal afferent activity responds to phonation-specific events during vocalization. This stereotyped afferent information might be used by the central nervous system to modulate vocalization.

Intrapericardial algogenic chemicals evoke cardiac-somatic motor reflexes in rats.

Many patients suffer from secondary muscle hyperalgesia after experiencing angina pectoris. Electrophysiological studies have also demonstrated electromyography (EMG) activities evoked during brief occlusion of the coronary artery in cats. Our objectives are: (1) to develop an animal model to study muscle changes resulting from cardiac dysfunction and (2) to investigate possible links between muscle hyperalgesia and EMG activities observed with ischemic heart diseases. Intrapericardial infusion of algogenic chemicals produced EMG activities in paraspinal muscles. Among these paraspinal muscles, the spinotrapezius produced the most consistent responses (38/53 animals). The evoked responses, in most cases, persisted longer than 90 s (29/53 muscles). The EMG activities consisted of three patterns of motor-unit discharge, namely single-unit, complex, and ventilation-related. The duration of the patterns, as well as the shape and amplitude of the motor-unit waveforms, are similar to the characteristics of muscle spasms. Since sensitization of muscle afferent fibers by noxious stimuli could produce angina-like pain, the spasm-like EMG activities might sensitize muscle afferent fibers that contribute to acute angina pain and secondary muscle hyperalgesia of cardiac patients.

Effects of abdominal or cardiopulmonary sympathetic afferents on upper cervical inspiratory neurons.

Responses of upper cervical inspiratory neurons (UCINs) to abdominal visceral or cardiopulmonary sympathetic stimulation were studied using extracellular recordings from 213 UCINs in 54 pentobarbital sodium-anesthetized and paralyzed rats. Phrenic nerve activity was used to assess inspiration. The UCINs discharging during inspiration only were mainly in the C(1) segment, whereas phase-spanning UCINs were mostly in the C(2) segment. Phase-spanning activity was typically retained after overventilation or vagotomy. When greater splanchnic nerve (GSN) or cardiopulmonary sympathetic afferent (CPSA) fibers were electrically stimulated, augmented UCIN activity was observed in 65% of cells responding to CPSA stimulation but in only 17% of cells responding to GSN. Response latencies were 10.7 +/- 0.5 and 20.6 +/- 1.5 (SE) ms, respectively. Many augmented responses to CPSA stimulation (64%) and all augmented responses to GSN stimulation were followed by suppression of UCIN discharge (biphasic response). Phrenic nerve activity was suppressed by both GSN and CPSA stimulation, but with shorter latency for the latter (29 +/- 0.7 vs. 14.0 +/- 0.7 ms). Excitation of UCINs using CPSA stimulation occurs more often and by a more direct pathway than for GSN input.

Recording from afferents in the intact recurrent laryngeal nerve during respiration and vocalization.

The goal of this study was to determine whether sensory fibers in an intact recurrent laryngeal nerve (RLN) are influenced by respiration or vocalization. Patterns of RLN afferent activity were examined during respiration and evoked vocalization by means of midbrain electrical stimulation in cats anesthetized with alpha-chloralose. Nerve bundles were dissected from an intact RLN, with motor function preserved. The bundles were cut and the laryngeal end was placed on floating bipolar electrodes. Fifteen right RLNs were examined. A total of 9 single and multiunit afferent fibers from 4 cats were isolated and examined during respiration. Four units, analyzed from 3 fibers, showed respiratory phase modulation. Eight units, analyzed from 4 fibers in 1 cat, were observed during vocalization and showed no vocalization phase modulation. The RLN afferents could contribute to reflex modulation of the respiratory cycle, but more extensive sampling would be necessary to preclude effects from vocalization.

Effects on breathing of medullary bicuculline microinjections in immature opossums.

