anaesthetized cat: role of the nucleus tractus solitarii

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1 3858 Journal of Physiology (1995), 487.3, pp Hypothalamic modulation of laryngeal reflexes in the anaesthetized cat: role of the nucleus tractus solitarii M. S. Dawid-Milner, L. Silva-Carvalho, G. E. Goldsmith and K. M. Spyer* Department of Physiology, Royal Free Hospital School of Medicine, Rowland Hill Street, London NW3 2PF, UK 1. This investigation was initiated because activation of laryngeal afferents, either by electrical stimulation of the superior laryngeal nerve (SLN) or by natural stimulation of receptors in the laryngeal mucosa, results in a cardiorespiratory response comprising bradycardia, hypotension and apnoea (phrenic nerve activity was suppressed). This pattern of response is qualitatively equivalent to the response that is evoked on activation of the arterial baroreceptors. 2. Preliminary studies indicated that the effects of activating the SLN were suppressed during stimulation in the hypothalamic defence area (HDA) at points that also blocked the effects of baroreceptor stimulation. 3. Recordings were taken from seventy-two neurones localized within the ipsilateral nucleus tractus solitarii (NTS) whose activity was modified by SLN stimulation. Sixty neurones responded with an EPSP on SLN stimulation; nine of these had an inspiratory firing pattern. Five neurones were seen to receive an IPSP on SLN stimulation. 4. Five respiratory SLN-activated neurones were unresponsive to stimulation of the other nerve inputs, whilst four received convergent EPSP inputs on sinus nerve (SN) stimulation. One cell of these four also received inputs from the aortic and the vagus nerves. Sixty-one non-respiratory SLN-activated neurones also received convergent inputs from the sinus nerve. Of these, fifty displayed an EPSP, four an IPSP and seven an EPSP-IPSP. Fifteen neurones also received inputs from the aortic nerve and seventeen from the vagus. 5. From the population of neurones affected by SLN stimulation, twenty-four of seventy were also influenced by HDA stimulation (3 were respiratory cells). Sixteen of these responses consisted of an EPSP (2 respiratory cells), five of an IPSP (1 respiratory cell) and three of an EPSP-IPSP. 6. In neurones receiving an IPSP on HDA stimulation, the SLN-evoked excitatory response was reduced throughout the period of HDA-evoked inhibition. These neurones were all shown to receive excitatory inputs from the arterial baroreceptors and laryngeal mechanoreceptors. 7. Additionally, in the thirty-seven neurones that were excited by SLN stimulation but received no direct synaptic input on HDA stimulation, a conditioning stimulus to the HDA evoked a block of SLN-evoked responses without an accompanying change in membrane potential. Several of these neurones were also affected by both baroreceptor and laryngeal mechanoreceptor stimulation. 8. These observations are discussed in the context of the role of the NTS in cardiorespiratory control. The potential importance of these interactions in respiratory distress are highlighted and the implications for the organization of central pathways for the control of autonomic and respiratory function are discussed. * To whom correspondence should be addressed.

2 74 M. S. Dawid-Milner and others J Physiol Selective mechanical or chemical stimulation of the laryngeal mucosa elicits several responses, including an apnoeic reflex, an expiratory reflex, cough or swallowing, together with associated cardiovascular responses. These responses can also be induced by electrical stimulation of the superior laryngeal nerve (SLN) and are prevented by lesioning the nucleus tractus solitarii (NTS) or sectioning the SLN (see Iscoe, 1988 for review). The apnoeic reflex is characterized by the presence of a classical triad of responses: apnoea, bradycardia and hypotension. The neuronal mechanism underlying the respiratory apnoea involves activation of medullary postinspiratory neurones with a resulting inhibition of inspiratory and expiratory cells within the ventral respiratory group (Jodowski & Berger, 1988; Lawson, Richter, Czyzyk-Krzeska, Bischoff & Rudesill, 1991), which is mediated by the release of excitatory amino acids in the medial NTS (Karius, Ling & Speck, 1993). The bradycardia and hypotension may be elicited by the convergence of SLN inputs onto 'baroreceptor-sensitive' neurones within the NTS (Mifflin, Spyer & Withington-Wray, 1988a). There is evidence that the stimulation of the hypothalamic defence area (HDA), which produces a sustained increase in blood pressure together with tachycardia and tachypnoea, is partly mediated by facilitation of the chemoreceptor reflex within