Serotonergic effects on hypoglossal neural activity and reflex responses

BRAIN RESEARCH ELSEVIER

Brain Research 726 (1996) 213-222

Research report

Serotonergic effects on hypoglossal neural activity and reflex responses M.A. Douse

*, D . P . W h i t e

Department of Medicine, UCHSC and Respirato~ Care, Denver VA Medical Center, 1055 Clermont Street, Denver, CO 80220, USA Accepted 12 March 1996

Abstract

We determined the effects of serotonin (5HT; 6 concentrations ranging from 0.005 to 500 jxM) pressure microinjection into the hypoglossal (XII) motor nucleus (100-500 nl; pH = 7.2-7.4) on XII whole nerve activity and reflex response to upper airway negative pressure in 15 decerebrated, vagotomized, paralyzed and artificially ventilated cats. Increasing 5HT concentration resulted in a concentration dependent increase in ipsilateral tonic XII activity, with no change in phasic XII activity. Threshold concentrations ranged from 0.005 to 0.5 ixM, with the maximal response reached at 5 txM. Increasing 5HT concentration also increased the duration of the XII response. This ranged from 50 s with 0.5 IxM, to over 10 min with 500 p.M 5HT. However, 5HT did not significantly change the XII whole nerve reflex response to upper airway negative pressure ( - 2 0 cm H20) at any 5HT concentration (n = 5). All 5HT effects were reversed by microinjection of 1.0 mM methysergide. We conclude that XII responses to 5HT are elicited at low concentrations of 5HT, which have a relatively short duration of effect, but that 5HT at the XII motor nucleus has no effect on the XII reflex response to upper airway negative pressure. Keywords: Control of breathing; Upper airway; Microinjection; Decerebrate cat

1. Introduction

Precise control of upper airway muscles is critical in maintaining airway patency and effective ventilation. A major dilator muscle of the upper airway is the genioglossus innervated by the hypoglossal (XII) nerve. One important aspect of XII motor control is the reflex response to negative pressure in the upper airway. This upper airway reflex is primarily mediated through the superior laryngeal nerve and reflexively increases XII neural output and genioglossal muscle activity, thereby defending airway patency [13,16,31-33,36]. Changes in upper airway reflex responses may therefore be an important factor in maintaining effective ventilation [52], yet little is known concerning neural mechanisms that modulate the upper airway reflex. Serotonin (5HT) is commonly regarded as an important neuromodulator (cf. [3,19]) that mediates changes in the excitability (gain) of neural responses to synaptic input [ 12,51 ]. In non-respiratory motoneurons, 5HT application increased the neural response to glutamate in both lumbar [54,55] and facial motoneurons [34]. Similarly, 5HT appli-

* Corresponding author. Fax: (1) (303) 393-4639; E-mail: [email protected] 0006-8993/96/$15.00 Published by Elsevier Science B.V. PH S 0 0 0 6 - 8 9 9 3 ( 9 6 ) 0 0 3 3 5 - 6

cation into the trigeminal motor nucleus augmented both spontaneous neural activity and the jaw-closure reflex [44]. In respiratory motoneurons, 5HT increased phrenic motoneuron activity [28,46] and enhanced phrenic responses to electrically evoked synaptic inputs [37]. Microinjection of 5HT into the XII motor nucleus increased XII motoneuron activity both in vitro [2] and in vivo [26], and potentiated the XII response to injected current in vitro [2]. To date, however, nothing is known about 5HT effects on XII motoneuron responses to upper airway negative pressure. In addition, the effects of 5HT in respiratory motor control have been reported to be long lasting, enhancing motoneuron activity for periods of minutes to hours [26,37,46]. The experiments described in these previous reports, however, have used mM concentrations of 5HT [26,37,46]. In vivo microdialysis studies show that the 5HT concentration in the XII motor nucleus extracellular space is ca. 8 nM [25]. Near this concentration, 5HT uptake mechanisms are rapid and effective [5], leading to the prediction that microinjection of lower 5HT concentrations will have an effect of limited duration. Thus, the long lasting effects of 5HT in respiratory motor control may be due to the relatively high concentrations used in the previous reports. Our goals in these experiments were therefore threefold. First, to determine the threshold concentration and maxi-

M.A. Douse, D.P. White~Brain Research 726 (1996) 213-222

214

mal effect of 5HT on XII motoneuron activity by performing a dose-response curve. Second, to determine the concentration dependent duration of 5HT effects at the XII motor nucleus. And third, to determine 5HT effects on the upper airway reflex mediated at the XII motor nucleus using the in vivo decerebrate cat preparation as an experimental model.

