In acute experiments on nembutal-urethan-anesthetized rats, structures selectively sensitive to capsaicin were found near the ventral surface of the medulla at ...
Neuroscience Letters 182 (1994) 129-132
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Capsaicin-sensitive area in the ventral surface of the rat medulla S t a n i s l a w V. K o u l c h i t s k y , O l e g A . A z e v , A l e x a n d e r V. G o u r i n e , V l a d i m i r A . K u l c h i t s k y Laboratory of Brainstem Physiology, Institute of Physiology, Academy of Sciences, 220725 Minsk, Beylorussia
Received 17 August 1994; Revised version received26 September 1994; Accepted 10 October 1994
Abstract
In acute experiments on nembutal-urethan-anesthetized rats, structures selectively sensitive to capsaicin were found near the ventral surface of the medulla at the exit of hypoglossal nerve roots. Microinjection of 5-50 nl 0.01% capsaicin to the rostral region of the capsaicin-sensitive area mostly activated respiration, arterial pressure and heart rate (HR) while that to the caudal region inhibited arterial pressure and HR. In chronic experiments on rats, injection of 25 nl 1% capsaicin to the caudal capsaicin-sensitive area led to a decrease in arterial pressure by 3545% and in HR by 10-15% within a week after operation. Arterial pressure and HR virtually reached the control level and the rostral and caudal ventral medulla showed asymmetric distribution of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d)-positive cells by the end of the 2nd week. It is suggested that nitric oxide may be involved in the mechanisms of neurochemical rearrangements in the brainstem after application of capsaicin to the caudal ventrolateral medulla (CVLM). Key words: Ventrolateral medulla; Capsaicin; NADPH-diaphorase; Arterial pressure; Heart rate; Phrenic EMG activity
It is believed [6] that most neurones of the CNS are insensitive to acute and prolonged action of capsaicin. However, a line of evidence favours a suggestion that hypothetical sensory neurones of the central chemo-sensitive areas of the medulla may selectively respond to capsaicin. The suggestion is supported by findings that among few capsaicin-sensitive areas of the CNS the drug may cause degeneration of nerve terminals in specific regions of the cat medulla involved in the control of the cardiovascular and respiratory systems [8]. Since central chemoreceptors of the ventral medulla possess high sensitivity to changes in blood and liquor H + ion concentration (pH) [2,3,9,10] and capsaicin and protons seem to activate sensory nerves and cells via a c o m m o n mechanism [1], we attempted to investigate the effect of capsaicin on the ventrolateral medullary structures including the so-called 'central chemoreceptors'. We suggested that prolonged application of capsaicin to the ventrolateral surface of the rat medulla in chronic experiments may result in redistribution of nitric oxide synthase (NOS)-positive bulbar structures since nitric oxide (NO)
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is known to be an intercellular/intracellular messenger in several areas of the CNS [4,7,11] and can be involved in the processes of nociceptive input [14]. Thus, another aim of the present study was to investigate the distribution of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) (apparently, identical with NOS [4,7])-positive cells in the rat medulla after unilateral application of capsaicin to the caudal ventrolateral medulla (CVLM). Experiments were performed on 19 Wistar male rats (230-270 g), which were anesthetized with urethane and nembutal (400 and 30 mg/kg i.p., respectively), and on conscious animals (n = 9). In acute experiments, the ventrolateral medullary surface was exposed and the dura was reflected and the arachnoid removed from the injection sites. Capsaicin injection vehicle (10% alcohol/10% Tween 80 in artificial cerebrospinal fluid) or capsaicin (Merck, Darmstadt, Germany) in volume 5 nl was unilaterally injected for 30 s to the medulla with a nanoliter pump via a glass micropipette with a tip diameter 50/lm in increasing concentration (0.5-5.0-50.0 nmol). Electromyogram (EMG) from the lateral costal diaphragm was recorded using bipolar steel wires as were blood pressure (BP) in the left common carotid artery and electrocardi-
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S. E Koulchitsky et al./Neuroscience Letters 182 (1994) 129-132
Integrated Diaphragmatic EMGActivity BP 130 ]
~
(ram Hg) 8O
l
HR 430 (b'min~) 400.11
CAPSAICIN(0.01%,5 nl)
5s
Fig. I. Changes in phrenic EMG, BP and HR after injection of 5 nl 0.01% capsaicin to RVLM 500/tm deep from surface. Horizontal bar indicates duration of injection.