the NTS (Silva-Carvalho, Dawid-Milner, Goldsmith & Spyer, 1993, 1995a) and an inhibition of the baroreceptor reflex that is due to hyperpolarization of baroreceptor-sensitive cells within the NTS and mediated by GABA acting at GABAA receptors (Miffin, Spyer & Withington-Wray, 1988b; Jordan, Mifflin & Spyer, 1988). Preliminary reports from this laboratory presented to the Physiological Society (Dawid-Milner, Silva-Carvalho, Goldsmith & Spyer, 1994) indicate that the stimulation of the HDA may counteract the cardiorespiratory responses induced by SLN stimulation, thus indicating an interaction between HDA and SLN inputs. In order to determine the locus of this interaction we now report studies involving intra- and extracellular recordings from NTS neurones activated by SLN stimulation, and have assessed the effects of HDA, sinus nerve (SN) and specific laryngeal mechanoreceptor, chemoreceptor and baroreceptor activation on these neurones to determine further the role of the NTS in these interactions. METHODS Experiments were carried out in nine cats (body weight, kg) anaesthetized with sodium pentobarbitone (Sagittal; 4 mg kg-', initial dose i.p., supplemented when necessary with 4-5 mg i.v.). A femoral artery was cannulated with a balloontipped catheter (Swan-Ganz; Baxter Healthcare Co., Thetford, UK), which was positioned in the descending aorta. The catheter lumen was used for continuous recording of arterial blood pressure and the inflation of the balloon produced a rise in arterial resistance to elicit a bilateral baroreceptor response. A catheter was inserted into a femoral vein for administration of drugs and supplementary anaesthetic. The trachea was cannulated caudal to the larynx and the animals breathed spontaneously a mixture of 2-enriched room air. In three animals a balloon-tipped catheter (Fogarty; Baxter Healthcare Co.) was positioned in the larynx so that its inflation produced a respiratory apnoea. A balloon-tipped catheter was introduced into the right carotid sinus via the external carotid artery and positioned so that its inflation produced an increase in baroreceptor activity. Selective carotid body chemoreceptor excitation was effected by injecting through the catheter lumen small amounts ( 1-2 ml) of saline saturated with CO2 or 1 ml of 1% sodium cyanide. Animals were paralysed with gallamine triethiodide (Flaxedil; 4 mg kg' iv., supplemented with 1-3 mg kg' h' iv.). The level of anaesthesia was assessed by observing the presence or absence of a significant withdrawal reflex to pinching a paw before paralysis and by the absence of alterations in blood pressure, phrenic nerve activity and heart rate. Throughout the experiment, a stable level of these variables was used as an indication of the anaesthetic level and any change under resting conditions was countered by supplemental anaesthetic doses as described above. All other experimental procedures are as described by Silva- Carvalho, Dawid-Milner & Spyer (1995 b). The effects on cardiorespiratory parameters (heart rate, systolic pressure, diastolic pressure and the area under the curve taken from the integrated and smoothed phrenic record) during SLN stimulation, HDA stimulation and SLN + HDA stimulation were analysed statistically by Student's paired t test. All data are given as means + S.D. Values of t corresponding to P< 5 were considered to be significant. RESULTS Peripheral recordings Electrical stimulation of SLN afferents (-1 ms, 3-1 Hz, 2-5 V, 5 s) resulted in a complete silencing of phrenic nerve activity with no discernible postinspiratory activity in most cases (Fig. 1A). In two NTS inspiratory cells studied in experiments in which this response was elicited, whose activity was recorded as a control, postinspiratory activity was clearly present. On a few occasions phrenic nerve activity was reduced in both intensity and frequency without eliciting apnoea. Both patterns of evoked respiratory response have been reported previously in response to SLN stimulation, together with a fall in arterial blood pressure and bradycardia (Widdicombe, 1986), although the fall in heart rate was partially prevented due to the vagolytic action of the paralysing agent gallamine, as is well documented. Conversely, electrical stimulation of the perifornical region of the hypothalamus (1 ms, 1-2 1sA, 1 Hz, 5 s) evoked the typical autonomic and respiratory features of the defence reaction (Fig. 1B and see Silva-Carvalho et al. 1995b). Increases in frequency or amplitude, or both, of phrenic bursts were evoked, but these terminated at the end of the stimulus, and phrenic nerve activity was then decreased in both frequency and amplitude for several cycles while blood pressure remained elevated.