2. Materials and methods

2.1. Animal preparation Experiments were performed on 15 decerebrate adult cats of either sex, weighing between 1.8 to 4.6 kgs (mean = 3.0 4-0.2, _+ S.E.M.). Initially the animals were surgically anesthetized with halothane (4% induction; 1-2% maintenance). Surgical levels of anesthesia were characterized by slow regular breathing, blood pressure less than 130 m m H g and pupil contraction. The femoral artery and vein were cannulated to measure blood pressure continuously (Harvard) and to give fluids (5% dextrose in lactated ringers; 5 m l / k g / h ) . Dexamethasone was given (0.2 m g / k g ; i.m.) to minimize brain edema. Rectal temperature was monitored and maintained at 38_+ 0.5°C with a servo-controlled heating pad (Harvard). A tracheostomy was performed and a tracheal cannula tied into place for ventilation. For negative pressure application, an upper tracheal cannula was tied into place with its tip at the vocal cords and a nasal cannula inserted > 1 cm into one nostril. Quick setting epoxy was then used to occlude the mouth and other nostril. The upper tracheal cannula was connected to the nasal cannula and a pressure transducer (Fig. 1). Thus, negative pressure was applied to the upper airway from both above and below. The animals' head was held in a fixed position in a stereotaxic frame (head inclination at 45 °) throughout the experiment to minimize positional effects on XII activity. Upper airway negative pressure changes were produced with a syringe while the animals were ventilated through the lower tracheal cannula (Fig. 1; cf. [31-33]).

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The animals were placed into a stereotaxic frame, paralyzed with pancuronium bromide (0.2-0.4 m g / k g ; i.v.) and ventilated (Harvard 683) with oxygen enriched air ( 3 5 - 4 0 % 02). End-tidal CO 2 was monitored (Datex Normocap 200) and maintained between 4 - 6 % . Bilateral pneumothoraces were done as an aid to recording stability and an end-expiratory pressure of 2 - 3 cm H 2 0 applied to maintain lung inflation. The vagus nerves were cut bilaterally. The genioglossal branches of both hypoglossal nerves and the C5 phrenic nerve were exposed and prepared for recording (Fig. 1). The forebrain and medulla dorsal surface were exposed, the dura reflected bilaterally and the pia mater removed from the recording locations. All exposed surfaces were covered with agar, paraffin oil or bathed in saline. Decerebration was performed to the midcollicullar level by transection and aspiration [23]. Halothane was then discontinued and there was a minimum of 2 h before the experimental protocols were performed.

2.2. Neural recording and stimulation Whole nerve activity was recorded with bipolar silver wire electrodes, amplified (Grass P15 or Neurolog NL100), filtered ( 1 0 0 - 1 0 0 0 0 Hz) and a moving time average obtained (time constant = 100 ms; Bak Electronics PF-1). To aid in identifying XII motoneurons in the medulla, the XII nerves were stimulated with the same electrodes using a stimulus isolation buffer ( 0 - 5 V; 0.2 ms duration; Digitimer D4030). Signals were displayed on oscilloscopes (Tektronix 2216; Nicolet 310), thermal array chart recorder (Graphtec WR4000) and stored on video tape (Vetter 4000A). Using stereotaxic co-ordinates with the obex as a reference, micropipettes were positioned into the XII motor nucleus. Extracellular neural activity was amplified (Neurolog NL102), filtered (125-8000 Hz) and action potentials discriminated with a time amplitude window gate (Bak Electronics DIS-l). The XII motor nucleus was located by recording inspiratory activity at the appropriate brainstem co-ordinates and a large field potential upon stimulation of the XII nerve ( 0 - 5 V; 0.2 ms duration). Additionally, spike-triggered averaging of the neural activity recorded from the whole XII nerve, using the centrally recorded XII motoneuron activity as a trigger, confirmed pipette placement [26]. A sharp peak in the averaged signal confirmed that the neurons recorded in the medulla have axons in the XII nerve recorded peripherally and was therefore a XII motoneuron.

