Laryngeal and diaphragmatic muscle activities after central nervous system lesions in cats.

AIM: To examine the central control and coordination of respiratory pump muscles and laryngeal valve muscles by systematic decerebration (DECER), cerebellectomy (CBELL), pontine respiratory group lesioning (PRG) and pontomedullary section (PMED). METHODS: Activities of posterior cricoarytenoid (PCA), thyroarytenoid (TA) and diaphragm (D) muscles and their responses to inspiratory (I) and expiratory (E) total occlusions were determined in 10 adult cats. RESULTS: INTACT anesthetized cats (n = 6) exhibited inspiratory PCA (PCA(I)) and D activities. Expiratory PCA (PCA(E)) was present but TA activity was absent. It was found that successive DECER, CBELL and PRG lesions attenuated PCA(E), the intact pattern being noted in 7/10, 4/10 and 0/6 cats, respectively. After PMED, variable PCA, TA and continuous D activities occurred only with blood gas abnormalities. Augmented PCA and D responses to I- and E-loads occurred after PRG lesions: the I-load PCA(I) and D responses resembled apneusis and the E-load PCA(E) and D responses resembled central apnea. CONCLUSION: The decreasing PCA(E) activity observed with successive DECER, CBELL and PRG lesions suggests that these areas influence laryngeal abductor control of glottic size. The synchronous activities after PMED transection suggest a role for more rostral structures in coordinating laryngeal and diaphragmatic muscle activities.

Evidence for medullary projections of the intercostal nerve-elicited inspiratory termination.

This study hypothesized that the ICN-elicited inspiratory termination reflex required synaptic activation in two distinct regions of the ventral respiratory group (VRG): (1) transitional (tVRG), and (2) pre-Bötzinger complex (pre-BötC). Data from adult cats indicate that axons of passage associated with the ICN-elicited termination reflex traverse tVRG, but that relevant synaptic processing does not occur in this region. Furthermore, data indicate that neither synaptic nor axonal transmission within the pre-BötC is required for the SLN- or ICN-elicited termination reflex.

Differential effects of excitatory amino acid receptor antagonism in the ventral respiratory group.

This study tested the hypothesis that selective antagonism of excitatory amino acid (EAA) receptors within the ventral respiratory group (VRG) would induce changes in both respiratory rhythm and pattern. In the paralyzed, decerebrate, vagotomized and ventilated cat, baseline values for respiratory (Ttot), inspiratory (Ti), and expiratory (Te) durations and peak integrated phrenic nerve (integralPN) amplitude were established. Microinjection of the non-NMDA (N-methyl-d-aspartate) receptor antagonist NBQX (2, 3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline) into rostral/inspiratory-modulated (iVRG) or caudal/expiratory-modulated VRG elicited an immediate apnea. When PN activity resumed, Ttot was significantly decreased, and integralPN amplitude was attenuated. NMDA receptor antagonism with microinjections of AP5 (2-amino-5-phosphonopentanoic acid) into iVRG decreased Te for more than 30 min. NMDA receptor antagonism in inspiratory/expiratory-modulated VRG (level of obex, transitional VRG) yielded either apnea or a significant reduction in Ttot, Ti and integralPN amplitude. Our data suggest that endogenous EAA receptor-mediated neurotransmission throughout the VRG is active in the determination of both respiratory timing and pattern. Our data further suggest that tVRG serves a unique function within the respiratory network.

Short-latency excitation of phrenic motor output mediated by non-NMDA receptors.

This study tested the hypothesis that the short-latency excitation of the phrenic motor output elicited by superior laryngeal nerve (SLN) stimulation requires non-NMDA receptor-mediated neurotransmission in the region of the dorsal respiratory group (DRG) of the adult cat. Injection of the non-NMDA receptor antagonist NBQX into the DRG severely attenuated or abolished the short-latency excitation, indicating that the short-latency excitation requires non-NMDA receptor-mediated neurotransmission within the DRG.

Role of N-methyl-D-aspartate receptors in the pontine pneumotaxic mechanism in the cat.

