Neurons in rostral ventrolateral medulla mediate vestibular inhibition of locus coeruleus in rats

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Neuroscience Vol. 77, No. 1, pp. 219–232, 1997 Copyright ? 1997 IBRO. Published by Elsevier Science Ltd Printed in Great Britain 0306–4522/97 $17.00+0.00 S0306-4522(96)00436-8

NEURONS IN ROSTRAL VENTROLATERAL MEDULLA MEDIATE VESTIBULAR INHIBITION OF LOCUS COERULEUS IN RATS S. NISHIIKE,*† N. TAKEDA,* T. KUBO* and S. NAKAMURA‡ *Department of Otolaryngology, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan ‡Department of Physiology, Yamaguchi University School of Medicine, Yamaguchi 755, Japan Abstract––The effects of caloric vestibular stimulation on the central noradrenergic neuron system were examined in the rat. In urethane-anesthetized rats, caloric stimulation inhibited the spontaneous activity of noradrenergic locus coeruleus neurons and increased systemic blood pressure. Electrical and chemical lesions in the ventrolateral medulla attenuated both the locus coeruleus inhibition and the blood pressure increase in response to caloric stimulation. Neither the neuronal inhibition nor the pressor effect was attenuated by any deafferentation of the forebrain or baroreceptors, or lesioning of the nucleus tractus solitarius. These findings indicate that the caloric stimulation-induced locus coeruleus inhibition is mediated by neurons in the ventrolateral medulla, and that these neurons also mediate the vestibulo-pressor responses. The locus coeruleus inhibition via the ventrolateral medulla is, however, considered to be independent of ventrolateral medulla-mediated systemic pressor effect. Collectively these findings suggest that the ventrolateral medulla is the major origin of inhibitory vestibular input to the noradrenergic neurons of the locus coeruleus, and that the ventrolateral medulla plays an important role in the vestibulo-autonomic response. Copyright ? 1997 IBRO. Published by Elsevier Science Ltd. Key words: caloric stimulation, nucleus prepositus hypoglossi, nucleus tractus solitarius, baroreceptors, decerebration, vestibulo-autonomic response.

Noradrenergic locus coeruleus (LC) neurons are reported to participate in the processing of sensory information. Various sensory stimuli, including vestibular otolith stimulation by body tilt, activate LC neurons.12,29 Vestibular caloric stimulation using hot-water irrigation of the middle ear, however, inhibits the spontaneous discharge of LC neurons.24,26 Middle ear irrigation with hot water induces ampulopetal endolymphatic flow in the horizontal semicircular canal, which increases the firing rate of vestibular afferents from the canal.2,15 These findings indicate that the activation of the horizontal semicircular canal inhibits LC neuronal activity. The LC inhibition occurs with a long latency, of approximately 80 s, after the commencement of caloric stimulation, and is long lasting (approximately 3 min).24 LC neuronal activity is not inhibited †To whom correspondence should be addressed at: Department of Physiology, Freı`e Universita¨t Berlin, Arnimallee 22, 14195 Berlin, Germany (present address). Abbreviations: ANOVA, analysis of variance; BP, blood pressure; DNB, dorsal noradrenergic bundle; FS, footpad stimuli; LC, locus coeruleus; MBP, mean blood pressure; NTS, nucleus tractus solitarius; PGi, nucleus paragigantocellularis; PrH, nucleus prepositus hypoglossi; PSTH, peristimulus time histogram; Rmag, magnitude of excitatory responses; VLM, ventrolateral medulla.

by non-specific stimuli, including irrigation of the middle ear with water at body temperature that does not cause endolymphatic flow in the semicircular canal, caloric stimulation of the middle ear after labyrinthectomy, and irrigation of the auricle with hot and cold water. These findings indicate that caloric vestibular stimulation specifically inhibits LC neuronal activity. It has been reported that vestibular stimulation produces a change in blood pressure (BP),18,33,35 and that it usually produces an increase in sympathetic nerve discharge.35,41 This indicates that caloric vestibular stimulation affects the sympathetic nervous system and BP, causing a vestibulo-autonomic response.27 All these findings suggest that caloric stimulation-induced LC neuronal inhibition is involved in the vestibulo-autonomic response. There are two possible pathways responsible for the LC neuronal inhibition: (i) vestibular inputs processed by some nucleus in the brain inhibit LC spontaneous discharge (primary pathway), and (ii) BP changes in response to vestibular stimulation secondarily inhibit LC spontaneous discharge (secondary pathway). We first examined the primary pathway. Using the retrograde tracing technique with wheatgerm agglutinin conjugated horseradish peroxidase, Aston-Jones et al.1 identified two major inputs to the

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LC: one from the nucleus paragigantocellularis (PGi) in the rostral ventrolateral medulla (rostral VLM) and the other from the nucleus prepositus hypoglossi (PrH). An inhibitory influence of the forebrain on LC neurons has also been reported.30 Our previous study, however, disclosed that a lesion in the PrH does not alter caloric stimulation-induced LC inhibition.25 In the present study, to localize the origin of the inhibitory input to the LC in response to caloric stimulation (primary pathway), we examined the effects of VLM lesions and deafferentation of the forebrain on caloric stimulation-induced LC neuronal inhibition. We then examined the secondary pathway. It has been demonstrated that changes in BP affect LC neuronal activity.22,34 It is thus possible that caloric stimulation-induced BP changes inhibit LC neuronal activity. Accordingly, to test the possible secondary influence of BP changes on LC neuronal activity, we examined the effects of both denervation of baroreceptors and lesions in the nucleus tractus solitarius (NTS), in which all baroreceptor afferents end,32,38 on the caloric stimulation-induced LC neuronal suppression. EXPERIMENTAL PROCEDURES

