Neonatal capsaicin treatment impairs functional properties of primary olfactory afferents in the rat

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Neuroscience Letters, 127 (1991) 251-254 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50

ADONIS O30439409100322Z NSL 07842

Neonatal capsaicin treatment impairs functional properties of primary olfactory afferents in the rat H. P6rez, S. Ruiz, H. Inostroza and M. Perretta Laboratory of Neurophysiology and Biophysics, Institute of Nutrition and Food Technology ( INTA ), University of Chile, Santiago (Chile) (Received 27 December 1991; Revised version received 28 March 1991; Accepted 3 April 1991)

Key words: Olfactory nerve; Olfactory bulb; Capsaicin; Nerve excitability; Evoked response; Rat Long-lasting influences of neonatal capsaicin treatment on functional properties of the olfactory nerve were studied in 30-day-old rats by determining excitability thresholds of nerve fibres by means of orthodromic field potentials elicited in the main olfactory bulb, as well as by analyzing the pattern of the low-frequency component of the bulbar electroencephalogram. In addition, body, brain and olfactory bulbar weights were measured. Neonatal capsaicin resulted in reduced excitability of primary olfactory afferents and reduced wave amplitude of the bulbar electroencephalogram. Capsaicin treated animals had reduced body, brain and bulbar weights, the latter being the most affected. The result indicates that capsaicin given early in life leads to altered transmission properties of olfactory nerve fibres at later age, suggesting that olfactory afferents are sensitive to the neurotoxic action of capsaicin as occurs in other chemosensory afferent systems.

It has recently been shown that there is substance Plike immunoreactivity in the olfactory nerve of gymnotid fish [22]. Substance P has also been reported to be present in the primary unmyelinated C-fibres of the nociceptive and the carotid chemosensory systems [3, 7], where it seems to play a role as excitatory neurotransmitter [14]. However, the presence of this neuropeptide has never been demonstrated in primary olfactory afferents of mammalian species. Capsaicin is an agent known to produce a selective and irreversible degeneration of large number of nociceptive and carotid chemosensory C-fibres, while leaving large myelinated fibres unaffected [8, 13]. Neonatal capsaicin results in depletion of substance P, cholecystokinin and somatostatin in rat dorsal root ganglia [1]. Capsaicin treatment also leads to reduced calcitonin gene related peptide-like immunoreactivity co-localized with substance P in vascular and visceral sensory neurons of guinea pigs [2]. In contrast, markers that are associated with central nervous system neurones and not primary afferents, such as neurotensin, Leu-enkephalin and serotonin immunoreactivities, are unchanged after neonatal capsaicin [9]. On the whole, these data point to a marked neurotoxic Correspondence: H. P6rez, Laboratory of Neurophysiology and Biophysics, Institute of Nutrition and Food Technology (INTA), University of Chile, Casilla 138-11, Santiago, Chile.

action of capsaicin on unmyelinated neuropeptide-containing primary afferents. Nevertheless, despite that olfactory afferents are constituted exclusively by unmyelinated axons [20], susceptibility of the olfactory nerve to capsaicin has not yet been investigated. In this study, we examined electrophysiologically whether neonatal capsaicin treatment resulted in functional changes of the olfactory input to the main olfactory bulb (MOB). As described previously [8], on the second day of postnatal life, pup rats of either sex were injected subcutaneously at the back of the neck with a single dose of 50 mg/kg capsaicin suspended in a vehicle consisting of 10 ethanol, 10% Tween 80 and 80% saline; control pups were given the solvent alone at the same age. Body weight was measured periodically. At 30 days of age, animals were subjected to a single dose of 50 mg/kg i.p. of sodium pentobarbital anesthesia and placed in a stereotaxic apparatus. After exposure of the left MOB, heart and respiratory rates were measured for 30 min. Then a bipolar enamel-coated stainless-steel electrode (two braided 100/~m diameter wires, 0.8 mm tip separation) was positioned on the mid-dorsal surface of the MOB (one electrode tip on the bulb surface and the other penetrated the bulb 0.8 mm). This electrode allowed recordings of the bulbar electroencephalographic (EEG) activity, as well as the orthodromically evoked field potentials produced in the MOB by stimulating the ipsilateral olfactory nerve (ON) [16]. To record just the