Systemic injection of MK-801, an N-methyl-D-aspartate (NMDA) receptor-associated channel blocker, induces an apneusis in vagotomized cats similar to that produced by pontine respiratory group (PRG) lesions, suggesting the possible involvement of NMDA receptors in the pontine pneumotaxic mechanism. Previous results from our laboratory indicate that the efferent limb of the pontine pneumotaxic mechanism is unlikely to require NMDA receptor-mediated neurotransmission. Therefore, the present study examined the potential involvement of PRG NMDA receptors in the pontine pneumotaxic mechanism. Experiments were conducted in decerebrate, paralyzed, and ventilated adult cats. The effects on inspiratory time (TI) of MK-801 microinjection into PRG were tested in 12 cats. Pressure microinjection of MK-801 (15 mM, 80-3,000 nl) significantly prolonged TI in all animals when lung inflation was withheld. TI progressively increased in most animals for > or = 30 min. After this period, partial recovery of the effect occurred in eight cats as TI shortened toward predrug levels. In three animals, microinjection of MK-801 induced a complete apneusis in the absence of lung inflation from which there was no detectable recovery. Microinjections into regions approximately 2 mm distant from PRG produced little or no effect. These results provide evidence that NMDA receptors located in the region of PRG play an important functional role in the control of the breathing cycle.

Nucleus tractus solitarius and excitatory amino acids in afferent-evoked inspiratory termination.

This study tested the hypothesis that excitatory amino acid (EAA) neurotransmission at non-N-methyl-D-aspartate (non-NMDA), but not NMDA, receptors within medial regions of the nucleus tractus solitarius (NTS) is required in the inspiratory termination elicited by vagal or intercostal nerve (ICN) stimulation. Adult cats were anesthetized, decerebrated, vagotomized, and ventilated. After control responses to stimulation of the superior laryngeal nerve (SLN), vagus, and ICN were obtained, EAA receptor antagonists were injected into the medial aspects of the NTS. Injections of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or 6,7-dinitro-quinoxaline-2,3-dione (DNQX), EAA receptor antagonists; (+/-)-2-amino-5-phosphonopentanoic acid (AP5), an NMDA antagonist; or 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX), a non-NMDA antagonist, ipsilateral to the vagus abolished the termination response. The SLN-elicited response persisted after AP5 injection but was abolished by NBQX injections. The ICN-elicited response persisted after bilateral injections of CNQX/DNQX or procaine. We conclude that the inspiratory termination elicited by ICN stimulation is independent of the regions medial to the NTS. Inspiratory termination elicited by vagal or SLN stimulation requires non-NMDA-mediated EAA neurotransmission within medial aspects of the NTS, but the vagally elicited response also requires NMDA receptors.

Substance P immunoreactive projections to the ventral respiratory group in the rat.

Retrograde tracing (rhodamine beads) combined with immunohistochemistry was used to determine the origin of neurons containing substance P that project to the rostral ventral respiratory group in the rat. Double-labeled neurons (rhodamine beads plus substance P immunoreactivity) were found in the midline caudal raphe nuclei (raphe obscurus, raphe pallidus, and raphe magnus) and in the ventrolateral medulla in the parapyramidal region. The findings of this study suggest that substance P-containing neurons in the caudal raphe nuclei and parapyramidal region project to inspiratory neurons in the rostral ventral respiratory group and may therefore influence their activity.

Excitatory amino acid neurotransmission in superior laryngeal nerve-evoked inspiratory termination.

Superior laryngeal nerve (SLN) stimulation elicits a transient inhibition of inspiration (single shocks) or inspiratory termination (stimulus trains). The neural pathways mediating these responses are unknown, but the medial nucleus tractus solitarius (mNTS) has been implicated in the termination reflex. This study tested the hypothesis that SLN-evoked inspiratory termination requires excitatory amino acid (EAA) neurotransmission in medial aspects of the NTS. Experiments were conducted in decerebrate, vagotomized, and paralyzed adult cats. Inspiratory motor output was recorded from the phrenic nerve. After control responses to SLN stimulation were recorded, a unilateral injection of the EAA antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX, 10 mM) was made into the mNTS. The transient inhibitions were not altered by DNQX. Inspiratory termination elicited by stimulation of the SLN contralateral to the injection persisted after DNQX (n = 4). Stimulation of the ipsilateral SLN no longer elicited termination (5 of 9 animals) or did so only at greatly elevated thresholds (4 of 9). We conclude that EAA neurotransmission in the mNTS is not required in the transient reflex but is necessary for the production of the SLN-evoked inspiratory termination.

Pontine-evoked inspiratory inhibitions after antagonism of NMDA, GABAA, or glycine receptor.