The animal experiments in this study were conducted according to the guidelines approved by the Animal Care Committee of Osaka University Medical School. Surgery Thirty-five male Sprague–Dawley rats (250–400 g, SLC, Japan) were used. The rats were anesthetized with urethane (1.3 g/kg, i.p.), supplemented as necessary during the experiments. One polyethylene catheter was inserted into a femoral vein for drug injections, and another was inserted into a femoral artery for the continuous recording of mean BP (MBP) in all experiments. The MBP data were collected using a strain-gauge transducer (DSK-101, Kawasumi) connected to the arterial catheter, and entered into an Apple computer (Macintosh, Quadra 700). Rats in which the MBP remained below 60 mmHg were excluded from the study. The rats were intubated with a tracheal cannula and then fixed in a stereotaxic apparatus so that the skull was horizontal. They were immobilized with gallamine triethiodide (15 mg/kg, i.v.; 30 mg/kg, i.p.). The body temperature was maintained at 37&1)C with a heating pad. We assessed the adequacy of anesthesia by monitoring the electroencephalographic patterns during the experiments. Stimulation electrode implantation For the electrophysiological identification of the location of the LC, bipolar stimulating electrodes were implanted into the frontal cortex and dorsal noradrenergic bundle (DNB) ipsilateral to the LC recording. The electrodes consisted of two insulated stainless steel wires (diameter 200 µm) with bared tips approximately 0.5 mm apart. The coordinates for these stimulation sites were: in the frontal cortex, 1.5 mm anterior to the bregma, 1.0 mm lateral to the midline, and 1.2 mm from the cortical surface; in the DNB, 1.5 mm anterior to the lambda suture point, 0.8 mm lateral to the midline, and 5.7–6.0 mm deep from the cortical surface.23 Stimuli applied to the frontal cortex were single square pulses of 1-ms duration, and those applied to the DNB were 0.5-ms duration. The current intensity range was 0.1–5 mA, and the frequency of stimulation 1 Hz, in all experiments.

Footpad stimuli (FS) were presented through a pair of 23-gauge needles placed subcutaneously in the medial rear footpad contralateral to the LC recording (n=3).6,9 These stimuli were single square pulses (0.5 ms in duration) and were applied at an intensity of 30 V and a frequency of 0.5 Hz in all experiments. A peristimulus time histogram (PSTH) of the neuronal response to FS was generated for 50 consecutive stimuli. Locus coeruleus recording The dorsal surface of the cerebellum was exposed by removing the overlying bone. The electrical activity of LC neurons was recorded extracellularly with glass micropipettes (5–10 MÙ) filled with 2 M NaCl or 0.5 M sodium acetate containing 2% Pontamine Sky Blue. A recording electrode was inserted anteriorly into the brain at an angle of 15–20) from a point 3.0 mm posterior to the lambda suture point and 1.2 mm lateral to the midline. The location of the LC was determined by the appearance of a field response of the LC evoked by DNB stimulation ipsilateral to the LC recording. As reported previously,23 when the DNB is stimulated, a field response is elicited in the dorsolateral tegmentum of the pons with a sharp localization in the LC (Fig. 1A). The single unit activity of LC neurons was recorded superposed on the field response when the recording electrode was adequately advanced. The spikes were antidromically evoked with low-stimulation intensity (0.1–1 mA) (Fig. 1B). The antidromic nature of DNBevoked responses was determined by collision extinction with spontaneously occurring spikes. The LC neurons thus identified revealed characteristic features, as described previously: wide spike duration, slow and tonic spontaneous firing, and excitation by tail pinches followed by long-lasting suppression of firing.13,23 Caloric stimulation The bulla tympani of the ear ipsilateral to the LC recording was opened by a retroauricular surgical approach.17 A polyethylene tube (outer diameter 1 mm) was inserted into the middle ear cavity. Through this tube, the middle ear was irrigated with 5 ml of hot water (44)C), at a rate of 0.1 ml/s, to cause vestibular activation.2,15,24,26 Caloric stimulation is most effective when the horizontal semicircular canal is in the vertical position. In our preparations, the rats were placed on the stereotaxic apparatus so that the horizontal canal was tilted approximately 30) with respect to the horizontal plane.4 Using the same preparations, we previously found that caloric stimulation altered the firing rate of vestibular nucleus complex neurons.26 In another study, we found that middle ear irrigation with hot and cold water induced caloric nystagmus in unanesthetized rats placed in the prone position.17 We therefore concluded that our procedure for caloric stimulation induced endolymphatic flow in the horizontal semicircular canal. In most experiments, the temperature of the middle ear was monitored with a thermistor. Section and lesion studies In section and lesion experiments, the rats were anesthetized with urethane (1.3 g/kg, i.p.) before the surgery. During the surgical procedure, the level of anesthesia was assessed by the absence of withdrawal or corneal reflex. The LC activity and MBP were recorded at least 1–2 h after the end of the surgical procedure. Most recordings were obtained 2 h after the procedure. After the VLM lesions, the MBP in all rats showed a brief increase followed by gradual and prolonged decrease. In our experiments, however, rats in which MBP remained below 60 mmHg were excluded

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Fig. 1. Electrophysiological method for (A,B) locating recording the electrode in the LC and (C) monitoring the response of LC neuron to caloric stimulation of the middle ear. (A) Field response of the LC evoked by electrical stimulation of the DNB. The stimulation intensity was 1 mA. (B) Antidromic responses of a LC neuron evoked by DNB stimulation with current intensity of 0.3 mA. Upward deflection is negative. Five sweeps are superimposed in each trace. (C) The response to caloric stimulation was characterized by transient excitation followed by prolonged inhibition. The inhibition occurred with a latency of 110 s and lasted for 160 s. Caloric stimulation increased MBP with a latency of 20 s and duration of 250 s. The black bar indicates the period of hot-water irrigation of the middle ear.