2.3. Neurochemical microinjections Fig. 1. Schematic of experimental preparation showing application of negative pressure to the isolated upper airways (Paw), whole nerve recordings for the left and right hypoglossal (L. and R. XII), and phrenic (Phr.) nerves, and pressure microinjection of neurochemicals via a pipette electrode into the XII motor nucleus of a decerebrate cat.

One to four barrel glass micropipettes suitable for extracellular neural recording and neurochemical injection were made (total tip diameter 5 - 1 0 ~m), filled with the appro-

M.A. Douse, D.P. White~Brain Research 726 (1996) 213-222

priate neurochemical(s) prepared in saline and pH corrected to 7.2-7.4 on the day of the experiment. The pipette holder has a side arm for neurochemical pressure injection and a silver wire for neural recording. All injection sites were positively identified electrophysiologically (see above). Neurochemical injection volume (100-500 nl) was directly determined by measuring the meniscus level in the pipette during pressure application (General Valve Picospritzer II). Control injections of the same volume of vehicle (165 mM saline; pH 7.2-7.4) controlled for volume and vehicle effect. 2.4. Protocols

The C O 2 thresholds for phrenic and phasic XII inspiratory-related neural activity were determined and end-tidal CO 2 was maintained at a constant 2 - 4 mmHg above the XII phasic activity threshold level. This corresponded to approx. 34 mmHg in Denver CO, approximately normal levels for cats. The neurochemical filled pipette was positioned into the XII motor nucleus and the experimental protocols were then performed. Two series of experimental protocols were performed. In the first series, a 5HT concentration (dose) vs. whole XII nerve response curve was obtained. This was achieved by microinjecting increasing concentrations of 5HT (100 hi) into the test XII motor nucleus and monitoring the ipsilateral whole nerve response. At least 2 rain separated microinjections of 5HT into the XII motor nucleus. Up to six concentrations of 5HT were used: 0.005, 0.05, 0.5, 5, 50 and 500 p~M. The point at which the largest increase in tonic XII neural activity was observed for each 5HT concentration was then used to construct a dose-response curve. The duration of 5HT effects at each concentration were obtained by measuring tonic XII activity levels every 10 s following microinjection of 5HT, for at least 120 s. At the highest 5HT concentration used (500 txM), tonic activity levels were monitored for over 10 rain following 5HT application into the XII motor nucleus. In addition, the XII whole nerve response to microinjection of 1.0 mM methysergide (non-selective 5HT2 antagonist; new nomenclature [15]) was monitored prior to and following microinjection of 5HT. In the second experimental series, test pressures of - 20 cm H 2 0 were applied to the upper airway, and the XII motor response monitored bilaterally before and after microinjection of 5HT (0.005 to 500 txM) into the test XII motor nucleus. The long lasting effects of 500 IxM 5HT, however, also allowed testing of multiple negative pressures (0, - 5 , - 1 0 , - 1 5 , and - 2 0 cm H 2 0 in random order). In addition, the XII reflex response to multiple negative pressures were obtained following microinjection of 1.0 mM methysergide (200-500 nl) into the test XII motor nucleus (n = 4). At least 2 min separated the negative pressure tests. The XII reflex response was quantified as the change in the phasic signal (expiratory trough to

215

inspiratory peak) of the integrated XII trace (time constant = 100 ms) of the five breaths obtained during application of negative pressure to the upper airway. Individual animals were used as their own control to minimize variability (i.e. determine the reflex response before and after each microinjection). 2.5. Data analysis

Hypoglossal whole nerve responses were quantified as integrated tonic activity during late expiration and as the phasic activity (expiratory trough to inspiratory peak) of the XII integrated trace (moving time average; time constant = 100 ms). Phasic XII nerve inspiratory peaks during control conditions were assigned a value of 100 arbitrary units (AUs). All XII neural responses to negative upper airway pressure and neurochemical microinjection were then expressed as a multiple of this control value for each animal. Each animal was used as its own control to obtain a paired response and to minimize variability between animals. In the first experimental series, reciprocal plots of 5HT concentration and XII whole nerve activity (i.e. 1/(5HT) vs. 1 / X I I activity) were constructed to obtain estimates of the 50% effective concentration (ECs0) and the 5HT concentration at the XII maximal response. Linear regression analysis of these plots produced estimates of the ECs0 and the maximum response. The significance of the concentration dependent time effects of 5HT were tested with a repeated measures ANOVA. The half-life of 5HT effects were calculated and compared with a paired t-test. In the second experimental series, the experimental design is of a matrix form, with pressure levels vs. neurochemical. Statistical analysis of the results was achieved with ANOVA techniques. An interaction term was used to identify changes in the XII response to upper airway negative pressure due to the neurochemical. The data obtained following application of the multiple negative pressures was analyzed using linear regression techniques. Comparing the effects of 5HT and methysergide on the XII reflex response was done by statistically comparing the slope terms with a paired t-test. In all cases, a P value < 0.05 was considered significant.