3 J Physiol Hypothalamic modulation of laryngeal reflexes 741 When SLN and HDA stimuli were delivered simultaneously, the effects of SLN stimulation were suppressed. The triad of cardiorespiratory responses to SLN stimulation (hypotension, bradyeardia and apnoea) were suppressed, so the typical features of the defence response predominated, although the evoked increase in blood pressure and heart rate were slightly smaller. The HDA-evoked increase in respiratory frequency was similar to controls, but the amplitude of the phrenic burst was somewhat decreased (Fig. 1 C). This reduction in the magnitude of the HDAevoked response was more clearly observed when the intensity of HDA stimulation was reduced close to A IC a Cc. L.. E a) I Phr _T a) U).. co Q Ecm ye '-E co 2 1 SLN B ci)' co e Phr ai) m a-.e 1 ~~~~~HDA C " E cii a) co 222 Phr jull"- -A-.kak..AkadbiLA6dLi&Amwkakmhi. I'"r Iyṟ I-11rrp".iI 1.- r' -- 'I )-. ml E oe m HDA + SLN 5 s Figure 1. Laryngeal and HDA interactions The effects of stimulation of the superior laryngeal nerve (SLN; 5 s, 1 Hz, 3 V, 1 ms), and of the hypothalamic defence area (HDA; 5 s, 1 ms, 1 1sA, 1 Hz). Phr, phrenic nerve activity. In each panel arterial blood pressure is also displayed. A, SLN stimulation provokes apnoea that is accompanied by a decrease in heart rate and blood pressure. B, HDA stimulation provokes an increase in phrenic nerve activity together with increases in heart rate and arterial blood pressure. C, simultaneous stimulation of SLN and HDA evokes a response similar to B, thus showing that the HDA is able to prevent the response to SLN stimulation.

4 742 M. S. Dawid-Milner and others J Physiol n 35- :L3 c > 3- S CZ o 15 c ai) i- 1 HDA SLN HDA + SLN 24 - 'c Q) n a) a) 215- I 21 I 35- E 3- -I) cx ci) c>> 1 ** E 19- E,t 17- a) : 15- n c, 13 -o HDA SLN HDA + SLN 5 5 HDA SLN HDA + SLN Figure 2. Cardiorespiratory responses to HDA and SLN stimulation Effects of HDA stimulation (5 s, 1 ms, 1 1sA, 1 Hz), SLN stimulation (5 s, 1 Hz, 3 V, -1 ms) and simultaneous stimulation of both (HDA + SLN) on mean values of phrenic activity (area under the curve taken from integrated and smoothed phrenic records), heart rate, systolic blood pressure and diastolic blood pressure. l, control;, stimulation. Each column shows the mean value (n = 6) with S.D. *P< -5; **P< -1. The statistical analysis between groups showed differences between HDA and SLN stimulation and between HDA + SLN and SLN for all parameters (P < -1). No differences were observed between HDA and HDA + SLN. A B SLN HDA SLN Figure 3. Intracellular recordings from NTS neurones on SLN stimulation A, effect of SLN stimulation (2 pulses, 1 khz, - Ims, 8 V, given at 1 Hz) on a neurone with a membrane potential of -78 mv with no on-going activity. Note the evoked EPSP and action potential discharge. B shows, in the same neurone, the increase in the evoked effect and the shortening in latency of SLN stimulation when preceded by a conditioning stimulus to the HDA (3 pulses, 1 ms, 5 Hz, 15 /ua, given at 1 Hz). Note that HDA stimulation provokes an EPSP with action potentials at short variable latencies. This cell also responded to sinus stimulation and was activated by the intracarotid injection of 1 ml of 1% sodium cyanide. Inflation of a balloon positioned in the larynx failed to evoke any change in discharge (not shown). Each record shows 2 superimposed traces.