from the study. The critical time for the recording was about 6 h after the procedure. All lesion sites were histologically verified after the experiments. Bilateral electrical lesions in the ventrolateral medulla. Bilateral electrical lesions of the VLM were made in eight rats with bipolar (n=4) or monopolar (n=4) insulated stainless-steel electrodes (diameter 200 µm). The coordinates for the lesion sites were 3.1–3.3 mm posterior to the lambda, 1.6–2.0 mm lateral to the midline, and 8.5–8.7 mm ventral from the cortical surface or 0.5–0.7 mm above the ventral brain surface.6,9,28 Through the monopolar electrodes, a direct current of 500 µA was applied from a constant current source for 20 s. The neck muscles of the rats were used as the ground for the circuit. Direct current of 1 mA was passed through the bipolar electrodes for 20–30 s. Unilateral electrical lesions in the ventrolateral medulla. Unilateral electrical lesions, ipsilateral to the LC recording, were made in the VLM in three rats, with bipolar insulated stainless-steel electrodes (diameter 200 µm). The stereotaxic coordinates, current intensity, and duration were identical to those described above for the bilateral electrical lesions in the VLM.

Unilateral lesions in the ventrolateral medulla with microinjection of kainic acid In three rats, kainic acid (0.5 nmol) or saline (0.9%), saturated with Pontamine Sky Blue, in a volume of 0.1 µl, was microinjected into the ipsilateral VLM at the coordinates given above. The microinjection was done by pressure ejection, using a microsyringe (Hamilton) attached to the micropipette (diameter 300 µm). The positions of dye spots were identified in brain sections. Deafferentation of the forebrain The internal carotid arteries were ligated bilaterally to minimize bleeding during decerebration. The decerebration was carried out using a combination of aspiration and transection at the rostral pole of the superior colliculus (n=2). The transection zone was packed with 3% agar in saline. Denervation of baroreceptors In five rats, the skin and neck muscles overlying the trachea and carotid arteries were excised. The vagal nerves,

Abbreviations used in the figures Amb AP Cu ECu Gr In IO LRt

ambiguus nucleus area postrema cuneate nucleus external cuneate nucleus gracile nucleus intercalated nucleus inferior olive lateral reticular nucleus

mlf MVe Sp5 SpVe 7n 10n 12n

medial longitudinal fasciculus medial vestibular nucleus nucleus of the spinal tract of the trigeminal nerve spinal vestibular nucleus facial nucleus dorsal motor nucleus of vagus hypoglossal nucleus

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carotid sinus nerves and aortic depressor nerves on both sides were dissected free from the surrounding structures, and were then severed bilaterally.22,34 Lesions in the nucleus tractus solitarius In five rats, the skin and neck muscles were excised, and the occipital plate was removed. The caudal cerebellum and dura were also removed. After the obex was exposed, bilateral aspiration lesions were made in the NTS under a surgical operating microscope. The lesions extended rostrolaterally from the caudal border of the area postrema along the inner wall of the IVth ventricle, and terminated approximately 1.5 mm rostral to the area postrema. They extended ventrally approximately 0.5–1.0 mm below the dorsal surface of the medulla.11,28 Histology The recording sites in the LC were marked by iontophoretic ejection of Pontamine Sky Blue ("10 µA, 5 min). At the end of each experiment, the brain was removed and fixed in 10% formalin for more than 24 h. Serial frozen sections (50 µm) were cut, mounted, and stained with Neutral Red. In all lesion experiments, the lesion sites and the positions of the dye spots were verified histologically. Data analysis Action potentials, converted to square waves by a window discriminator and summed by a rate-meter over 10-s epochs, were displayed on the monitor of the Apple computer. The signals and MBP were also stored on the computer or on digital audio tape for off-line analysis. The spontaneous activity of LC neurons was recorded for at least 3–4 min before caloric stimulation. The mean and standard deviation (S.D.) of firing rate were determined by averaging spontaneous firing for 1 min (10-s bin width) before caloric stimulation. Excitation and inhibition were considered significant whenever the changes in firing rate were at least twice the S.D. of the mean background activity at two consecutive bins (20 s). The response offset was determined as the time at which the firing rate had returned to a consistent level within 2 S.D. of the baseline. The mean and S.D. of MBP were calculated by averaging the values for the last 1 min (six samples/min) before caloric stimulation. Increase and decrease were assumed to be significant whenever the changes in MBP were at least twice the S.D. of the baseline at two consecutive samples. The LC firing rate and MBP were averaged every minute, and the statistical significance of differences between the normal and bilateral VLM lesion groups was analysed by two-way analysis of variance (ANOVA) with repeated measures (group and time as factors). PSTH was analysed using the computer to determine excitatory and inhibitory epochs in response to FS.6,9 The baseline period was defined as the 500-ms epoch preceding stimulation, and the mean and S.D. of counts per baseline bin were determined. The onset of significant excitation was defined as the first of four consecutive bins (10-ms bin width) whose mean value exceeded the mean baseline activity by 2 S.D., and response offset was determined as the time at which activity had returned to a consistent level within 2 S.D. of the baseline. The following equation was used to normalize response magnitudes for differing baseline activity and to calculate the magnitude of excitatory responses (Rmag):6,9Rmag=(counts in excitatory epoch)" (mean counts per baseline bin# number of bins in excitatory epoch). Inhibition was defined using the computer as an epoch of at least 12 bins in which the mean count per bin was less than 35% of that during baseline. Statistical comparison of data was conducted using Student’s t-test or two-way repeated-measures ANOVA with post hoc Scheffe´’s F-test. The difference in type re-

sponse of LC neurons to caloric stimulation between two groups was compared using Chi-square statistics. RESULTS