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olfactory input-dependent low frequency component of the bulbar EEG [4], the bandwidth of the amplifier was adjusted to 0.3-30 Hz. Electrical stimulation of the ON was carried out near its site of emergence from the cribriform plate with a bipolar, enamel-coated steel electrode (side-by-side pair of 100 ~tm diameter each). The recordings were displayed on an oscilloscope and digitized at a rate of 400/s by an A/D converter interfaced to a microcomputer. The stimulating current was conventionally measured by inserting a 4.7 k(2 resistor in the circuit. Strength~luration data for threshold activation of the olfactory bulb (responses of 0.5 mV amplitude) were obtained by varying both the current intensity and pulse duration of the stimuli. The threshold current for 50-/zs duration pulses as well as chronaxie were chosen as significant parameters for quantitative characterization of ON excitability [17]. Chronaxie was determined by linear regression analysis of current x duration versus duration plotting according to the procedure reported elsewhere [5]. Body temperature was monitored and maintained at 38°C by a heating blanket. The right femoral artery was cannulated for monitoring mean blood pressure and in most experiments blood pressure remained stable (100130 mmHg) throughout the experiment; in those few instances where blood pressure decreased below 100 mmHg, the experiment was stopped and the data collected were discarded from the study. Capsaicin treatment caused a decrease of ON excitability, as revealed by the significantly higher chronaxie values observed in capsaicin-treated animals when compared to controls; just as well, the mean current intensity required to evoke threshold responses from the MOB was also significantly higher in the capsaicin treated rats (Table I). In control rats under pentobarbital anesthesia the low frequency component of the bulbar EEG consisted of 3-

TABLE I E F F E C T OF N E O N A T A L C A P S A I C I N T R E A T M E N T ON SOME ELECTROPHYSIOLOGICAL PARAMETERS ~To evoke a 0.5 mV response recorded from the olfactory bulb using 50/~s test stimuli delivered to the olfactory nerve, bStudent's t-test. Values are means ± S.D.

Olfactory nerve chronaxie (~s) Threshold current (uA) a Bulbar EEG frequency (Hz) Bulbar EEG amplitude (mV)

Normals

Capsaicinized

(n = 8)

(n = 8)

176 ± 38 239 + 39 3.7+0.5 1.9 ± 0.7

352 ± 57 343 ± 49 2.8±0.7 0.9 ± 0.3

pb

< 0.005 < 0.005 <0.010 < 0.005

T A B L E II E F F E C T OF N E O N A T A L CAPSAICIN T R E A T M E N T ON SOME PHYSICAL AND PHYSIOLOGICAL PARAMETERS aStudent's t-test. Values are means ± S.D.

Body weight (g) Brain weight (mg) Bulbar weight (mg) Heart rate (l/min) Respiratory rate (1/min)

Normals

Capsaicinized

(n = 8)

(n = 8)

92. I + 1.4 1605+47 58.2__+3.3 435+45 102.0+6.6

75.2 + 6.6 1473+48 48.0+1.1 376__+24 93.2+6.1

P~

< 0.005 <0.005 <0.005 <0.025 <0.05

5 Hz oscillatory waves of about 2 mV amplitude. In contrast, the capsaicin treated rats displayed a bulbar EEG pattern showing waves of a markedly lower amplitude (about one half) and of a slightly but significantly lower frequency than controls (Table I). Table II shows that neonatal capsaicin treatment of pup rats resulted in decreased body, brain and bulb weights at 30 days of age, as compared to matched controis. Bulbar weight deficit (17.5%) was significantly higher ( P < 0.025) than whole brain deficit (8.2%). Both heart and respiratory rates were significantly decreased in capsaicin treated rats. At 30 days of age capsaicin treated animals showed significantly increased ON chronaxie values, as well as increased stimulation strengths required to evoke threshold responses in the MOB. It has been established that chronaxie is a useful index to evaluate both peripheral nerve and central white matter excitability (see review by Ranck [18]), a greater chronaxie indicating a lower excitability of neural structures involved in the generation of the responses elicited. With respect to this, it is known that weak electrical stimulation of the cribriform plate sufficient to evoke MOB responses involves excitation of olfactory primary afferents that synapse within the gtomeruli onto mitral/tufted (M/T) cells dendrites [20]. It follows, therefore, that capsaicin given early in life will lead to altered transmission properties of the olfactory nerves at later age, and also to altered synaptic processing within the olfactory bulbs. Bulbar EEG amplitude was diminished in the capsaicin treated animals. These surface potentials are thought to be associated to the respiratory cycle [4], the amplitude of the wave likely being dependent on the number of primary afferents excited during inspiration. On this basis, the reduced wave amplitude of the low-frequency bulbar EEG component points to a reduced number of olfactory nerve fibres able to be excited in capsaicintreated rats. Whether centrifugal inputs to the MOB