3. Results 3.1. General

The end-tidal CO 2 thresholds in the experiments described below using the decerebrate cat preparation were 4.4 _+ 0.2% (±S.E.M.) for phasic phrenic activity and 5.3 _+ 0.3% for phasic XII activity. In Denver, CO this corresponds to approx. 26 mmHg and approx. 31 mmHg for phrenic and phasic XII thresholds respectively. Endtidal CO 2 was then set at 0.5% above the XII threshold

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M.A. Douse, D.P. White/Brain Research 726 (1996)213-222

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Fig. 3. Duration of serotonin effects on ipsilateral tonic Xll activity after microinjection into the XII motor nucleus. A: Mean XII responses to 0.005 ~M (open triangles), 0.05 ~ M (filled triangle), 0.5 ~ M (open box), 5.0 ~M (filled box), 50 ~M (open circle) and 500 ~M (filled circle) serotonin. Standard error bars have been omitted for clarity. B: Mean XII response ( ± S.E.M.) to 0.5 txM (open box) and 500 p~M (filled circle) to serotonin. AUs: arbitrary units•

level (approx. 34 mmHg) and maintained at this level for the duration of the experiment. Pipettes were placed into the XII motor nucleus, just rostral to the obex where the majority of the genioglossal motoneurons are located [49]. XII motoneurons were recorded 0.3 mm rostral and 1.2 mm medial to the obex at depths ranging from 1.6 to 2.3 mm (mean = 1.9_+ 0.1 ram). All XII motoneurons had an inspiratory discharge pattern and were confirmed as XII motoneurons by their antidromic activation during stimulation of the medial (genioglossal) branch of the ipsilateral XII nerve. In the 11 cats examined, spike triggered averaging of whole XII nerve activity, using the centrally recorded XII motoneuron discharge as a trigger, revealed sharp peaks in the averaged signal, further confirming pipette placement.

significant changes in phasic XII activity levels when quantified as the expiratory trough to inspiratory peak integrated XII trace. There were also no significant changes in contralateral XII activity, which served as a control for the test side, and also monitored for neurochemical spread beyond the test XII motor nucleus. In each animal tested, the relationship between the peak amplitude of the tonic component of whole XII nerve response and the logarithm of the 5HT concentration was plotted (Fig. 2A). There was considerable variation between preparations in the magnitude and duration of effect at each 5HT concentration. The threshold concentration that just produced a detectable response ranged from 0.005 to 0.5 >M. As a group, the increase in XII tonic activity reached significance at 5HT concentrations above 5 ~M (Fig. 2B; P < 0 . 0 1 ) . Above 5 ~M, the dose-response curve tended to approach a plateau (Fig. 2B). Linear regression analysis of the reciprocals of the XI1 response vs. 5HT concentration ( 1 / X I I vs. I/[5HT]) for each animal (R 2 = 70 _+ 8) produced estimates of the maximal response and the effective 5HT concentration to pro-

3.2. Dose response

Pressure microinjection of 5HT into the test XII motor nucleus (100 nl; up to 6 concentrations ranging from 0.005 to 500 p~M; pH = 7.2-7.4) resulted in a concentration dependent increase in the tonic component of the ipsilateral XII whole nerve activity (Fig. 2). There were no