Effects of caloric vestibular stimulation on locus coeruleus neuronal activity and mean blood pressure The activity of 23 neuronal units in the LC was recorded in three normal rats. The spontaneous discharge rate of these units ranged between 1.1 and 5.1 spikes/s (mean&S.E., 2.6&0.2 spikes/s). Responses of LC neurons to caloric stimulation of the middle ear were classified as pure inhibition, excitation followed by inhibition, pure excitation, or no effect (Table 1). The predominant effects of caloric vestibular stimulation on the spontaneous activity of LC neurons are inhibitory24 (Fig. 1C, top). The inhibition response and the subsequent inhibition of the excitation–inhibition response are attributed to specific vestibular activation.24 The excitation response and the excitation of the excitation–inhibition response are due to thermal somatosensory stimulation, stimuli that uniformly activate LC neurons.24,26 The inhibition, i.e. inhibition and excitation– inhibition responses, occurred with a long latency from the start of stimulation (Table 1). The period of inhibition persisted long after caloric stimulation. The firing rate in both was decreased to about 60% of the baseline activity. In comparison with the inhibition, the excitation, i.e. the excitation and excitation–inhibition responses, to caloric stimulation of the middle ear was characterized by a transient increase in firing with a short latency. The mean value for MBP recorded simultaneously with the recording of these LC neuronal units was 75.4&1.9 mmHg (n=23). The majority (78%; 18/23) of the MBP recordings demonstrated change in response to hot-water irrigation of the middle ear. In most instances, caloric stimulation elicited pure increase (Fig. 1C, bottom), but other responses of MBP were an increase followed by a decrease, a decrease followed by an increase, a decrease, and no effect (Table 1). The MBP increased by 7.1&0.9 mmHg in the increase response. The increase–decrease and decrease–increase responses (n=3) were characterized by a transient increase followed by a transient decrease and a transient decrease followed by a transient increase, respectively. Effects of bilateral electrical lesions in the ventrolateral medulla on the locus coeruleus inhibition in response to caloric stimulation Histological analysis of the lesions in the VLM in eight rats revealed that the bilateral electrical lesions covered a region approximately 500–1500 µm in diameter. The extent of the lesion in the VLM in a representative rat is shown in Fig. 2. The activity of 40 LC neuronal units was recorded in the eight rats after the bilateral electrical lesions

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Table 1. Response of locus coeruleus neurons and mean blood pressure to caloric stimulation in normal rats Excitation LC activity Inhibition Excitation followed by inhibition Excitation No effect Total

n 7 (30) 10 (44) 3 (13) 3 (13) 23

Inhibition

Latency (s)

Duration (s)

Change (%)

11&3 13&9

73&7 67&3

131.4&5.7 151.3&17.3

Latency (s)

Duration (s)

Change (%)

83&17 112&13

103&35 163&54

60.5&10.9 60.1&8.6

Latency (s)

Duration (s)

Change (mmHg)

29&12 160 20&10

109&21 50 25&5

7.1&0.9 3.6 8.1&2.8

Decrease MBP

n

Increase Decrease followed by increase Increase followed by decrease Decrease No effect Total

13 (56) 1 (4) 2 (9) 2 (9) 5 (22) 23

Increase

Latency (s)

Duration (s)

Change (mmHg)

100 95&15.0 70&40

30 55&25 110&60

"1.4 "4.9&0.2 "4.4&2.8

Latency (s), duration (s) and change (LC activity, % of baseline activity; MBP, mmHg) are shown as mean&S.E. The number (percentage) of units showing each response type to caloric stimulation is also given.

Fig. 2. Effects of electrical bilateral lesions in the VLM on LC neuronal inhibition in response to caloric stimulation. The coronal sections and neuronal activity are from a single rat. (A) Caloric stimulation produced increase of LC neuronal activity, with a latency of 10 s and duration of 110 s. The inhibition of the neuronal activity in response to caloric stimulation was abolished by the bilateral VLM lesions. MBP showed a slight decrease followed by an increase in response to caloric stimulation. The black bar indicates the period of hot-water irrigation of the middle ear. The change in the temperature of the middle ear is shown in the lower trace. (B) Schematic representation of bilateral lesion of VLM (plates from the atlas of Paxinos and Watson28). The right side of the scheme is ipsilateral to the LC recording.

were produced in the VLM. The firing rate of the recorded units was 3.1&0.3 spikes/s. Hot-water irrigation altered the spontaneous firing of 32 of the 40 LC units. In contrast to the results in normal rats,

half of the units recorded showed an excitation response to hot-water irrigation of the middle ear (Table 2, Fig. 2). Chi-square analysis indicated a significant difference in the frequency of response

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Table 2. Response of locus coeruleus neurons and mean blood pressure to caloric stimulation in rats with bilateral lesions in the ventrolateral medulla Excitation LC activity Inhibition Excitation followed by inhibition Excitation No effect Total

n 6 (15) 6 (15) 20 (50) 8 (20) 40

Inhibition

Latency (s)

Duration (s)

Change (%)

3&4 8&2

58&13 89&20

142.0&8.4 147.0&8.5

Latency (s)

Duration (s)

Change (%)

98&16 118&17

83&16 40&9

86.4&2.3 73.7&6.1

Decrease MBP

n

Increase Decrease followed by increase Decrease No effect Total

14 (34) 8 (20) 13 (33) 5 (13) 40

Increase

Latency (s)

Duration (s)

Change (mmHg)

14&2 38&11

28&3 83&22

"5.4&1.8 "4.0&0.7

Latency (s)

Duration (s)

Change (mmHg)

48&4 70&7

72&12 99&26

3.5&0.6 2.5&0.4

Latency (s), duration (s) and change (LC activity, % of baseline activity; MBP, mmHg) are shown as mean&S.E. The number (percentage) of units showing each response type to caloric stimulation is also given.