ogram to calculate the heart rate (HR). The E M G signal was processed by full-wave rectifying and smoothing with a moving-average digital filter. Respiratory rate was calculated from breath-to-breath intervals. A relative measure of the amplitude of each breath was obtained by subtraction of the maximal and minimal amplitudes of the EMG. From these values, a relative measure of minute ventilation could be calculated. Rectal temperature was maintained close to 37.5°C by a servocontrolled blanket. A day before chronic experiments, nembutal-urethan-aneslhetized (20 and 400 mg/kg, respectively, i.p.) rats were unilaterally (CVLM) treated with 25 nl 1% capsaicin or 25 nl saline in accordance with atlas coordinates [12]. Arterial pressure and HR were recorded with a cuff at a tail artery. 2 weeks after unilateral injection of 1% capsaicin to the CVLM all rats were given lethal overdoses of nembutal and perfused transcardially with 0.9% saline followed by 4% paraformaldehyde. NADPH-d was detected in the brainstem histochemically using tetrazolium salt technique [13]. Experimental data were analysed with Student's t test. Three principal conclusions can be drawn from analysis of the results. (1) The CVLM of rats possesses structures selectively sensitive to capsaicin. Topographically, the capsaicin-sensitive area is located lateral from the pyramidal tracts at the level of the exit of hypoglossal nerve roots and spreads rostrally and caudally 1.0-1.5 mm beyond the exit. Dorsally, the area spreads up to 2 mm deep from the ventral medullary surface. Microinjection of 5-50 nl 0.01% capsaicin to different depths from the ventral surface revealed the maximal effect on BP and phrenic E M G at a depth of 1 mm. These effects on respiration, BP and HR varied in strength and direction; however, injection of 0.01% capsaicin to the rostral capsaicin-sensitive area (n = 11) led to a prevailing rise in the amplitude of integrated phrenic E M G (+53.7 + 4.9%), an increase in BP by 32.6 _+ 7.1 mm
Hg (P < 0.01), with HR virtually unchanged. Microinjection of 0.01% capsaicin to the caudal capsaicin-sensitive area ( n = 8 ) depressed BP by 41.5+ 5.9 mm Hg (P < 0.01) and decreased HR by 57 _+ 6 beats/min (P < 0.05), without changing respiration. Original recording from representative experiments is shown in Figs. 1 and 2. It is noteworthy that repeated (10-15 min after) injections of capsaicin in the same (0.5 nmol) or in increasing dosage up to 50 nmol to its most effective sites of action were considerably less effective on respiration and BP as compared with the previous application of the drug to the same site or, like in case with the injection vehicle, were completely ineffective. The rostral capsaicin-sensitive area is located within the caudal region of the rostral ventrolateral medulla (RVLM) containing sympathoexcitatory neurones which, via descending pathways to sympathetic preganglionic spinal neurones [2,10], are capable of exerting tonic sympathetic drive on functions of the autonomic nervous system. In contrast, the inhibitory effect of capsaicin on BP and HR after its injections to the caudal capsaicin-sensitive area which topographically corresponds to the CVLM may be accounted for by that this area contains sympathoinhibitory neurones [2,10]. (2) Chronic experiments with injection of 25 nl 1% capsaicin (n = 6) to the caudoventrolateral reticular nucleus (CVL) of the medulla [12] localized in the caudal capsaicin-sensitive area showed significant (as compared with control animals (n -- 3) which received 25 nl saline to the CVL) decrease in arterial pressure from 86.3 + 3.7 mm Hg by 35~,5% and in HR from 403.4 + 9.6/min by 10-15% in the 1st week after operation. These indices tended to recover by the end of the 2nd week. It may be suggested that the known capsaicin-induced reflex fall in rat BP [5] is mediated via the caudal capsaicin-sensitive area of the ventral medulla. (3) At the end of the 2nd week after injection of 1% capsaicin to the CVL, NADPH-d-positive cells were shown to appear in the caudal capsaicin-sensitive area at the side contralateral to the injection (Fig. 3b,d). The mean number of NADPH-d-positive cells in this area/ section was 41 + 5 (n = 6). Injections of 25 nl saline to the CVL in control experiments revealed just single 4sol Heart rate J {beats min') J 350J II
200] Blood Pressure | (ram Hg) I 5OJ
t
mi,,,,,~n 1
CAPSAICIN(0.01%,5 nlI Fig. 2. Changes in BP and HR alter injection of 5 nl 0.01% capsaicin to CVLM 500/2m deep from surface. Arrow indicates beginning of l-min-long injection.
S. 1I..Koulchitsky et al./Neuroscience Letters 182 (1994) 129-132
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Fig. 3. Distribution of NADPH-d staining in RVLM (a,c) and CVLM (b,d) 2 weeks after unilateral injection of 25 nl 1% capsaicin to caudoventrolateral reticular nucleus of medulla, a,b, contralateral; c,d, unilateral side to injection. Bar a,b, 50 pm; c, 40/lm; d, 100/.tm.
NADPH-d-positive neurones in this brain region which does not disagree with available literature data [15]. The asymmetric distribution of NADPH-d-positive cells after capsaicin was found also in the RVLM (n = 6) (Fig. 3a,c) which contained 53 + 6 cells/section at the side contralateral and 4 + 2 cells at the side ipsilateral to the injection. Although by the time of appearance of asymmetric distribution of NADPH-d-positive cells in the ventral medulla arterial pressure and HR reach virtually the control level, we are not able to unequivocally answer whether the neurochemical rearrangements revealed in the brainstem reflect either repair or destructive processes in response to capsaicin. However, the results suggest a probable involvement of NO in the neurochemical rearrangements in the brainstem after application of capsaicin to the CVLM.
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