5 J Physiol Hypothalamic modulation of laryngeal reflexes 743 threshold values, and the intensity of SLN stimulation was enhanced; however, the effects of HDA stimulation always predominated. If SLN stimulation preceded the HDA stimulus, the HDA response overcame the SLN-evoked 'inhibition'. Conversely, if the SLN stimulus continued beyond the HDA stimulus, apnoea often occurred, although the cardiovascular effects of SLN stimulation were then not observed. Statistically, the effects of HDA stimulation on SLN-evoked responses may represent a summation of opposite effects, in which HDA stimulation at threshold intensities is more powerful than SLN stimulation at just submaximal intensities (Fig. 2). Intracellular studies in NTS neurones influenced by SLN stimulation Recordings were taken from seventy-two neurones whose activity was modified by SLN stimulation (range of resting membrane potentials, -49 to -72 mv). Seventy-one neurones were shown to receive synaptic inputs from the SLN; the other one neurone (non-respiratory modulated; resting membrane potential, -59 mv), whilst tonically active (discharge varying between 2 and 3 Hz), was not affected synaptically but showed a significant reduction of on-going activity to -5 Hz (P < 5) for 12 ms after the stimulation (disfacilitation). Sixty neurones displayed an EPSP with onset latencies of 3-12 ms (mean, ms) on SLN stimulation (Figs 3A, 4B, 5B and 6C) and an amplitude in the range of mv (mean, P3 mv). The amplitude of the EPSP was measured by abolishing action potential discharge by the passage of hyperpolarizing DC current or by reduction in stimulus intensity. Nine of these neurones had an inspiratory firing pattern, their discharge being in phase with phrenic bursts. Fifty-one neurones were not respiratory modulated; the latency of the evoked response to SLN stimulation was 3-12 ms (mean, ms) and the amplitude was mv (mean, P8 mv). In the neurones tested the threshold for evoking neuronal responses was 5-5 V; increasing the intensity of stimulation decreased the latency to onset and increased the amplitude of these synaptic responses. Five neurones exhibited an IPSP with onset latencies of 4-12 ms (mean, ms) and an amplitude of -1-8 to -59 mv (mean, P4 mv). Threshold values for evoking IPSPs were similar to those for evoking EPSPs (1-6 V). In two cells the application of hyperpolarizing DC current (2-5 na) reversed the IPSP to a depolarizing potential (-8, -85 mv), suggesting that it resulted from postsynaptic inhibition. Six neurones displayed an EPSP-IPSP, with onset latencies for the EPSP of 7-12 ms (mean, ms) and amplitudes of 1P4-3-8 mv (mean, P3 mv); for the IPSP the onset latencies were 12-2 ms (mean, ms) and amplitudes were -1-6 to -5-7 mv (mean, mv). In one cell the application of hyperpolarizing DC current (2 na) reversed the IPSP (-78 mv). Changing the intensity of stimulation (n = 2) altered the pattern of EPSP-IPSP response. This was due to thresholds for EPSPs being generally lower ( V) than those for IPSPs. On increasing the intensity of the stimulation, EPSPs increased in amplitude and decreased in latency, but when an IPSP was evoked further increases in intensity produced a decrease in the duration of the EPSP due to an overlapping of the IPSP in the later portion of the EPSP (not illustrated). Properties of non-respiratory SLN-activated neurones Other peripheral inputs. Sixty-one neurones also received convergent inputs from the SN (2 were not tested). In fiftyseven neurones the pattern of response to SN stimulation was identical to that elicited by SLN stimulation (Figs 4, 5 and 6). Fifty neurones exhibited an EPSP, the onset latency of which was 1-5 ms shorter than for their SLN inputs. Latencies in response to SN stimulation were 1-1 ms (mean, ms) and amplitudes were 1P7-8-1 mv (mean, mv). In the neurones tested, the threshold intensity was always higher than for SLNevoked responses (3-12 V). Four cells displayed an IPSP with latencies of 2-1 ms (mean, P4 ms) and amplitudes of -18 to -5-9 mv (mean, P4 mv); the latency for a SN-evoked IPSP was always shorter than that for a SLN-evoked IPSP. Seven cells exhibited an EPSP-IPSP with latencies of 3-1 ms (mean, ms) and amplitudes of mv (mean, mv) for the EPSP; and latencies of 8-15 ms (mean, 11P ms) and amplitudes of -1-4 to -9-8 mv (mean, mv) for the IPSP. Fourteen neurones (13 were not tested) were shown to receive inputs from the aortic nerve (AN) (12 EPSP) and sixteen from the vagus (14 EPSP). Apart from two neurones, the responses to AN and VN stimulation were similar to those evoked by SLN stimulation. In these two neurones, in which SLN stimulation evoked an EPSP-IPSP, both AN and VN elicited IPSPs. HDA inputs. Twenty-one (31 %) SLN-activated neurones also received inputs from the HDA (2 were not tested), whilst forty-five (66 %) were unaffected. Fourteen neurones received an EPSP with latencies of 1-2 ms (mean, ms; Figs 3B, 5A and B) and amplitudes of mv (mean, P7 mv). In four neurones IPSPs were elicited with latencies of 2-4 ms (mean, ms; Fig. 4A) and amplitudes of mv (mean, 5* mv) and in three neurones an EPSP-IPSP was evoked, with latencies for the EPSP of 3-5 ms (mean, 4' + 8 ms) and amplitudes of mv (mean, P1 mv); and, for the IPSP, latencies of 2-3 ms (mean ms) and amplitudes of mv (mean, 5*3 + 1'2 mv). In the four cells exhibiting an IPSP in response to HDA stimulation, the effect was seen as a long-lasting hyperpolarization of the membrane potential, as described by

6 744 M. S. Dawid-Milner and others J Physiol Silva-Carvalho et al. (1995 b). The hyperpolarization developed 2-4 ms after the onset of HDA stimulation, reached its maximum 2-3 ms later and returned to control membrane potential values 15-2 ms after the stimulation (Fig. 4A). During the initial and later phases of the IPSP, a significant decrease in the number of spikes evoked by SLN stimulation was observed (from to ) P < 5), whilst at more negative membrane potentials the SLN-evoked EPSP was reduced in amplitude (from to mv, P< 5) and failed to depolarize the membrane for spike discharge (Fig. 4E). In three of these cells, hyperpolarizing DC current (2 na) was applied, which reversed the evoked IPSP to depolarizing potentials (reversal potentials between -7 and -85 mv), suggesting that they resulted from postsynaptic inhibition. The pattern of the HDA-evoked IPSPs was dependent on the number of HDA pulses given (2 cells A studied). Increasing the number of pulses (from 1 to 1) increased the amplitude ( 9 and 1P5 mv, respectively) and duration of the IPSP (14 and 22 ms, respectively) without modifying its onset. With longer bursts of many (1-2) stimuli no change was observed in amplitude, but there was a change in the duration of the IPSP. In the three cells receiving an EPSP-IPSP the effects were similar to those previously described for IPSPs, although if EPSPs evoked by SLN stimulation were timed to coincide with the HDAevoked EPSPs the pattern of convergence resulted in an initial facilitation. In thirty-seven cells that were seen not to receive any synaptic input on HDA stimulation (see above), a conditioning stimulus to the HDA evoked a block of SLNevoked responses without any change in membrane potential, i.e. a disfacilitation (Fig. 6B and D). This effect B_ C SLN 1 mv n 1 mv L... 1 ms SN nc a16 'oe m 6 I lty swwru r- 'RI -.I "I ssr-wsrl". MY y E SLN + SN Phr -- Barotest 1 s HDA SLN + SN Figure 4. Additional evidence for HDA inhibitory actions on SLN-evoked responses Intracellular recording of a cell within the NTS (membrane potential, -62 mv). A, this neurone responded to stimulation of the HDA (5 pulses, 5 Hz, -1Ims, 2 1sA, given at 1 Hz) with an IPSP (upper traces). The unit was baroreceptive, as shown in the lower left panel, since inflation of the ipsilateral carotid sinus (Barotest) evoked a burst of action potentials. B, SLN stimulation (1 pulse, -1 ms, 7 V at 1 Hz) evoked an excitatory response. C, EPSP and action potentials evoked on stimulation of the SN (2 pulses, a1 ms, 1 khz, 9 V at 1 Hz). D, simultaneous stimulation of both nerves (SN + SLN) evoked an enhanced response. The latency was shortened and a third spike was evoked in 8% of the stimulations. Stimulation parameters as in B and C. E, conditioning stimulus to the hypothalamus (HDA) suppressed the combined effects of SLN + SN stimulation (compare with D). Neuronal recordings in A (upper traces) B, C, D and E are shown as 2 superimposed traces.