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M.A. Douse, D.P. White/Brain Research 726 (1996) 213-222

duce 50% of the whole XII nerve maximal response (ECs0). The maximal response was estimated to be 109 _+ 26 AUs and occurred at approximately 5 ptM (cf. Fig. 2). The ECs0 (a measure of the potency of 5HTs' effects on XII motoneuron activity [10]) was highly variable and estimated to be at 1.9 _ 0.9 ptM 5HT (cf. Fig. 2). Increasing 5HT concentration also increased the duration of the XII response (Fig. 3). At the lowest concentration where a consistent 5HT effect was seen (0.5 p,M), XII activity returned to baseline within 50 s (Fig. 3B and 4B). At the highest concentration (500 p,M) the effects lasted for over 2 min (Fig. 3B), and, when followed (n = 6), for over 10 min. This resulted in a significant difference between the calculated half-lives (25.6 + 7.1 s at 0.5 o,M vs. 464.6_+ 257.2 s at 500 p,M; P < 0 . 0 5 ) . All 5HT effects were reversed by microinjection of 1.0 mM methysergide (100 nl; pH = 7.2-7.4; n = 4). Prior microinjection of 1.0 mM methysergide into the test XII motor nucleus (200-500 nl; pH = 7.2-7.4; n = 4) resulted in a significant 42.7 +_ 9.2% ( P < 0.05) decrease in phasic ipsilateral XII activity (see Fig. 6). Subsequent microinjection of 5HT into the XII motor nucleus had no further effect. Control pressure microinjections of saline (100-500 nl; 165 mM) into the test XII motor nucleus had no apparent effect on ipsilateral XII activity.

3.3. Reflex response While all cats responded to pressure microinjection of 5HT into the XII motor nucleus with an increase in tonic XII activity, not all animals had a XII reflex response to upper airway negative pressure (cf. [21]). Therefore we report only those experiments in which a complete reflex protocol was performed (n = 5). Applying - 2 0 cm H 2 0 negative pressure to the upper airway resulted in a significant 4 7 . 8 _ 8.8% increase in peak phasic XII activity ( P < 0.01), with no change in tonic activity (cf. [16,31-33]). Microinjection of 5HT (100 nl) into the XII motor nucleus tended to increase the upper airway reflex response in some cases (Fig. 4). However,

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this effect was small and highly variable between animals. Thus, as a group (n = 5), 5HT did not significantly change the XII whole nerve response to upper airway negative pressure ( - 2 0 cm H20) at any 5HT concentration (Fig. 5). Indeed, with 500 IoM 5HT (100 nl) there was a slight decrease in the XII reflex response to - 2 0 c m H 2 0 applied to the upper airway. The long lasting effects of 500 IxM 5HT made possible determination of the slope or gain of the XII reflex response to multiple upper airway negative pressures (0, - 5 , - 1 0 , - 1 5 and - 2 0 c m H 2 0 in random order). Microinjection of 500 p,M 5HT (100 nl) into the XII motor nucleus resulted in a slight increase in the slope of the response curve from 3.0 _+ 0.7 (R 2 = 94 _+ 2) to 5.4 _+ 1.1 A U s / c m H 2 0 ( R 2 = 58 ± 12). However, this increase was highly variable and not significant. Indeed, pressure microinjection of large volumes (200-500 nl) of 500 I,M 5HT into the XII motor nucleus actually decreased the slope of the XII neural response to negative pressure from 2.7 _+ 0.5 to 0.8 _+ 0.9 A U s / c m H 2 0 ( P > 0.05). In contrast, prior pressure microinjection of 1.0 mM methysergide into the test XII motor nucleus (n = 4; 200-

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M.A. Douse, D.P. White~Brain Research 726 (1996) 213-222

500 nl; pH = 7.2-7.4) resulted in a significant 42.7 _+ 9.2% ( P < 0.05) decrease in phasic XII activity and a significant 44.6_+ 11.3% ( P < 0.05) decrease in the XII reflex response to - 2 0 cm H 2 0 upper airway pressure (Fig. 6). Methysergide (1.0 mM) also significantly decreased the slope of the response curve from 2.8 _+ 0.5 to 1.0 _+ 0.2 A U s / c m H 2 0 ( P < 0.05).

4. Discussion The major findings of this study are the short time dependence of serotonins' effects on XII tonic activity and lack of a modulatory effect of serotonin on XII reflex response to negative pressure applied to the upper airway. In contrast to previous studies where serotonins' effects have been reported to be long lasting (minutes to hours [26,37,44,46,55]), application of a lower concentration of 5HT resulted in a much shorter duration of action on XII activity (seconds). Surprisingly, however, 5HT did not augment the XII reflex response to upper airway negative pressure at any concentration. This is in contrast to previous studies where 5HT increased the responsiveness of other motoneurons to synaptic input [34,37,44,54,55]. Possible reasons for these differences will be discussed below.

