Anatomical and electrophysiological evidence for a glycinergic inhibitory innervation of the rat locus coeruleus

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Neuroscience Letters, 128 (1991) 33-36 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030439409100324R NSL 07841

Anatomical and electrophysiological evidence for a glycinergic inhibitory innervation of the rat locus coeruleus P.-H. Luppi 1, P.J. Charlety2, P. Fort 1, H. Akaoka 2, G. Chouvet 2 and M. Jouvet 1 1D@artement de M~decine Expkrimentale, INSERM U52, CNRS URA 1195, Facultb de Mbdecine, Universitk Claude Bernard, Lyon (France) and 21NSERM U171, Centre Hospitalier Lyon-Sud, Pierre-Bknite (France) (Received 12 February 1991; Revised version received 3 April 1991; Accepted 3 April 199 I)

Key words: Glycine; Immunohistochemistry; Electrophysiology; Locus coeruleus; Rat Using a highly specific antiserum to glycine and a very sensitive immunohistochemical technique with streptavidin-horseradish peroxidase, we visualized for the first time a dense plexus of glycine varicose fibers in the locus ceruleus (LC) of the rat. We further demonstrated that iontophoretically applied glycine inhibits the spontaneous LC noradrenergic cell discharge and that this inhibition is blocked by co-iontophoresis of strychnine. These anatomical and electrophysiological results indicate that the rat locus ceruleus receives an inhibitory glycinergic input.

The rat locus coeruleus (LC) is a nucleus of the pontine reticular formation composed of a compact noradrenergic cell group innervating nearly all the neuroaxes [11, 14]. These neurons present a regular tonic discharge during waking, decrease their activity during slow wave sleep and stop their firing during paradoxical sleep [3] (PS). During waking or under anesthesia, these neurons present a phasic activation in response to sensory stimuli [3, 4, 7]. These and other data obtained by electrophysiological and pharmacological experiments suggest an important role of these cells in the maintenance of cortical waking [4, 14, 16]. Recent electrophysiological and anatomical studies [4, 6] in the rat further suggest that two major afferents control the electrical activity of these cells: a system localized in the nucleus paragigantocellularis (PGi) containing excitatory amino acids responsible for the occurrence of the phasic responses to sensory stimuli [12] and a y-amino butyric acid (GABA)-ergic inhibitory system localized in the nucleus prepositus hypoglossi (PrH) [13]. We report anatomical and electrophysiological evidence for another inhibitory input to the LC mediated by glycine. Five Sprague-Dawley rats were perfused, under deep anesthesia, through the ascending aorta with 100 ml of Ringer's lactate solution, followed by 400 ml of 4% paraformaldehyde, 0.25% glutaraldehyde and 0.2% picric acid in cold phosphate buffer (PB, 0.1 M, pH 7.4). The Correspondence: P.-H. Luppi, D6partement de M6decine Exp6rimentale, INSERM U52, CNRS URA 1195, Universit6 Claude Bernard, 8 avenue Rockefeller, 69373 Lyon Cedex 08, France.

brains were postfixed overnight at 4°C in the same fixative solution without glutaraldehyde and then rinsed for 48 h in 0.1 M PB containing 30% sucrose. Coronal 20/tm sections were cut on a freezing cryostat. The immunohistochemical procedure is described elsewhere [15]. Immunohistochemical detection of glycine (Gly) was carried out by sequential incubations of the free-floating sections in 'rabbit' antiserum to Gly (1:2000) for 72 h at 4°C, biotinylated 'donkey anti-rabbit' (1:2000, Jackson Immunoresearch Lab.) and streptavidin conjugated with horseradish peroxidase (1:40000, Jackson Immunoresearch Lab.) both overnight at 4°C. Sections were treated with 0.02 % 3,3'-diaminobenzidine tetrahydrochloride (DAB) solution containing 0.003 % hydrogen peroxide ( H 2 0 2 ) and 0.6% nickel ammonium sulfate in 0.05 M Tris-HC1 buffer (pH 7.6). Recording from LC neurons was performed as described previously [5, 9]. Briefly, extracellular recordings were obtained from single neurons physiologically and histologically identified in the LC of 4 halothane-anesthetized rats. Iontophoretic applications of drugs were performed through a 7-barrel pipette, broken to a tip diameter of 15-20 /~m and glued alongside the glass recording electrode which protruded 15-20/lm. Four of the 7 barrels were filled with the following solutions: Gly (400 mM, pH 4, Sigma), GABA (400 mM, pH 4, Sigma), strychnine HC1 (25 mM, pH 4, Sigma) and bicuculline methiodide (25 mM, pH 4, Sigma). The remaining barrels, filled with saline, were used for automatic current balancing and current test under balanced conditions. Filtered (0.3-10 kHz band pass signal) and unfltered