Fig. 3. Effects of electrical bilateral VLM lesions on the response of LC neurons and MBP to caloric stimulation. (A) In the VLM-lesioned group (n=23), caloric stimulation produced significantly less inhibition of LC neuronal activity than in the normal group (n=40) (two-way ANOVA with repeated measures, P<0.05). (B) In the VLM lesioned group, caloric stimulation had significantly less effect on MBP than in the normal group (two-way ANOVA with repeated measures, P<0.01). Mean values were compared between normal and VLM-lesioned rats every 1 min after caloric stimulation, using the unpaired t-test (**P<0.01; ***P<0.001). Prestimulus mean values were compared with each poststimulus value using Scheffe´’s F-test (*P<0.05). The error bars indicate S.E. Black bars indicate the period of hot-water irrigation of the middle ear.

type to caloric stimulation between the rats with electrical bilateral VLM lesion and normal rats (÷2=11.99, d.f.=2, P<0.01). The inhibition of LC neurons in response to hotwater irrigation of the middle ear was significantly attenuated in rats with bilateral lesions of the VLM (n=40), compared with that in normal rats (n=23; Fig. 3). Two-way ANOVA with repeated measures revealed significant effects of group (F1,183=6.6,

P<0.05), time (F3,183=32.9, P<0.0001) and group#time interaction (F3,183=2.1, P<0.05). A significant difference of LC firing between two groups was observed 3 min after the start of stimulation (unpaired t-test, P<0.01). After the VLM lesions, MBP recordings showed rebound increase and subsequent gradual uniform decrease of BP; the same results were obtained with unilateral electrical and chemical VLM lesions. After

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Fig. 4. Effects of electrical unilateral VLM lesion on LC neuronal inhibition in response to caloric stimulation. The coronal sections and neuronal activity are from a single animal. (A) Caloric stimulation did not produce any response in LC neuronal activity. MBP showed no response to caloric stimulation. The black bar indicates the period of hot-water irrigation of the middle ear. The change in the temperature of the middle ear is shown in the lower trace. (B) Schematic representation of unilateral lesion of the VLM. The right side of the scheme is ipsilateral to the LC recording.

the exclusion of rats in which MBP remained below 60 mmHg, the mean MBP in the bilateral VLM lesions was 85.1&3.3 mmHg. The MBP recordings showed various responses to caloric stimulation in the rats with bilateral VLM lesions (Table 2). The response of MBP to hot-water irrigation was significantly less pronounced in rats with bilateral lesions of the VLM (n=40) than in normal rats (n=23) (Fig. 3). Analysis revealed significant effects of group (F1,183=7.8, P<0.01), time (F3,183=6.7, P<0.001) and group#time interaction (F3,183=7.3, P<0.0001). A significant difference of MBP between two groups was observed 1 min after the stimulation (unpaired t-test, P<0.001).

Effects of unilateral electrical or chemical lesions in the ventrolateral medulla on the locus coeruleus inhibition In the three rats with unilateral electrical lesion in the VLM ipsilateral to the recording, 15 LC neuronal units were recorded after the lesion. The extent of the lesion site in a representative animal is shown in Fig. 4. Similar to the effects in the rats with bilateral VLM lesions, hot-water irrigation activated the majority (excitation response: 67%, n=10) of the LC units (Fig. 4). An inhibitory response was observed in 20% (n=3; excitation–inhibition response: 13%, n=2; inhibition response: 7%, n=1), and no effect in 13% (n=2). Caloric stimulation elicited an increase

response in 40% (n=6) of MBP recordings, a decrease response in 20% (n=3), and no effect in 40% (n=6). The extent of electrical lesions was clearly defined by light microscopy. After the data for the extent of each lesion site were stored in the computer, the region responsible for the inhibition of LC neurons was precisely localized by constructing using the computer overlapping lesions ipsilateral to the recording (bilateral lesions, eight rats; unilateral lesions, three rats). The site of overlap thus obtained from data for 11 lesions is shown in Fig. 5. The overlapping site involved the ventromedial PGi, just lateral to the inferior olive and centered approximately 300–500 µm above the ventral surface of the medulla. Electrical lesions of the facial nucleus, parvocellular reticular nucleus, and lateral reticular nucleus did not attenuate the LC neuronal inhibition in response to caloric stimulation (data not shown). In two rats, 16 LC neuronal activity was recorded after the microinjection of kainic acid into the unilateral VLM. The site of kainic acid injection in one of them is shown in Fig. 6. The majority (63%; 10/16, no effect) of the units did not respond to caloric stimulation (Fig. 6). Five neurons showed an excitation response and the remaining one showed an inhibition response. In 38% (6/16) MBP recordings, caloric stimulation evoked the decrease response

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The excitation response was observed in 18% (n=4), and there was no effect in 32% (n=7). The mean value of the MBP recordings was 84.7&1.7 mmHg (n=22). Caloric stimulation increased most (increase response: 54%, 12/22) of MBP recordings (Fig. 8A). Other MBP responses were an increase–decrease response in 18% (n=4), a decrease response in 5% (n=1), and no effect in 23% (n=5). Fig. 5. Computer overlap reconstruction of lesions in the VLM. Lesions are ipsilateral to the LC recording from 11 rats (bilateral, eight rats; unilateral, three rats). The sites where more than three lesions overlap are shaded. The right side of the scheme is ipsilateral to the LC recording. The location of the PGi is indicated on the left side of the scheme. Note the consistent overlap of lesions with the ventromedial PGi in the rostral VLM.