7 ~~~~~~~~~~~~~~~~Barotest J Physiol Hypothalamic modulation of laryngeal reflexes 745 was caused by a reduction in the number of evoked action very short-latency inputs (1-3 ms) and in six cases this potentials (from to 4 + 7, P< 1) or a excitatory influence was associated with a later HDAinduced disfacilitation of SLN inputs seen as a decrease in decrease in the amplitude of the EPSPs evoked by SLN stimulation (from P6 to 1P2 + 9 mv, P< 1), or the number of spikes (from to , both. The timing of this effect was similar to that observed P < 1; Fig. 5). In the other five cells, when the timing for HDA-evoked IPSPs (2-2 ms). The disfacilitation thus of an SLN-evoked EPSP coincided with the HDA-evoked reflects an inhibitory action of the HDA on an antecedent response there was an increase in the number of spikes neurone in the SLN reflex pathway within the NTS. (from 1P66 + X51 to , P< 1) or amplitude of All those cells that were either inhibited or disfacilitated by the SLN-evoked EPSPs and a decrease in the latency of the HDA stimulation received convergent inputs from the SN response (see Fig. 3A and B). Additionally, in a further and when tested (n = 13) for baroreceptor sensitivity, three cells that had not been shown to receive synaptic either by inflating a balloon in the descending aorta or in input on HDA stimulation, a conditioning stimulus to the the carotid sinus, they responded with a depolarization or HDA facilitated the SLN-evoked response. This included an increase in activity (Figs 4 and 5). Three of these cells an increase in the number of action potentials (from were also activated by stimulation of laryngeal to , P< 5) or the amplitude of mechanoreceptors on inflating a balloon located in the the EPSPs evoked by SLN stimulation (from to larynx (Fig. 5). 3*4 + 3, P< 5), or both. The timing of this influence was similar to that observed for HDA-evoked EPSPs, In fourteen neurones HDA stimulation evoked an EPSP although no changes were observed in the membrane with onset latency of 1-2 ms. Nine of these cells showed potential of these cells. This suggests that the facilitation A C 1 mv.nd _ a -.&MMmbld& -,.h J U.A 1. i AA&&M. " - -A- jk-- - lip- orlooom.1 1. Pr--Mmpprr------F-v - -F * * *!I SN 1 a _ 16 SN B D I L- ---k-.- 'd- l--. --M_ 1 mv, W- M-ploop- T) :3 16 U) -ae - 6 NN mv 1 ms t Larynx stimulation 1 s Figure 5. Hypothalamically evoked inhibitory actions Intracellular recording of a neurone located in the NTS (membrane potential, -45 mv). A, stimulation of SN (2 pulses, 1 khz, -1 ms, 12 V at 1 Hz; upper panel) evoked an excitatory response, which was reduced (lower panel) if SN stimulation was preceded by a conditioning stimulus of the HDA (5 pulses at 5 Hz, t1 ms, 1 1uA at 1 Hz). B, this neurone was also excited by stimulation of the SLN (1 pulse at -1Ims, 8 V, 1 Hz; upper panel). HDA stimulation, as in A, also decreased the effect of SLN stimulation. HDA stimulation excited this neurone at short latency (*). In A and B, 2 traces are superimposed. This neurone was activated during the rise in blood pressure induced by the inflation of a balloon in the descending aorta (C). Furthermore, the inflation of a balloon in the larynx also excited the cell (D).

8 746 M. S. Dawid-Milner and others J Physiol was produced as a consequence of an excitatory input to an antecedent neurone in the SLN reflex pathway in the NTS. All those cells that were facilitated or received longer latency (more than 4 ms) excitatory inputs on HDA stimulation, received convergent excitatory inputs from the SN. When tested (n = 15), these neurones failed to respond to inflation of a balloon in either the carotid sinus or the aorta, or were inhibited. In one case a clear membrane hyperpolarization accompanied baroreceptor stimulation. In contrast, the seven cells tested for their responses to intracarotid injection of either CO2-saturated saline or sodium cyanide ( 1%) all showed either membrane depolarization or bursts of action potentials. None of these 'chemosensitive' neurones responded to mechanical stimulation of the larynx (n = 7). A B * SN C 5 mv 1 ms ll D SLN I t Figure 6. Hypothalamically evoked inhibitory actions Intracellular recording of a NTS neurone (membrane potential, -45 mv). A, neurone excited by SN stimulation (2 pulses at 1 khz, 1 ms, 1 V given at 1 Hz). C, stimulation of the SLN (1 pulse, 1 ms, 8 V, 1 Hz) also evokes an excitatory response. B and D, a conditioning stimulus from the HDA (5 pulses, 5 Hz, Ims, 1,tA given at 1 Hz) results in a reduced response to SN (B) and SLN stimulation (D). Each record shows 2 superimposed traces. Only the last HDA stimulus is shown (*).