The role of medullary gamma-aminobutyric acid A (GABAA)-receptor activation in the expression of breathing was studied in suckling opossums (Didelphis virginiana) from 4 to 8 wk of age. Animals were anesthetized with a thiobarbiturate, and the ventral medulla was exposed so that drugs could be microinjected into the medulla with a two-barrel glass micropipette. The GABAA antagonist bicuculline at approximately 1-3 pmol total dose was injected in volumes < 1 nl; injections of saline with pontamine sky blue dye were used as controls and to mark location. Breathing pattern was assessed using diaphragm electromyography (EMG). Effective sites for bicuculline microinjection were obtained in the ventrolateral medulla, with both lateral reticular and inferior olive nuclei present as rostrocaudal markers. Responses among 18 tested animals included increased duration of breaths (Ttot) and duration of inspiration (Ti). Peak amplitude of the diaphragm EMG and peak amplitude of the diaphragm/Ti were not consistently affected. Transient apnea, lasting at least 8 s, occurred in five of the animals. Control solutions did not elicit respiratory responses. These results suggest that neural circuitry in the medulla, using the neurotransmitter GABA, can have an early role in the modulation of breathing pattern.

Maximum discharge rates of respiratory neurons during opossum development.

The observed low frequencies of action potentials observed in medullary respiratory neurons of immature opossums (Didelphis virginiana) could occur because these cells are incapable of achieving higher sustained firing rates. Nonsustainability of firing might also help explain why the inspired breath is brief (approximately 0.1 s) in the youngest opossums and rises very slowly during postnatal life. Firing frequencies of medullary respiratory neurons were examined in spontaneously breathing thiobarbiturate-anesthetized opossums before and after stimulation by the glutamate agonists, N-methyl-D-aspartate (NMDA; 20 mM) or kainic acid (KA; 0.5 mM). Drugs were applied using progressively larger pressure injections through a micropipette; animals were tested from the 5th postnatal wk to adulthood. With a sufficient injection volume, stimulation of cell firing would be followed by apparent suppression of action potentials. A maximum "sustained" firing frequency was obtained from the last injection where discharge remained elevated for at least 0.5 s. Inspiratory and expiratory neurons tested with either drug showed the lowest rates of firing in opossums at 5-9 wk of age compared with 10- to 14-wk-old animals and/or adults. Despite higher rates of discharge in 10- to 14-wk-old animals and/or adults, maximum sustained neuronal firing in the youngest animals was at a higher frequency than during spontaneous breathing and, at least in the cell population tested, does not represent a limitation that might affect breathing pattern.

GABAergic effects on respiratory neuronal discharge during opossum development.

Changes in breathing pattern between immature and adult animals could be due in part to changing postsynaptic sensitivity to particular neurotransmitters by respiratory neurons and/or to the fate of these neurotransmitters after release. To probe for such effects, gamma-aminobutyric acid (GABA) and the GABAA antagonist, bicuculline, were pressure injected by micropipette in very small volumes (approximately 25 pl) near identified medullary respiratory neurons in Inactin-anesthetized adult and suckling opossums. At a concentration of 10 mM, GABA induced suppression of respiratory neurons firing in animals from about 3 wk of age (the youngest animals tested) onward, with the largest responses occurring in adults. For those age groups tested with 0.5 and 50 mM GABA, shorter and longer responses, respectively, were observed. Bicuculline increased the discharge of respiratory units at all ages tested, but responses normalized to initial firing rates did not systematically differ between sucklings down to 4 wk of age and adults. Bicuculline also influenced the onset and cessation of firing in both inspiratory and expiratory neurons. Discharge of respiratory neurons in immature suckling opossums is characterized by few spikes and low firing rates with each breath. However, recovery of neuronal firing from an exogenous load of GABA and release of neuronal firing after antagonism of GABAA receptors does not show a developmental pattern that would implicate GABA as the crucial mediator of these effects.

Short latency excitation of upper cervical respiratory neurons by vagal stimulation in the rat.

Extracellular recordings were made from 29 respiration-phased neurons in the upper cervical spinal cord (C1-C3) in nine anesthetized rats while ipsilateral and contralateral vagi were stimulated via platinum hook electrodes. Neuronal responses to vagal stimulation were recorded using peristimulus histograms. An accumulation of spikes with an average latency of 4.0 +/- 0.8 (S.D.) ms occurred in 11 cells after ipsilateral stimulation. These results indicate that there are fibers in the vagus which oligosynaptically excite respiratory neurons in the upper cervical spinal cord.