(Fig. 6). Three MBP showed an increase response, one showed decrease–increase and the remaining six showed no effect. Microinjection of saline into the VLM did not attenuate LC neuronal inhibition in response to caloric stimulation (data not shown). Effects of bilateral electrical lesions in the ventrolateral medulla on the neuronal response to footpad stimuli In two normal rats, data for PSTH evoked by FS were obtained from nine LC neurons. FS activated all LC neurons recorded, at a mean onset latency of 27&3 ms and with a duration of 317&26 ms (Fig. 7A). In seven of the nine LC neurons, the FS-evoked excitation was typically followed by inhibition lasting 323&85 ms, a characteristic observed in LC neurons following activation by synaptic or antidromic stimuli.6,9 In two of the rats with bilateral VLM lesions (Fig. 7B), the neuronal response of 10 LC neurons to FS was recorded. Most of the neurons (60%; 6/10) showed no response to FS (Fig. 7C). The remaining four showed excitation, and in one of the four, this was followed by inhibition. The excitation of LC neurons in response to FS was significantly less pronounced in rats with bilateral lesions of the VLM (Rmag=28&10) than in normal rats (Rmag=108&16) (unpaired t-test, P<0.01). The VLM lesions did not alter the spontaneous discharge of LC neurons (normal rats: 2.6&0.5 spikes/s; rats with VLM lesions: 2.4&0.3 spikes/s, unpaired t-test, P>0.7).

Effects of baroreceptor denervation Twenty LC neuronal units were recorded in five rats after sectioning of vagal nerves, carotid sinus nerves and aortic depressor nerves. The spontaneous discharge of the units was 3.2&0.3 spikes/s. Despite there being no baroreceptor afferents, the inhibitory response was observed in the majority (55%, 11/20) of LC neurons recorded (inhibition response: 30%, n=6; excitation–inhibition response: 25%, n=5) (Fig. 8B). The excitation response was observed in 15% (n=3), and there was no effect in 30% (n=6). The mean value of MBP recordings was 89.8&2.1 mmHg (n=20). In most units, the MBP was increased by caloric stimulation (increase response, 55%, n=11/20) (Fig. 8B). Other responses of the MBP were a decrease–increase response in 10% (n=2), a decrease response in 5% (n=1), and no effect in 30% (n=6).

Effects of lesions in the nucleus tractus solitarius Histological analysis of the coronal sections indicated that, in these five rats, most of the area of the NTS was lesioned. The extent of the lesion around the area of the NTS in a representative rat is shown in Fig. 9. In these five rats, 23 LC neuronal units were recorded after the NTS lesions. The discharge rate of the neurons was 2.6&0.3 spikes/s. All of the 23 LC neurons showed changes in response to hot-water irrigation of the middle ear. Hot-water irrigation produced the inhibitory response in most (70%, n=16) of the neurons recorded (inhibition response: 17%, n=4; excitation–inhibition response: 53%, n=12) (Fig. 9). The excitation response was observed in 30% (n=7) of the neurons. The mean value of MBP recordings was 93.6&3.0 mmHg. The responses of the MBP were an increase response in 83% (n=19) (Fig. 9), an increase– decrease response in 9% (n=2), a decrease response in 4% (n=1), and no effect in 4% (n=1).

Effects of deafferentation of forebrain The activity of 22 LC neurons was recorded in two rats after precollicular decerebration. The firing rate of the LC neurons recorded was 2.8&0.4 spikes/s. The inhibitory response occurred in 50% (n=11) of the neurons recorded (inhibition response: 32%, n=7; excitation–inhibition response: 18%, n=4) (Fig. 8A).

DISCUSSION

Our results with lesion studies demonstrate that the VLM, particularly its PGi regions, is an important part of the pathway conducting vestibular information to the LC. Bilateral and unilateral electrical

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227

Fig. 6. Effects of chemical unilateral VLM lesion on LC neuronal inhibition in response to caloric stimulation. The coronal section and neuronal activity are from a single animal. (A) After microinjection of kainic acid (0.5 nmol/0.1 µl) into the unilateral VLM, this LC neuron did not respond to caloric stimulation, nor did the MBP. The black bar indicates the period of hot-water irrigation of the middle ear. The change in the temperature of the middle ear is shown in the lower trace. (B) Schematic representation of unilateral injection site (arrow) of the VLM. The right side of the scheme is ipsilateral to the LC recording.

lesions and unilateral chemical lesions all attenuated LC neuronal inhibition induced by caloric vestibular stimulation. Moreover, the VLM lesions also attenuated caloric stimulation-induced pressor effects. None of the deafferentation of the forebrain, deafferentation of baroreceptors, and lesions of the NTS attenuated either the neuronal inhibition or the pressor effects. Methodological considerations In the present study, the majority (74%) of LC neurons recorded in normal rats were inhibited by irrigation of the middle ear with hot water. The inhibition occurred with a long latency and persisted long after caloric stimulation (Table 1, Fig. 1C). Our previous findings that neither non-specific stimulation of the middle ear and auricle nor caloric stimulation in the labyrinthectomized rats caused LC inhibition indicate that caloric vestibular stimulation specifically inhibits LC neuronal activity.24 In comparison with the inhibition, the excitatory response of some LC neurons to caloric stimulation of the middle ear was characterized by a transient increase in firing with a short latency (Table 1, Fig. 1C). The excitatory responses of LC neurons to this caloric stimulation are similar to those of LC neurons to sensory stimuli,12 suggesting that these excitatory responses can be attributed predominantly to thermal