9 J Physiol Hypothalamic modulation of laryngeal reflexes 747 Properties of respiratory SLN-activated neurones Other peripheral inputs. Nine neurones had an inspiratory firing pattern, their discharge being in phase with phrenic bursts. Five SLN-activated neurones did not show any other peripheral inputs (i.e. SN, AN and VN). The latency of the excitatory responses of these neurones to SLN stimulation, ranging between 3 and 6 ms (mean, 4*3 + 1P2 ms), was significantly shorter than that of the other SLN-activated cells receiving peripheral inputs and showing respiratory modulation (7' ms, n = 4, P< 5). The amplitude of the EPSPs was 1P3-3-9 mv (mean, mv) and was not significantly different from the amplitude of the other group of respiratory SLNactivated cells (mean, 3*4 + 1P6 mv, n = 4). These cells exhibited inspiratory-related discharge, and latency fluctuations (-2--4 ms) were observed during the central respiratory cycle. These neurones are equivalent to those postsynaptic to SLN afferents described by Bellingham & Lipski (1992). The threshold intensity for evoking responses was not different from that observed for the full population of the SLN-activated cells ( 5-5 V). Four respiratory SLN-activated neurones (latency, 4-1 ms; mean, ms; and amplitude, mv; mean, 3* mv) also received convergent inputs from the SN (latency, 3-8 ms; mean, ms; and amplitude, mv; mean, mv). One of these also received convergent inputs from the AN and from the VN. This group of cells showed no differences in the latency and amplitude of the response to SLN stimulation when compared with the full population of non-respiratory neurones receiving EPSPs on SLN stimulation. HDA inputs. Three respiratory SLN-activated cells also received inputs on HDA stimulation. These cells also received SN inputs. In two cells the effect of HDA stimulation was seen as an EPSP (3 and 7 ms latency; 2-6 and 3 6 mv amplitude). In the other, the effect of HDA stimulation was seen as an IPSP (latency, 2 ms; amplitude, 6-1 mv and duration, 4 ms). DISCUSSION The present study has shown that stimulation in the hypothalamic defence area is able to produce suppression of reflex responses that are elicited by electrical stimulation of the SLN. The triad of cardiorespiratory responses that are observed in this reflex comprise apnoea, bradycardia and a fall in arterial blood pressure. When the HDA is stimulated simultaneously with the SLN, the cardiorespiratory features of the defence response predominate, even if the HDA is stimulated at a low intensity, which is just above the threshold for evoking cardiorespiratory responses. This pattern of interaction is shown to involve synaptic mechanisms within the NTS. A group of NTS neurones has been identified that are excited by the stimulation of the SLN, and in three cases by mechanical stimulation of the larynx also and are inhibited on HDA stimulation. Such neurones also receive excitatory inputs from the SN and arterial baroreceptors. Additionally, a second group of neurones was identified that also received convergent excitatory inputs from the SN and SLN stimulation, but these were reduced during HDA stimulation, although a direct synaptic input was not observed. This indicates an HDA-evoked disfacilitation. Previous studies have indicated a convergence of SLN and SN inputs onto NTS neurones (Biscoe & Sampson, 197; Mifflin et al. 1988b; Mifflin, 1993) and the present study has extended these. It has, furthermore, provided indications of a functional organization of these inputs. Baroreceptor inputs evoke a bradycardia and fall in blood pressure as well as inhibiting respiration. This pattern of response is qualitatively identical to the effects that may be produced on mechanical stimulation of the larynx. Conversely, non-mechanosensitive laryngeal inputs have also been identified in the present study, but these were shown to be directed to NTS neurones that were excited on activation of the arterial chemoreceptors. In the former situation the effect of HDA stimulation was inhibitory, in the latter case excitatory. Our observations on the interactions between laryngeal activation and the defence response are not in agreement with those of Lopes & Palmer (1978). These authors substantiated the earlier observations that HDA stimulation could suppress the bradycardia that normally accompanies baroreceptor stimulation (see Hilton, 1966). However, their experiments suggested that if the SLN was stimulated simultaneously a bradycardia persisted. They suggested that this was a consequence of the SLN-evoked apnoea that removed a 'respiratory gating' of vagal cardioinhibitory neurones, both on-going and HDA evoked. This would allow the baroreceptors to exert their normal excitatory drive to these vagal neurones. This implied that the major effect of HDA stimulation was mediated by actions in the ventrolateral medulla, partly on the vagal cardio-inhibitory neurones of the nucleus ambiguus (McAllen & Spyer, 1978; Gilbey, Jordan, Richter & Spyer, 1984), but presumably also on the premotor sympathetic neurones that have since been identified (McAllen, 1986; see Guyenet, 199 for review). Their assertion is no longer tenable, since we have provided compelling evidence, in this and previous studies, for hypothalamic actions at the level of the NTS that have a major role in modulating baroreceptor function (Miffin et al. 1988b; Jordan et al. 1988; Silva-Carvalho et al. 1995b; reviewed by Spyer, 1994). Furthermore, in the present study we demonstrate that the triad of cardiorespiratory responses evoked on SLN stimulation are themselves suppressed by HDA