Carbon monoxide and lethal arrhythmias.

The effect of acute exposure to carbon monoxide on ventricular arrhythmias was studied in a previously described chronically maintained animal model of sudden cardiac death. In 60 percent of dogs with a healed anterior myocardial infarction, the combination of mild exercise and acute myocardial ischemia induces ventricular fibrillation. The events in this model are highly reproducible, thus allowing study by internal control analysis. Dogs that develop ventricular fibrillation during the test of exercise and acute myocardial ischemia are considered at high risk for sudden death and are defined as "susceptible"; dogs that survive the test without a fatal arrhythmia are considered at low risk for sudden death and are defined as "resistant." In the current study, the effects of carboxyhemoglobin levels ranging from 5 to 15 percent were tested in resistant and susceptible dogs. A trend toward higher heart rates was observed at all levels of carboxyhemoglobin, although significant differences were observed only with 15 percent carboxyhemoglobin. This trend was observed at rest and during exercise in both resistant and susceptible dogs. In resistant animals, in which acute myocardial ischemia is typically associated with bradycardia even under the control condition, this reflex response occurred earlier and was augmented after exposure to carbon monoxide. This effect may depend on the increased hypoxic challenge caused by carbon monoxide, and thus on an augmentation of the neural reflex activation or a sensitization of the sinus node to acetylcholine induced by hypoxia. In both resistant and susceptible dogs, carbon monoxide exposure induced a worsening of ventricular arrhythmias in a minority of cases. This worsening was not reproducible in subsequent trials. These data indicate that acute exposure to carbon monoxide is seldom arrhythmogenic in dogs that have survived myocardial infarction. Nevertheless, the observation that carbon monoxide exposure increases heart rate at rest and during moderate exercise may have clinical implications relevant to patients with coronary artery disease.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects on breathing of rostral pons glutamate injection during opossum development.

To determine whether pathways from the rostral pons, capable of influencing breathing, were present in immature mammals, the excitatory amino acid glutamate (sodium salt) was pressure injected in very small volumes into the rostral pons of suckling and adult opossums. The youngest animals tested were approximately 3 wk old (1.5-2.9 g). Animals were anesthetized with the thiobarbituric acid derivative, Inactin, and the electromyogram of the diaphragm was used to assess changes in breathing rhythm and ventilatory output. Glutamate concentrations of 50, 150, and 1,000 mM were injected into the rostral pons. Active sites were generally located between parabrachial and either lateral lemniscal or trigeminal nuclei. Effects of glutamate in opossums of all ages included changes in diaphragm activity and respiratory timing over several breaths. In the youngest animals, a very high incidence of apnea occurred as an initial response (17 of 20 sites) at the 1,000 mM concentration. The high incidence of apneic response in the youngest animals suggests that strong activation of rostral pontine neurons can more easily disrupt respiratory output; a physiological circumstance of such activation might include a diving response stimulated by trigeminal afferents.

Medullary expiratory activity during opossum development.

Medullary expiration-phased action potentials were obtained from 5-sec-butyl-5-ethyl-2-thiobarbituric acid- (Inactin) anesthetized suckling opossums from approximately 30 days of age to near weaning (80-90 days of age). The medulla was explored for expiratory neuronal action potentials during positive pressure breathing (PPB) (approximately 3 cmH2O) because many expiratory cells had little or no discharge during spontaneous breathing off load. After finding an expiratory unit, the response of the cell to removal (30-60 s) and reinstatement of PPB was measured. Projections to the spinal cord were also examined, and position of the cell was assessed. Results were compared with those obtained from rostral and caudal medullary expiratory neurons in adult opossums. Mean discharge rate of expiratory cells during PPB increased as a function of age. Discharge started with a delay after the cessation of inspiration; this delay decreased as a function of age. Cells averaged 7.5 spikes/breath during PPB and 2.2 spikes/breath off load. Forty-four percent of cells discharged less than once per breath when PPB was removed. Of cells becoming completely silent off load, 73% lost their discharge within the first two unloaded breaths. Only 5% of caudal and 15% of rostral medullary expiratory cells in adult opossums became silent after the end-expiratory transpulmonary pressure was reduced to normal. Neurons in sucklings were most often bulbospinal as were caudal medullary cells in adults; neurons were found in the region of the nucleus ambiguus.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon monoxide and lethal arrhythmias in conscious dogs with a healed myocardial infarction.