Single-shock stimulation of the pontine respiratory group (PRG) produces a transient short-latency inhibition of inspiratory motor activity. Stimulus trains delivered to the PRG can elicit a premature termination of inspiration. This study examined the involvement of N-methyl-D-aspartate (NMDA), gamma-aminobutyrateA (GABAA), or glycine receptors in these inhibitory responses. Experiments were conducted in decerebrate, paralyzed, and ventilated cats. Control responses to PRG stimulation were obtained from recordings of the left phrenic nerve activity. After systemic injection of MK-801, bicuculline, or strychnine (antagonists to NMDA, GABAA, or glycine receptors, respectively), responses to stimulation were again recorded. Inspiratory termination elicited by the PRG stimulation persisted after antagonism of NMDA, GABAA, or glycine receptors. The onset latency and duration of the transient inhibition were not changed after administration of bicuculline, but MK-801 administration did significantly prolong the duration of the transient inhibition. Strychnine significantly prolonged both the onset latency and the duration. These data suggest that none of the three receptor types is required in the inspiratory termination response elicited by electrical stimulation of the PRG region and that NMDA, GABAA, or glycine receptor-mediated neurotransmission is not solely responsible for the transient inhibitory response. However, the prolonged onset and duration of the transient inhibition after strychnine administration suggest that glycine does normally participate in this response.

Endogenous nitric oxide required for an integrative respiratory function in the cat brain.

1. The involvement of nitric oxide (NO) in the respiratory function of the pons was examined by microinjecting NO synthase-related drugs into discrete regions of the pontine respiratory group (PRG) in decerebrate and decerebellate cats. 2. Microinjection of N omega-nitro-L-arginine (L-NNA, inhibitor of NO synthase), but not D-NNA (the inactive enantiomer), significantly prolonged the duration of inspiration in all 10 cats when lung inflation was withheld. 3. The prolongation of inspiration produced by L-NNA was partially reversed in three cats by microinjections of L-arginine (NO synthase substrate) at the same sites. 4. We conclude that endogenous production of NO from L-arginine in the PRG region is involved in the normal function of the pontine pneumotaxic mechanism. These findings provide the first conclusive evidence that endogenous NO formation is involved in the mechanisms associated with respiratory rhythm generation.

Effects of intravenous bicuculline and strychnine on inspiratory inhibitory responses in the cat.

Single shock stimulation of the superior laryngeal nerve (SLN), intercostal nerve (ICN), phrenic nerve (PN) or within the medullary respiratory groups (DRG-VRG) produces a transient, short-latency attenuation of inspiratory motor activity. Trains of stimuli delivered to SLN and ICN cause premature termination of inspiration. This study examined involvement of glycine and GABAA receptors in these reflex inhibitions. Experiments were conducted in decerebrate, vagotomized, and paralyzed cats. Control responses of left PN activity to threshold single shock stimulation of SLN, PN, ICN and the DRG-VRG were recorded and the thresholds for SLN- and ICN-evoked inspiratory termination were determined. Five min after intravenous injection of bicuculline (1 mg/kg) or strychnine (50 micrograms/kg), the responses to stimulation were again recorded. This procedure was reiterated until the cumulative dose elicited marked convulsions. Neither drug affected the inspiratory terminating reflexes. Systemic bicuculline had no effect on transient inspiratory inhibition. However strychnine prolonged the onset latency and the duration of all four inhibitory responses. Since the degree of transient inhibition was not lessened (only delayed), it appears that these inspiratory inhibitory reflexes do not rely exclusively on actions of glycine or GABAA receptors.

Blockade of N-methyl-D-aspartate receptors has no effect on certain inspiratory reflexes.

Previous studies have indicated that excitatory amino acids are involved in many afferent pathways. This study investigated the effects of intravenous MK-801 [an N-methyl-D-aspartate (NMDA) receptor-associated channel blocker] on several well-known respiratory reflexes elicited by afferent stimulation of the superior laryngeal (SLN), the intercostal (ICN), and the phrenic (PN) nerves. Control responses to stimulation were obtained from recordings of phrenic nerve activity in decerebrate, paralyzed cats. Inspiratory termination elicited by the delivery of stimulus trains to either the SLN or the ICN persisted after MK-801. The onset latency or duration of the short-latency excitations produced by SLN or ICN stimulation were unchanged. The transient inhibitions produced by SLN, ICN, PN, or medullary stimulation showed no significant changes in threshold, onset latency, or duration. Withholding lung inflation produced apneusis after administration of MK-801, indicating a central effect of the drug. Higher doses of MK-801 did not alter the parameters of these reflexes. These data indicate that NMDA-dependent neurotransmission is not required for the production of these reflexes.

Lesions of the rostral dorsolateral pons have no effect on afferent-evoked inhibition of inspiration.