10 748 M. S. Dawid-Milner and others J Physiol stimulation. The present observations of an interaction of all three inputs (baroreceptor, laryngeal and HDA) in the NTS lends further support to the notion that the activity of vagal neurones is determined by a regulation of the efficacy of NTS inputs (see Spyer, 199 for discussion) as well as a direct inspiratory-mediated inhibitory control of their activity (Gilbey et al. 1984; Richter & Spyer, 199), the latter corresponding to the 'gate' inferred by Lopes & Palmer (1978). Bellingham & Lipski (1992) and Mifflin (1993) have recently described the patterns of neuronal response generated in the NTS on SLN stimulation. The pattern of response observed in the present study coincides quite closely with those data, although a much larger proportion of neurones showed a level of mechanical sensitivity on laryngeal stimulation in Mifflin's (1993) study than in the present investigation. We also observed a group of monosynaptically activated neurones and a portion of these in our study were unresponsive to other inputs. A large proportion of NTS neurones (63%) that were excited by SLN stimulation were either directly inhibited or the inputs onto them were effectively reduced during HDA stimulation. Our data argue strongly for a complex intranuclear chain of connections mediating SLN inputs and, as with baroreceptor inputs, these appear to be under the control of an intrinsic group of GABA-containing neurones that mediate inhibitory actions exerted by HDA stimulation (see Silva-Carvalho et al. 1995a, b for discussion). Interestingly, a further group of NTS neurones that were excited by HDA stimulation were activated by SLN and SN stimulation. These were not excited by mechanical stimulation of the larynx or baroreceptor activation, but showed excitatory responses to arterial chemoreceptor stimulation. The potential role of these neurones is discussed at further length by Silva-Carvalho et al. 1995a). The present results concerning the description of the synaptic interactions within the NTS and SLN, SN and HDA inputs may have considerable clinical significance in the context of the sudden infant death syndrome. The stimulation of mechanolaryngeal and other upper airway receptors is a major cause of apnoea in newborn infants (Davies, Koenig & Thach, 1988). This is accompanied by bradycardia and systemic hypotension. There is evidence that this apnoeic reflex is particularly profound in fetuses and neonates (Downing & Lee, 1975) and that this respiratory inhibition cannot be overcome by arterial chemoreceptor excitation, producing death by suffocation (Lee, Stoll & Downing, 1977). The complex patterns of interaction within the NTS of arterial baroreceptor and chemoreceptor inputs, laryngeal mechanoreceptor and HDA inputs may provide a possible explanation for the abnormal sensitivity of the neonate to the effects of laryngeal-induced apnoea. Apnoea produces a transient hypoxaemia that, in normal circumstances, would activate the arterial chemoreceptors and so would counteract reflexly the apnoea and the accompanying hypotension. Furthermore, this afferent input is a significant stressful signal to excite the HDA, the activation of which leads to an inhibition of the baroreceptor reflex and facilitates the chemoreceptor reflex (see Spyer, 1994, for discussion) that would thus reverse the actions of laryngeal stimulation. Indeed, the activation of the HDA has been shown in the present report to suppress the effects of SLN stimulation. If the reciprocal connections between the NTS and HDA remain ineffective through immaturity in some neonates this restorative mechanism would be absent. In these circumstances SLN inputs activated by upper airway stimulation would evoke apnoea, bradycardia and hypotension and, without the facilitating influence of the HDA on respiratory activity and the chemoreceptor reflex (see Silva-Carvalho et al. 1995a), only the direct chemoreceptor-induced bradycardia would be observed (Daly, 1985). This would be expected to potentiate the existing bradycardia and could induce cardiac arrest. BELLINGHAM, M. C. & LIPSKI, J. (1992). Morphology and electrophysiology of superior laryngeal nerve afferents and postsynaptic neurones in the medulla oblongata of the cat. Neuroscience 48, BIscoE, T. J. & SAMPSON, S. R. (197). Response of cells in the brainstem of the cat to stimulation of the sinus, glossopharyngeal, aortic and superior laryngeal nerves. Journal of Physiology 29, DALY, M. DE B. (1985). 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