34 recording pipette signals were amplified, displayed and discriminated with conventional electronics. For quantitative analysis, the spontaneous firing rate and the firing rate during Gly iontophoresis were determined for at least 3 separate epochs (10-s duration) between and during pulses of Gly (10-60s). Comparisons of the same cells before and during Gly iontophoresis were performed using the two-tailed paired Student's t-test. Results are expressed as the mean firing rates + S.E.M., relative to basal firing rates. In accord with previous studies in the rat using Gly antisera from sources other than ours [1, 17], intensely Gly-immunoreactive cell bodies were observed on LCcontaining sections, in the granular layer of the cerebellar cortex (Fig. I D), the cochlear nuclei, the superior olivary complex, the medial nucleus of trapezoid body (Fig. 1C) and the nucleus of the lateral lemniscus. We observed an additional group of Gly-immunoreactive cells in the principal trigeminal sensory nucleus. As already described [15, 22] in the cat, the highest density

of Gly-immunoreactive fibers was found in the superior olivary complex and the cochlear nuclei. We also observed numerous Gly-immunoreactive fibers in the trigeminal nuclei, the K611iker-Fuse and parabrachial nuclei, and the LC. In addition, a moderate number of fibers were located in the pontine central gray and reticular formation as well as in the vestibular nuclei. As shown in Fig. 1A and B, the fibers located in the LC presented a morphology different from those found in other pontine nuclei, particularly in the adjacent superior vestibular nucleus (Fig. 1A). Indeed, the LC displayed coarse varicosities and terminal-like structures clearly surrounding all the cell bodies within the nucleus, while the adjacent vestibular nucleus contained principally passing fibers without evident varicosities (see arrows in Fig. 1A). The mean firing rate of the 15 LC neurons recorded (2.14+0.26 Hz) is in agreement with previous reports using halothane anesthesia [5]. Gly microiontophoresis (65 ___13 nA, 10-60 s pulses) induced a reversible, reproducible and significant inhibition of all LC neurons

Fig. 1. A and B: low (A) and high (B) powerphotomicrographshowingGly-immunoreactivefibersin the LC. Note the punctate aspect (terminal-like structures) of the fibers in the LC well contrasting with the non-varicosefibersin surrounding regions particularlythe adjacent superior vestibular nucleus (arrows). C: photomicrographillustrating the intenselyGly-immunoreactiveneurons and fibersin the medial nucleus of the trapezoid body. D: photomicrograph of the cerebellarcortex showing the intense staining of the Golgi cells as well as the presence of Gly-immunoreactivefibers in the granular layer. Bars: A,B, 50/~m; C,D, 100/zm. GC, granular layer; LC, locus coeruleus; MO, molecular layer; V4, fourth ventricle; VS, superior vestibular nucleus.