This study investigated a possible role of the rostral dorsolateral pons (including nucleus parabrachialis medialis and Kölliker-Fuse nucleus) in mediating several inspiratory inhibitions. These inhibitions included the transient inhibition of phrenic inspiratory motor output produced by stimulation of the superior laryngeal nerve (SLN), the intercostal nerve (ICN) or the phrenic nerve (PN), as well as the inspiratory termination produced by trains of stimuli delivered to the SLN or ICN. In decerebrate, paralyzed, and artificially ventilated cats, the inhibitions produced by stimulation of these nerves were observed before and after lesioning (either radiofrequency, n = 8, or electrolytic, n = 9) the dorsolateral pons. Delivery of stimulus trains to the SLN or the ICN continued to elicit inspiratory termination following pontine lesions with no significant change in the threshold. There were no significant effects of bilateral dorsolateral pontine lesions on the threshold, onset latency, or duration of the short-latency, transient inhibitions produced by SLN, ICN or PN stimulation. From these data, we conclude that the rostral dorsolateral pons is not required in the production of any of these inhibitory reflexes.

Experience with a physiology workshop for high school and college teachers.

Science education in the United States at all academic levels is widely perceived to need direct assistance from professional scientists. The current dearth of quality applicants from this country to medical and graduate schools suggests that our existing undergraduate and high school science curriculum is failing to provide the necessary stimulus for gifted students to seek careers in the health sciences. Recognizing the need to become more directly helpful to high school and college science teachers, members of the faculty of the Department of Physiology and Biophysics at the University of Kentucky College of Medicine held a 5.5-day Physiology Summer Workshop during June, 1989. Participants included 25 college teachers from Kentucky and 5 other states plus 22 Kentucky high school teachers. The presence of the two levels of educators provided communication about curricular concerns that would be best addressed by mutual action and/or interaction. Each day's activities included morning lectures on selected aspects of organ system and cellular physiology, a series on integrative physiology, and afternoon laboratory sessions. The laboratory setting allowed the instructor to expand on principles covered in lecture as well as provided the opportunity for in-depth discussion. A selection of evening sessions was presented on 1) grants available for research projects, 2) obtaining funds for laboratory equipment, and 3) graduate education in physiology.

Origin of serotonin-containing projections to the ventral respiratory group in the rat.

The major purpose of the present study was to determine the origin of the serotonin-containing neurons which project to the rostral ventral respiratory group in the rat. This was accomplished by using the technique of retrograde tracing with rhodamine-labeled latex microspheres (beads) combined with immunochemistry. The rhodamine-labeled beads were microinjected into electrophysiologically identified groups of inspiratory neurons in the rostral ventral respiratory group to retrogradely label neurons projecting to this site. Immunohistochemical processing of the tissue was then done to determine if serotonin was present in the retrogradely-labeled neurons. Serotonin-containing neurons projecting to the rostral ventral respiratory group were found in the raphe magnus, raphe obscurus, raphe pallidus and in the paraolivary region extending to the ventral medullary surface. No serotonin-containing neurons in more rostrally located raphe nuclei were found to project to the rostral ventral respiratory group. The findings suggest that caudal raphe serotonergic projections may affect the activity of respiratory neurons in the rostral ventral respiratory group. Projections to the rostral ventral respiratory group from other pontomedullary nuclei were also identified. Rhodamine-labeled neurons were found in the area of the Kölliker-Fuse nucleus, lateral and medial parabrachial nuclei, retrofacial nucleus, nucleus ambiguus/retroambigualis, nucleus tractus solitarius, A5 region, nucleus paragigantocellularis lateralis, retrotrapezoid nucleus, area postrema and spinal trigeminal nucleus. The projections to the rostral ventral respiratory group in the rat are similar to those previously described in the cat and suggest a common circuitry for the CNS control of breathing.

Bötzinger complex region role in phrenic-to-phrenic inhibitory reflex of cat.

Neuronal recordings, microstimulation, and electrolytic and chemical lesions were used to examine the involvement of the Bötzinger Complex (BötC) in the bilateral phrenic-to-phrenic inhibitory reflex. Experiments were conducted in decerebrate cats that were paralyzed, ventilated, thoracotomized, and vagotomized. Microelectrode recordings within the BötC region revealed that some neurons were activated by phrenic nerve stimulation (15 of 69 expiratory units, 9 of 67 inspiratory units, and 19 nonrespiratory-modulated units) at average latencies similar to the onset latency of the phrenic-to-phrenic inhibition. In addition, microstimulation within the BötC caused a short latency transient inhibition of phrenic motor activity. In 17 cats phrenic neurogram responses to threshold and supramaximal (15 mA) stimulation of phrenic nerve afferents were recorded before and after electrolytic BötC lesions. In 15 animals the inhibitory reflex was attenuated by bilateral lesions. Because lesion of either BötC neurons or axons of passage could account for this attenuation, in eight experiments the phrenic-to-phrenic inhibitory responses were recorded before and after bilateral injections of 5 microM kainic acid (30-150 nl) into the BötC. After chemical lesions, the inhibitory response to phrenic nerve stimulation remained; however, neuronal activity typical of the BötC could not be located. These results suggest that axons important in producing the phrenic-to-phrenic reflex pass through the region of the BötC, but that BötC neurons themselves are not necessary for this reflex.