253 regulating M / T - g r a n u l a r cells interactions, and thereby bulbar E E G amplitude, were also influenced by capsaicin treatment remains to be determined; however, no long-lasting neurotoxic effects o f systematically administered neonatal capsaicin on the central nervous system have been reported so far. As far as we know, the effects o f neonatal capsaicin administration on b o d y weight, total brain weight as well as on weights o f different brain regions during rat development have not systematically been studied so far. Weight deficits could be explained in terms o f a neurotoxic effect o f capsaicin on the olfactory nerve. C o m p a r able deficits in b o d y and brain weights have been reported after olfactory deafferentation by bulbectomy [6] or chemical destruction o f olfactory receptors [12] in neonatal rats, and here the losses were t h o u g h t to be the result o f feeding problems (mild undernutrition) subsequent to the loss o f olfactory cues which are essential for the suckling behavior o f the pups. There is evidence that capsaicin administration in the adult animal fails to produce deficits in o d o r detection performance [1 1, 21]; however, there is general agreement in that capsaicin subcutaneously administered during a d u l t h o o d does not cause degeneration o f central C-fibre terminals as occurs after neonatal capsaicin treatment, and also in that peripheral C-fibre d a m a g e is never as extensive in adults as in neonates (see review by Russell and Burchiel [19]). The fact that capsaicin treatment affected bulbar weight more than brain weight further supports the contention that neonatal capsaicin actually resulted in a decreased olfactory input. In this regard, the bulbs o f 4-week-old rats, whose right external nares were sealed by burning after birth, show a weight deficit o f a b o u t 20% [12], which is comparable with the 17.5 % bulbar weight deficit f o u n d in capsaicin-treated rats in the present study. In conclusion, the present findings indicate that capsaicin, when administered neonatally in the rat, exerts a deleterious effect on the functional properties o f the ON. However, further m o r p h o m e t r i c studies are required to demonstrate conclusively the neurotoxic action o f capsaicin on primary olfactory afferents. Interestingly, neonatal capsaicin p r o d u c e d a decrease o f both heart and respiratory rates in 30-day-old rats. Decrease o f heart rate after neonatal capsaicin treatment has already been reported [10]. It is k n o w n that the C-fibre contingent conveying baro- and chemosensory inputs to the central nervous system undergoes degenerative processes after neonatal capsaicin [8, 10], and this provides a possible neuroanatomical explanation for the long-lasting changes in cardiovascular and respiratory function observed in the capsaicin treated animals. Just as well, however, it has been reported that neonatal capsaicin depletes substance P immunoreactivity from axon termi-

nals innervating the guinea pig heart [15]. T h o u g h at present it is u n k n o w n whether these fibres are m o t o r or sensory, this could constitute an alternative way to explain the effects o f capsaicin administration during early life on cardiovascular function. The authors thank Dr. A. Hern~idez for his helpful c o m m e n t s on the manuscript. This work was supported by G r a n t 90-1035 f r o m F O N D E C Y T . 1 Fitzgerald, M., Capsaicin and sensory neurones - a review, Pain, 15 (1983) 109-130. 2 Gibbins, I.L., Furness, J.B., Costa, M., Mclntyre, I., Hillard, C.J. and Girgis, S., Co-localization of calcitonin-gene related peptidelike immunoreactivity with substance P in cutaneous, vascular and visceral sensory neurones of guinea-pigs, Neurosci. Lett., 57 (1985) 125-130.

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