Environmental studies suggested that exposure to carbon monoxide (CO) increases cardiovascular mortality among patients with coronary artery disease. We investigated whether, in dogs with a healed anterior myocardial infarction at low and high risk for ventricular fibrillation, acute exposure to CO has adverse effects during acute myocardial ischemia combined with exercise. One month after myocardial infarction, 17 dogs had ventricular fibrillation and 16 survived during the combined exercise and ischemia test. These tests were then repeated in all dogs with different concentrations of carboxyhemoglobin (COHb) (from 5% to 15%). With 15% COHb, heart rate (HR) at rest and during exercise was higher (p less than 0.05) than in the control tests. Surprisingly, the reflex HR response to acute ischemia was also altered; namely, the HR reduction characteristic of the low-risk animals was anticipated and accentuated (-31 +/- 25 versus 2 +/- 30 beats/min, p less than 0.05). Conversely, the HR increase characteristic of the high-risk group was reduced by CO (44 +/- 52 versus 72 +/- 43 beats/min, p less than 0.05). With 15% COHb, malignant arrhythmias occurred in two of the low-risk dogs and in none of the high-risk dogs. In the latter, CO was tested with a combination of exercise work load and myocardial ischemia duration not associated with ventricular fibrillation (VF) in the control condition. This study demonstrated that brief exposure to CO (1) profoundly alters the reflex HR response to exercise and to acute myocardial ischemia and (2) does not enhance the occurrence of malignant arrhythmias in conscious dogs with a healed myocardial infarction.

Medullary inspiratory activity during opossum development.

Discharge properties of medullary neurons associated with inspiration were assessed during opossum development. Location of these neurons and projections to the spinal cord were also evaluated. Studies were performed on suckling opossums, from 15-20 days of age to weaning (approximately 100 days), as well as adult animals, anesthetized with Inactin. Most cells either discharged during inspiration (I cells) or were phase-spanning between late expiration and inspiration (EI cells). Number of spikes per breath for I and EI cells significantly increased as the animal matured. For younger opossums (up to approximately 60 days of age), bulbospinal I and EI cells discharged an average of 1.9 spikes per breath, with a minimum interspike interval of 59 ms. Their conduction velocities, determined by use of antidromic stimulation, were in the unmyelinated range (less than 2 m/s). The occurrence of few spikes per breath with long interspike interval, in bulbospinal cells of the younger opossums, suggests that inspiration is not subject to fine feedback control and that rate of high-frequency motor bursts is relatively slow.

Expiratory effects of cerebellar stimulation in developing opossums.

Positive-pressure breathing (PPB) causes expiratory activation of abdominal (ABD) and intercostal (IC) muscles in anesthetized opossums. Developmentally these responses begin to appear at about the 30th postnatal day and become prevalent over the next 3-4 wk. The purpose of the present study was to examine effects of constant-current cerebellar stimulation, applied over a single breath, on respiratory timing and expiratory electromyogram (EMG) activity in ABD and IC muscles during PPB in anesthetized opossums from 30-35 days of age to weaning. Electrode placements in the region of the deep cerebellar nuclei showed strong expiration-phased effects. For animals showing expiratory activation of both ABD and IC muscles during PPB, the response at low-current density consisted of suppression of the IC and ABD EMGs; relative inhibition often differed, with reduction of the ABD EMG being greater than for IC muscles. In animals of all ages tested, cerebellar stimulation prolonged the expiratory phase of the stimulated breath. Frequency of cerebellar stimulation needed to achieve maximum prolongation of the breath was significantly lower for younger vs. older animals. Pathways from deep cerebellar nuclei may differentially influence the activity of motor groups with comparable respiratory functions; this may be related to a cerebellar function of coordinating respiratory and nonrespiratory (i.e., movement and posture) functions of specific motor groups.