Respiratory rhythmicity after extensive lesions of the dorsal and ventral respiratory groups in the decerebrate cat.

It was previously demonstrated that extensive destruction of the regions of the dorsal (DRG) and rostral portions of the ventral respiratory groups (VRG) in the medulla does not disrupt respiratory rhythmicity in the anesthetized cat. The present experiments examined if either higher CNS structures or the caudal expiratory VRG might have been responsible for preserving rhythm in those studies. Results indicate that the DRG and VRG are not required for respiratory rhythmicity in the midcollicularly decerebrated cat.

Respiratory resetting induced by spinal cord stimulation in the cat.

Electrical stimulation (50-150 microA, 0.5-ms duration, 3-300 Hz) was performed within three different regions (lateral, ventrolateral, and ventral) of the C2-C3 spinal cord of decerebrate, vagotomized, paralyzed, and artificially ventilated cats. Spinal cord stimulation sites were located by inserting monopolar or bipolar stimulating electrodes either at the dorsolateral sulcus or at least 1 mm medial or lateral to the sulcus. With stimulation at each site, alterations in respiratory rhythm, orthodromic phrenic nerve responses, and antidromic activation of medullary respiratory-modulated neurons were examined. Phrenic nerve responses to cervical spinal cord stimulation consisted of an early excitation (2-4 ms) and/or a late excitation (4-8 ms). Stimulation of the lateral region evoked the greatest amplitude early response and stimulation of the ventrolateral region produced the greatest late excitation. All three stimulus sites elicited antidromic activation of some respiratory-modulated neurons in the dorsal (DRG) and ventral respiratory groups (VRG). The lateral region was the least effective resetting site, and it had the highest incidence of antidromic activation of both DRG and VRG neurons. The ventrolateral region of the cervical spinal cord was the most effective resetting site, but it had the lowest incidence of antidromic activation of DRG respiratory-modulated neurons. In addition, resetting responses were observed with spinal cord stimulation at similar sites in the thoracic and lumbar spinal cord regions thought to be devoid of inspiratory bulbospinal axons.(ABSTRACT TRUNCATED AT 250 WORDS)

Supraspinal involvement in the phrenic-to-phrenic inhibitory reflex.

Phrenic afferents are capable of attenuating the phrenic motor response elicited by the intercostal-to-phrenic excitatory reflex in decerebrate, paralyzed cats. High spinal transection eliminates the attenuating effect of the bilateral phrenic-to-phrenic inhibitory reflex. These results indicate that although phrenic nerve afferents do exert an inhibitory influence in the cervical spinal cord, some of the inhibitory effects are likely to involve supraspinal mechanisms.

Attenuation of phrenic motor discharge by phrenic nerve afferents.

Short latency phrenic motor responses to phrenic nerve stimulation were studied in anesthetized, paralyzed cats. Electrical stimulation (0.2 ms, 0.01-10 mA, 2 Hz) of the right C5 phrenic rootlet during inspiration consistently elicited a transient reduction in the phrenic motor discharge. This attenuation occurred bilaterally with an onset latency of 8-12 ms and a duration of 8-30 ms. Section of the ipsilateral C4-C6 dorsal roots abolished the response to stimulation, thereby confirming the involvement of phrenic nerve afferent activity. Stimulation of the left C5 phrenic rootlet or the right thoracic phrenic nerve usually elicited similar inhibitory responses. The difference in onset latency of responses to cervical vs. thoracic phrenic nerve stimulation indicates activation of group III afferents with a peripheral conduction velocity of approximately 10 m/s. A much shorter latency response (5 ms) was evoked ipsilaterally by thoracic phrenic nerve stimulation. Section of either the C5 or C6 dorsal root altered the ipsilateral response so that it resembled the longer latency contralateral response. The low-stimulus threshold and short latency for the ipsilateral response to thoracic phrenic nerve stimulation suggest that it involves larger diameter fibers. Decerebration, decerebellation, and transection of the dorsal columns at C2 do not abolish the inhibitory phrenic-to-phrenic reflex.