somatosensory responses.24,26 However, caloric stimulation with hot water (44)C) can elicit nociception in rats. In our previous study, during prolonged LC inhibition induced by caloric stimulation, nociceptive tail pinch stimuli activated LC neuronal activity24. This indicates that the LC inhibition following caloric stimulation is not due to the nociceptive effects of this stimulation. In urethane-anesthetized rats, nociceptive stimuli invariably excite LC neuronal activity, rather than inhibit it.13,23 It is possible that the inhibition following the excitation of LC neurons in this study was due to the postactivation inhibition mediated by á2-adrenoceptors.5,7 However, this appears unlikely, because the iontophoretic application of GABAA receptor antagonist blocks the inhibitory component without affecting the excitatory component of these responses.24 Moreover, the caloric stimulationinduced LC inhibition lasted much longer than the inhibition mediated by á2-adrenoceptors, that is, a few hundred milliseconds6,9 (Fig. 7A). We previously found that caloric vestibular stimulation immediately changes the neuronal activity in the vestibular nucleus complex.26 The LC neuronal activity, however, responds to caloric stimulation with a long latency.24 Our previous study disclosed that electrical vestibular stimulation caused shortlatency LC neuronal suppression. We therefore

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Fig. 7. Blockade of FS-evoked excitation of LC neurons by VLM lesion. (A) Representative example of a PSTH generated during single-pulse electrical stimulation of the footpad in a normal rat. FS yielded a potent, short-latency excitation of this neuron. Excitation was followed by inhibition of impulse activity. (B) Schematic representation of bilateral lesion of the VLM. The right side of the scheme is ipsilateral to the LC recording. (C) PSTH for FS after electrical bilateral VLM lesion (as shown in B). FS-induced excitation was attenuated in this neuron. Arrows indicate the time of FS. Stimulation intensity in (A) and (C) was 30 V. PSTH was generated for 50 consecutive stimuli, presented at 0.5 Hz. (D) Caloric stimulation caused excitation of the same neuron as shown in (C). An inhibitory response to caloric stimulation was not observed in the neuron. MBP showed decrease followed by prolonged increase. The black bar indicates the period of hot-water irrigation of the middle ear.

concluded that the long latency of LC neuronal inhibition after caloric stimulation is a result of the superposition of short-duration LC somatosensory excitation on the vestibular LC inhibition with a short latency.24 In most instances, MBP was increased by caloric stimulation (Fig. 1C). It has been reported that vestibular stimulation usually produces an increase in sympathetic nerve discharge.35,41 The increased sympathetic responses may cause an increased BP. However, other past studies demonstrated that low-intensity vestibular nerve stimulation produces BP decrease in anesthetized and decerebrated animals.18,33 It has been considered that the direction of the BP response to vestibular stimulation depends on the intensity of the vestibular stimulation41 or on the depth of anesthesia.14,33 It is possible that the caloric stimulation-induced BP increase represents an arousal response to noxious stimulation. However, this does not seem likely, because the BP increase response was also observed in precollicular decerebrated rats in this study.

The pathway responsible for locus coeruleus neuronal inhibition in response to caloric stimulation In this study, we first examined the origin of the primary inhibitory pathway to the LC. It has been reported that the PrH is one of the major inputs to the LC,1 and that it contributes GABAergic inhibitory input to the LC.10,11 We previously found that caloric stimulation-induced LC inhibition was attenuated by intravenous and microiontophoretic application of GABAA antagonists, suggesting that the inhibition is mediated by GABAA receptors.24 However, in another study we demonstrated that, in PrH-lesioned rats, caloric stimulation suppressed LC neuronal firing.25 Moreover, PrH neurons antidromically activated from the LC do not respond to caloric stimulation. These findings suggest that the PrH does not take part in the caloric stimulation-induced LC suppression. It has been reported that the PGi in the rostral VLM is another major input to the LC.1 Electrical stimulation of the PGi excited the majority of LC

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229

Fig. 8. Effects of (A) deafferentation of the forebrain and (B) bilateral sectioning of vagal nerves, carotid sinus nerves and aortic depressor nerves on the inhibition of LC neurons in response to caloric stimulation. (A) Caloric stimulation produced LC neuronal inhibition and MBP increase in the precollicular decerebrated rat. (B) Even though the influence of baroreceptors was excluded, caloric stimulation still produced LC neuronal inhibition and MBP increase. The black bars indicate the period of hot-water irrigation of the middle ear. The change in the temperature of the middle ear is shown in the lower traces.

neurons, and inhibited the minority of the neurons.9 The excitatory response of LC neurons is considered to occur by a non-N-methyl--aspartate-mediated pathway, and the inhibitory response by an adrenaline-mediated pathway. In the present study, bilateral electrical lesions of the VLM involving the PGi significantly attenuated the LC neuronal inhibition caused by caloric stimulation (Fig. 2). This finding suggests that, in addition to the non-Nmethyl--aspartate- and adrenaline-mediated pathways, there is a GABA-mediated inhibitory pathway from the VLM to the LC. The possible pathways for the GABAergic input are direct projection from GABAergic neurons in the VLM31 to the LC and indirect projections from the VLM to GABA neurons in the periventricular regions lateral and caudal to the LC, which could have an inhibitory influence on LC neurons.19 The same result obtained in the rats with bilateral VLM lesions was observed in rats with electrical unilateral VLM lesions ipsilateral to the LC recordings (Fig. 4). This finding is consistent with the observation of Aston-Jones et al.,1 who used retrograde tracer experiments to show that the main projection of the VLM to the LC is ipsilateral. Microinjection of kainic acid into the unilateral VLM also blocked the caloric stimulation-induced LC inhibition (Fig. 6). All of these findings indicate that the neurons in the ipsilateral VLM inhibit LC neuronal activity in response to caloric stimulation.