4.1. Critique of methods' Decerebrate cats were used in these experiments for two reasons. First, serotonergic brainstem neurons are more active in this preparation than in anesthetized preparations [6], allowing determination of the effects of blocking endogenous 5HT on XII motor activity and reflex responses. Second, XII motoneurons are depressed by anesthesia to a greater extent than spinal respiratory motoneurons [ 17,18]. Indeed, the XII reflex response to upper airway negative pressure is substantially diminished by anesthesia [18]. As pointed out by Kubin et al. [26], anesthesia may also depress motoneuron response to 5HT microinjection. In the decerebrate cat preparation this is less of a problem. Thus, the end-tidal CO 2 thresholds for phrenic and XII activity were 4.4 _+ 0.2% and 5.3 _+ 0.3%, respectively, approximately normal levels for cats. Indeed, phasic respiratory related XII motoneuron activity has been recorded from freely behaving cats [45], supporting the validity of the decerebrate cat preparation. Finding the balance between microinjecting an adequate volume to affect all of the XII neurons in the motor nucleus vs. neurochemical spread beyond the nucleus is a concern. Estimates of the effective spread of 5HT are difficult without direct measurements. Theoretical and experimental considerations suggest that a volume of 100 nl microinjected into the brainstem forms a sphere of approx. 1 mm radius [29,47]. This volume would encompass much of the XII motor nucleus without excessive spread into adjacent brainstem sites. Monitoring both XII nerves and

the phrenic nerve aided in assessing the spread. Additionally, spread of 5HT into the nearby dorsal respiratory group located in the ventro-lateral nucleus tractus solitarius could have decreased the phasic component of XII activity [7]. This was not observed. Thus, although we cannot rule out 5HT effects on nearby interneurons, we believe that 5HT was essentially confined to the XII motor nucleus, as argued previously by Kubin et al. [26]. There was some variability in the XII response to microinjection of 5HT into the XII motor nucleus. This is probably due to biological variability, condition of the preparation and the precise location in the XII motor nucleus where the micropipette was placed. The variability of XII activity and the upper airway reflex over time and between animals may have potentially obscured 5HT effects on XII reflex responses. However, each animal was used as its own control to obtain a paired response (XII reflex response before and after 5HT application) and to aid in minimizing variability between animals. Monitoring both the XII nerves and the phrenic nerve also helped avoid non-specific changes in animal state. Finally, methysergide is a relatively non-selective 5HT2 antagonist (new nomenclature [15]). The use of methysergide, therefore, describes only a broad spectrum antagonism of endogenous 5HT. Our goal, however, was to determine if 5HT was directly exciting XII motoneurons through 5HT receptor activation and to block endogenous 5HT effects on XII motoneuron sensitivity to the upper airway reflex. We did not intend to perform a thorough dissection of 5HT receptor subtypes and their effects on XII activity and reflex responses.

4.2. Dose response Serotonin increased XII tonic activity, but had no apparent effect on phasic XII activity. This is similar to a previous report, although substantially higher 5HT concentrations were used in that study [26]. Increasing the 5HT concentration produced a graded increase in XII tonic activity and increased the duration of the XII response. Threshold effects on XII activity were obtained at 0.005 to 0.5 ~ M 5HT. Maximal XII responses were observed at 5 txM 5HT, well below the high concentrations of 5HT used in previous motor control studies [26,37,44,46]. The results obtained following microinjection of methysergide suggest that endogenous 5HT excites XII motoneutons (cf. [26]). The results also suggest that 5HT was directly increasing XII motoneuron activity via 5HT receptor activation. As shown previously [26], this activation was probably mediated by the 5HT2 receptor subtype. Thus, the estimated ECs0 values correspond better with the affinity of 5HT for the 5HT2 receptor than with the 5 H T I A receptor [42]. The estimated ECs0 for the increase in XII activity was higher than that reported tbr 5HT excitation of spinal motoneurons [48], but comparable to that reported for other brainstem neurons [4].