35 tested ( - 6 8 % + 6 % relative to the prior spontaneous firing rate, n = 15, P<0.01) (Fig. 2). Iontophoresis of strychnine (64 + 6 nA), a selective Gly-receptor antagonist, induced a slight but significant tonic increase in LC neuron firing rate (+23%___ 10%, n = 13, P<0.05). Glyinduced inhibition ( - 80% ___26%, n = 10, P < 0.01) was reversibly antagonized by strychnine co-iontophoresis (-0.98%+0.67%, n = 10, P>0.20) (Fig. 2). Moreover, strychnine did not antagonize GABA-induced inhibitions of LC neurons (Fig. 2) while on the same neurons (n=2) bicuculline blocked GABA-induced inhibitions but did not antagonize the Gly-induced inhibitions. In these experiments, we observed highly varicose Gly-immunoreactive fibers clearly surrounding all the LC cell bodies. This staining is likely to be specific to Gly. Indeed, the specificity of the present antiserum has been previously examined in detail [15, 22] and we were able to demonstrate glycinergic cell bodies and fibers in the well-known glycinergic systems like the auditory nuclei and the cerebellar cortex. In addition, we noted that the LC noradrenergic neurons are inhibited by Gly, as already anecdotally described [7]. We further demonstrated that this inhibition is blocked by co-iontophoresis of strychnine, its specific antagonist, and not by bicuculline, thus indicating that Gly-induced inhibition is mediated by glycinergic receptors. These combined anatomical and electrophysiological findings indicate that the LC noradrenergic cells receive an inhibitory glycinergic input. It remains to be determined what the cells of origin of this innervation are and under what physiological conditions they release Gly. It has been reported that the LC receives only two signifiStrychnine GABA O

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cant afferent projections [4, 6], both from the rostral medulla: the nuclei paragigantocellularis (PGi) and prepositus hypoglossi (PrH). Further physiological and pharmacological experiments revealed a potent excitatory amino acid [12] and a weak adrenergic inhibitory input from the PGi [2] and a strong GABA-ergic input from PrH [13] to the LC. It has been further hypothesized that the excitatory amino acid pathway from PGi is a phasically active afferent conveying polymodal sensory information whereas the GABA-ergic projection from the PrH is a more tonically inhibitory input [6]. Interestingly, Gly has been recently shown to coexist with GABA in the spinal cord, the cochlear nuclei, the cerebellum and the vestibular nuclei [17, 20-23]. Glyimmunoreactive cell bodies have been localized in the PrH of the cat [15, 19] and rat (unpublished observations). The morphology of GABA- and Gly-immunoreactive terminals is very similar in the LC of the rat (unpublished results). In addition, using retrograde transport of WGA-apo-HRP-gold injected in the LC combined with GABA immunohistochemistry, Pieribone et al. [18] recently reported the presence of doublelabeled cells in the PrH. However, while the GABA antagonist bicuculline blocked the PrH-evoked inhibition of the LC cells, strychnine, a Gly antagonist, did not affect the PrH inhibition, thereby indicating that Gly may not contribute to the innervation of LC by PrH [6, 13]. Further anatomical studies using retrograde tracing with immunohistochemistry are needed to determine the cells of origin and the physiological role of the glycinergic innervation of the LC. In addition to GABA, the glycinergic input described here might be responsible for the inhibition of LC cells during paradoxical sleep (PS). Indeed, Gly is a very potent inhibitory neurotransmitter in the medulla oblongata and spinal cord [10] and has been demonstrated to be responsible for the hyperpolarization of the somatic motoneurones during PS [8]. This work was supported by INSERM U52 and U171, CNRS U R A 1195 and DRET 90/1615. We thank Dr. R.J. Wenthold for providing the Gly-antiserum and Dr. J. Carew for correction of the English.

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Fig. 2. Rate histogram showing Gly- (solid squares, 15 nA) and GABA- (empty squares, 40 nA) induced inhibition. Gly but not GABA inhibition was antagonized by strychnine co-iontophoresis (solid bar, 60 nA). Note that the inhibitoryeffectof Gly recoversprogressively after the end of strychnineco-iontophoresis. Note also the slight increasein basal firingrate by strychnine,possiblyindicatingthe blockade of some spontaneous Gly release. Ordinate, time; abscissa, firingrate in spikesper second.

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