It has been reported that, in cats, VLM neurons respond to vestibular electrical and body tilt stimulation.42,43 The majority of VLM neurons are considered to receive otolith inputs. However, the present study suggests that the canal inputs induced by caloric vestibular stimulation affect VLM neurons. In rats, it has been morphologically demonstrated that the PGi in the VLM receives the afferents from the medial vestibular nucleus,36,37 to which the canal inputs are mainly transmitted. This morphological evidence is consistent with our present data. Rasmussen and Aghajanian30 demonstrated that the serotoninergic system in the frontal cortex polysynaptically controls LC neurons. In precollicular decerebrated rats, however, we observed LC neuronal inhibition and MBP increase in response to caloric stimulation (Fig. 8A), indicating that the caloric stimulation-induced inhibition of LC neurons is not mediated by the forebrain. This finding is consistent with the observation that decerebration has little effect on the vestibulo-sympathetic response.18,33 Cardiovascular stimulation

aspects

of

caloric

vestibular

The bilateral electrical VLM lesions significantly attenuated the MBP increase, as well as the LC inhibition, following caloric stimulation (Fig. 2). The VLM is a major sympathoexcitatory nucleus in the

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Fig. 9. Effects of bilateral NTS lesions on LC neuronal inhibition in response to caloric stimulation. Coronal sections and neuronal recordings are from a single rat. (A) Caloric stimulation produced long-lasting inhibition preceded by transient increase of LC neuronal activity after bilateral NTS lesions. MBP was increased by caloric stimulation. The black bar indicates the period of hot-water irrigation of the middle ear. The change in the temperature of the middle ear is shown in the lower trace. (B) Schematic representation of bilateral lesion of the NTS. The right side of the scheme is ipsilateral to the LC recording.

brain,20,21 and has been reported to provide not only tonic but also phasic excitation of sympathetic preganglionic neurons, contributing to the maintenance and reflex control of BP.16 The VLM is also considered to mediate the vestibulo-sympathetic response.41 These findings suggest that the VLM contributes to the increased BP in response to caloric vestibular stimulation. LC neuronal activity is suppressed by increased BP.22,34 It is possible that the LC neuronal inhibition induced by caloric stimulation is a secondary effect of the vestibulo-pressor response (the secondary pathway). This is unlikely, however, since neither denervation of baroreceptors nor lesions in the NTS, in which all baroreceptors terminate,32,38 attenuated the LC neuronal inhibition induced by caloric stimulation (Figs 8B, 9). These findings indicate that caloric stimulation-induced inhibition of LC neuronal activity via the VLM is not due to a VLM-mediated pressor effect of vestibular stimulation. Moreover, it was demonstrated that LC neurons respond more to changes in blood volume than to changes in BP.8 Locus coeruleus stimulation

excitation

induced

by

caloric

Computer overlap reconstruction of the VLM lesions indicated that the lesions seemed to involve

the ventromedial PGi (Fig. 5), which is reported to mediate LC excitatory response to FS.6,9 Indeed, FS-evoked LC excitation was significantly attenuated by the VLM lesions (Fig. 7B,C). In the VLMlesioned rats, however, the LC neuron, which was not excited by FS, showed an excitatory response to caloric stimulation (Fig. 7D). It is therefore possible that the caloric stimulation-induced excitation of LC neurons is mediated by nuclei other than the PGi. The middle ear cavity is supplied by the tympanic nerve, a branch of the glossopharyngeal nerve.39 The mastoid cells and tympanic membrane are supplied by the meatal nerves, which are derived from the auriculotemporal branch of the trigeminal nerve. It is possible that the caloric stimulation of irrigation of the middle ear activates these nerves, leading to LC excitation. Some fibers of the glossopharyngeal nerve terminate in the NTS, whereas other fibers of this nerve and fibers of the auriculotemporal branch terminate in the trigeminal nucleus.39 The trigeminal nucleus may excite LC neurons in response to caloric stimulation, and the LC excitation via the trigeminal nucleus may not be mediated by the VLM. It is possible the caloric stimulation-induced LC excitation is due to the contralateral VLM neurons (in the unilateral lesion study) or surviving neurons from bilateral electrical VLM lesioning.

Medulla mediates vestibulo-LC response

Physiological significance

231 CONCLUSIONS

The vestibulo-autonomic response consists of a cardiovascular sympathetic component and an emetic parasympathetic component.3,41 Amphetamine, which facilitates noradrenergic neural transmission with an increase in noradrenaline release from the nerve terminals, is effective in preventing the emetic parasympathetic component of motion sickness.40 Therefore, it is suggested that the inhibitory VLM-LC circuit, which transmits and processes vestibular information, contributes to the development of motion sickness. We found in this study that lesions in the VLM abolished the inhibitory response of LC neuronal activity as well as the pressor response to caloric stimulation, indicating that VLM neurons may be a common locus of the vestibular effects on the sympathetic and parasympathetic nervous system.

These data demonstrate that caloric stimulationinduced LC inhibition is mediated by the VLM. The VLM also mediates increases in BP in response to caloric stimulation. Caloric stimulation-induced LC inhibition is not a secondary effect of the VLM-mediated pressor response. It is suggested that the VLM is involved in both the cardiovascular sympathetic component of vestibulo-autonomic response and the emetic parasympathetic component, the latter through VLM-mediated LC inhibition.

Acknowledgements—The authors thank Dr M. Umemoto for his technical advice. This study was partly supported by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan.

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