M.A. Douse, D.P. White/Brain Research 726 (1996) 213-222

Previous studies of 5HT effects on respiratory motor control have been reported to be long lasting (minutes to hours [26,37,46]). However, the experiments described in the previous reports have used mM concentrations of 5HT [26,37,46]. In contrast, 5HT effects on brainstem cardiovascular control have been elicited by nM concentrations of 5HT [27,56]. Additionally, the duration of the cardiovascular responses varied with the 5HT concentration used [27,56]. The data of Ribeiro-do-Valle et al. [44] also shows a concentration dependent duration of 5HT effects on the jaw-closure reflex. Our results demonstrate that 5HT effects on XII activity may be elicited with nM concentrations. At this concentration, 5HT effects last less than 50 s (Fig. 3). This is consistent with rates of 5HT reuptake mechanisms investigated in other parts of the brain [5]. Thus, our results suggest that the reported long lasting effects of 5HT are due to the relatively high 5HT concentrations used in the previous studies. Our data support the general finding of 5HT excitation of XII motoneuron activity [2,26]. However, Morin and co-workers [38,39] consistently observed a decrease in XII motoneuron activity upon application of 5HT into an in vitro neonate rat brainstem preparation. Concurrent with decreased XII motoneuron activity was membrane depolarization (mediated by 5HT2 receptors) and a decreased central respiratory drive potential [38,39]. The reasons for the differences between the results remains unclear, but may involve species or age specific differences in the balance of 5HT receptor subtypes present (e.g. 5HT2 depolarizing the neuron while 5HT1 decreased respiratory drive). Serotonin does have multiple and potentially opposing effects on respiratory motoneuron sensitivity to synaptic inputs, depending on the receptor subtypes involved (cf. [28]). Alternatively, spread of 5HT into the nearby dorsal respiratory group in the ventrolateral nucleus tractus solitarius could also result in a decreased phasic XII motor output [7].

4.3. Reflex response To clarify the role of neuromodulators such as 5HT in motor control it is important to not only assess their effects on basal motor activity but to also evoke a specific reflex or integrative pathway (cf. [35]). In non-respiratory motoneurons, 5HT application increased the neural response to glutamate in both lumbar [54,55] and facial motoneurons [34]. Similarly, 5HT application into the trigeminal motor nucleus enhanced both spontaneous neural activity and the jaw closure reflex [44]. In respiratory motoneurons, 5HT increased phrenic motoneuron neural activity [28,46] and augmented phrenic responses to electrically evoked synaptic inputs [37]. Application of 5HT into other motor nuclei therefore enhances motoneuron response to synaptic input. In an in vitro neonatal rat slice preparation, 5HT excited XII motoneurons via a non-specific XII motoneuron depo-

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larization and an increased slope of the XII motoneuron steady-state firing frequency vs. injected current relationship [2]. Serotonin effects on XII neural responses to synaptic or reflex input have not been examined in vivo, but the in vitro results suggest that 5HT would increase XII motoneuron sensitivity to reflex input. Surprisingly, however, application of exogenous 5HT into the XII motor nucleus did not augment the upper airway reflex response in our experiments. Indeed, at the highest concentration, reflex gain apparently decreased. This latter result may be due to the large increase in tonic activity obscuring the reflex increase in phasic XII activity. Thus, progressive saturation of XII motor output may have led to a diminished XII reflex response (cf. [8]). The reasons for the differences between our results and the previous studies are unclear, but may be due to: 1) activation of multiple 5HT receptor subtypes; 2) the method of motoneuron reflex activation; or 3) 5HT does not modulate the upper airway reflex at the XII motor nucleus level. Each will be discussed below. Exogenous 5HT at the XII motor nucleus may have opposing effects on reflex response, activating both excitatory 5HT2 and inhibitory 5HT1 receptor subtypes simultaneously. For example, 5HT applied to an in vitro neonatal rat brainstem/spinal cord preparation resulted in phrenic motoneuron depolarization and increased respiratory related synaptic drive. This response was mediated by 5HT2 receptors. When the 5HT2 receptors were blocked, a reduction of respiratory synaptic potentials was revealed, mediated by another, unidentified 5HT receptor subtype (probably 5HT1 [28]). However, in a recent study using decerebrate cats [26], increases in XII tonic activity were due to 5HT2 receptor activation, while application of 5HT1A agonists had no apparent effect. This suggests that the 5HT1A receptor subtype does not make an important contribution to 5HT modulation of XII motoneuron activity in the cat. Therefore, activation of multiple receptor subtypes may not have a significant effect on XII motor activity and reflex response in our experiments. However, further experiments are planned to examine this possibility. An important difference between our experiments and the previous studies [2,34,37,44,54,55] is the method of motoneuron reflex activation (i.e. negative pressure applied to the upper airways vs. chemical or electrical excitation of motoneurons). The upper airway reflex is primarily mediated by the superior laryngeal nerve which has afferent projections to the nucleus tractus solitarius (NTS, [22,30]). The NTS is an important site of respiratory sensory integration [24]. Electrophysiological studies show that the superior laryngeal afferents make monosynaptic connections with respiratory neurons in the ventrolateral NTS [1,20]. Recent electrophysiological data suggest that monosynaptic connections from these respiratory neurons are made onto XII motoneurons [41]. Thus, the upper airway reflex activation of XII motoneurons may be indirect and negative pressure may not be as strong an excita-

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tory input as direct chemical or electrical excitation. However, our use of negative pressure to asses 5HT modulation of XII motoneuron reflex effects may be more physiologically relevant. In contrast, the results obtained following microinjection of methysergide indicate that endogenous 5HT at the XII motor nucleus does provide an excitatory stimulus to XII motoneurons and also enhances upper airway reflex gain. This suggests important differences between exogenous and endogenous 5HT. However, methysergide decreased baseline XII activity, thus XII responses to sensory input would occur at a different portion of the input-output relationship between sensory input and XII motor activity (cf. [8,50,57]). Indeed, baseline XII activity and the XII response to the upper airway reflex were both reduced by approx. 45%, suggesting that the decrease in the XII reflex response due to methysergide was simply due to the decrease in baseline XII activity levels. This may occur by a non-specific effect on XII motoneurons, e.g. by changing XII motoneuron membrane potential (cf. [2]). In a strict sense, therefore, endogenous 5HT may not specifically augment XII reflex responses. We conclude that 5HT does not apparently modulate the upper airway reflex at the XII motor nucleus. Modulation of the upper airway reflex may, however, occur at other sensory integration sites in the hrainstem.

4.4. Implications The short lasting effects of low concentrations of 5HT on XII activity suggests that the role of 5HT in producing long term effects on motor control must be re-examined. Our results suggest that the previously reported long lasting effects of 5HT on respiratory motor control [26,37,46] may be due to the use of relatively high 5HT concentrations either overwhelming re-uptake mechanisms a n d / o r resulting in a tremendous stimulation of motoneuron activity with the induction of other mechanisms that produce long term effects on motoneuron activity. Our results also show that 5HT at the motor nucleus did not apparently augment the upper airway reflex. This is in contrast to previous studies [2,34,37,44,54,55]. We believe this difference is due to the different methods of reflex motoneuron activation and the lack of upper airway reflex modulation at the XII motor nucleus. This difference suggests the importance of using a physiologically relevant sensory input when assessing 5HT modulation of motoneuron reflex responses. Serotonin modulation of XII motor control may also have clinical implications. The serotonergic raphe system neural activities are remarkably state dependent, with reduced activity asleep compared to awake [9,11,19]. Current evidence suggests that the upper airway reflex is largely lost during sleep in human subjects [14,52,53]. Indeed, the upper airway reflex is believed to be critically important in maintaining airway patency in the awake obstructive sleep

apneic patient [36], with its loss during sleep leading to falling dilator muscle activity (e.g. genioglossus muscle innervated by XII motor nerve) and ultimately upper airway collapse in patients at risk due to inadequate airway anatomy [40,43]. Therefore, the loss of the upper airway reflex during sleep may be an important event in the pathophysiology of obstructive sleep apnea in those patients at risk due to inadequate airway anatomy. How serotonergic effects on XII motor control will apply to conditions during sleep is unclear. However, our hope is that studies concerning neurochemical modulation of XII motor activity and upper airway reflex response will provide new insights and, possibly, the rational for future investigations. In conclusion, the maximum XII response to 5HT was obtained at 5 IxM, well below previously tested 5HT concentrations in respiratory motor control. This led to a much shorter duration of 5HT effects than previously reported [26,37,46]. Surprisingly, 5HT had no effect on the XII reflex response to negative pressure applied to the upper airway. Serotonin modulation of the upper airway reflex may, however, occur at other brainstem sites.

Acknowledgements We are grateful to Drs. R.O. Davies and L. Kubin for their help and advice, and E.J. Puglisi for excellent technical and artistic assistance. This study was supported by NIH Grant HL48531.

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