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NSL 07844

Neuroscience Letters, 128 (1991) 52-56 ~} 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50

ADONIS 030439409100326J

Further evidence for the absence of a descending cholinergic projection from the brainstem to the spinal cord in the rat

F.E. Sherriff, Z. Henderson and J.F.B. Morr i son

Department of Physiology, University of Leeds, Leeds (U.K.)

(Received 12 December 1990; Revised version received 20 March 1991; Accepted 3 April 1991)

Key words: Choline acetyltransferase; Immunocytochemistry; Brainstem; Spinal cord; Rat

Serotonergic and catecholaminergic neurons are known to project from the brainstem to the spinal cord. However, evidence for a bulbo-spinal projection that is cholinergic is sparse despite immunocytochemical and physiological evidence for a cholinergic influence on the cord. In this study we examined the possibility of a direct cholinergic bulbo-spinal projection in the rat using a combination of retrograde axonal tracing techniques and choline acetyltransferase immunocytochemistry. Although many cells were found to project to the cord from the brainstem, none were identified as being cholinergic, confirming previous evidence that the cholinergic innervation of the cord is intrinsic.

The best known examples of direct projections from the brainstem to the spinal cord which are not directly involved in motor pathways are serotonergic (from the caudal raphe nuclei) [14] and catecholaminergic (from the locus coeruleus and sub-coeruleus) [18]. Both sys- tems appear to have an overall facilitatory effect on motoneurons and an inhibitory influence on pain trans- mission [8]. Little is known about whether there is a des- cending cholinergic projection from the brainstem, although there is ample evidence for a cholinergic influence on the spinal cord from immunocytochemical and physiological studies [2, 12, 16]. The brainstem is well endowed with cholinergic cells bodies, and as well as the cranial nerve motor nuclei, there are two major groups there which correspond to the pedunculopontine (PPTg) and laterodorsal tegmental (LDTg) nuclei re- spectively. The PPTg and LDTg have been found to pro- vide ascending cholinergic projections to many areas of the brain, particularly the thalamus [6, 11]. Recently, putative cholinergic cells have been found in other parts of the brainstem, eg. the vestibular nuclei and the mid- line reticular formation [3, 15]. In this study we examine the possibility of a direct cholinergic bulbo-spinal pro- jection from any of these cells using a combination of the retrograde axonal transport method and choline acetyl- transferase (CHAT) immunocytochemistry.

Thirteen Wistar rats (250-350g) were used in this

Correspondence: F.E. Sherriff, Department of Physiology, Worsley Medical and Dental Building, University of Leeds, Leeds LS2 9NQ, U.K.

study. Under Halothane anaesthesia and Buprenorphine analgesia (0.1 mg/kg, s.c.) a laminectomy was performed. Unilateral or bilateral injections of retrogradely trans- portable t racer - 1/A 20% HRP (5 animals), 20% wheat germ agglutinin-conjugated HRP (WGA-HRP) (4 ani- mals) or 2% True Blue (3 animals) - were made into the lumbar enlargement using a Hamilton syringe. In the remaining case, 1/11 2 % True Blue was injected into the mid-thoracic spinal cord. After 48h, the rats were injected i.p. with a lethal dose of sodium pentobarbital and perfused transcardially with 100 ml 0.9% saline fol- lowed by 400 ml 3 % paraformaldehyde in 0.1 M phos- phate buffer (pH 7.4). The brain and a portion of spinal cord at the injection site were removed and allowed to sink in 30% sucrose in 0.1 M phosphate buffer (pH 7.4) overnight at 4°C. Coronal sections were cut at 50/~m using a freezing microtome.

Retrogradely transported HRP or WGA- HRP was visualised using tetramethylbenzidine (TMB) to provide a map of cells in the brainstem that project to the spinal cord, for comparison with the distribution of CHAT- immunoreactive cells. For the demonstration of neurons double-labelled for ChAT-immunoreactivity and retro- gradely transported tracer, two approaches were used. In the experiments involving the use of W G A - H R P as a tracer, the retrogradely labelled cells were visualised with cobalt intensified diaminobenzidine (DAB) [I 7], be- fore the sections were processed for ChAT as described below. For the True Blue experiments, the sections were temporarily mounted under coverslips in phosphate buffer and the fluorescent cells were photographed under

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Fig. 1. The distribution of retrogradely labelled cells (open circles) after a lumbar injection of HRP, in areas where ChAT-positive cells (filled circles) are found.

54

Fig. 2. True Blue-labelled cells are compared with immunocytochemically labelled cells in the same section. Retrogradely labelled cells are shown amongst those of the cholinergic cells of the midline reticular formation (at the level of Fig. 1C) in A,B and C,D. Retrogradely labelled cells ventral to TH-positive cells in the locus coeruleus (LC) are shown in E and F; a double-labelled cell is arrowed. G and H show separate populations of cholinergic and spinally projecting cells intermingled in the PPTg (at the level of Fig. 1A). This is also shown at low power in I and J with respect to the LDTg (at the level of Fig. IB). All examples resulted from lumbar injections of True Blue except A,B and G,H which resulted from a thoracic

injection. Stars indicate blood vessels which were used as landmarks. Bar = 200/Lm for A-H and 500 pm for I,J.

UV optics. This was followed by ChAT staining and the sections were coverslipped in glycerol immediately after mounting. The photographs of True Blue-labelled cells were compared with the same sections stained for ChAT using a camera lucida. Adjacent sections were stained for Nissl substance using Cresyl Violet.

For the immunocytochemistry, the sections were washed in 0.1% Triton X-100 in phosphate buffered saline (pH 7.4), which was used as a wash solution and diluent throughout. The sections were incubated in 2 % bovine serum albumin for 1 hour then 1/50 rat anti- ChAT antibody overnight at 4°C. They were then incu- bated with 1/30 sheep anti-rat IgG for 1 h, then 1/100 monoclonal rat PAP for 1 h. Some sections were pro- cessed with the Vectastain biotinylated anti-rat IgG and avidin-biotin-HRP. Another group of sections, mostly at the level of the locus coeruleus, were stained for tyro- sine hydroxylase (TH) with a 1/20 mouse anti-TH anti- body (Medicorp), 1/50 goat anti-mouse IgG and 1/100 mouse PAP. Peroxidase activity was revealed using 0.1% DAB and 0.04% H202 for 10-20 min. After the reaction, the sections were mounted and coverslipped in DPX or glycerol.

After the lumbar injections, retrogradely labelled cells were seen in the red nucleus, dorsolateral pontine teg- mentum, vestibular nuclei, raphe nuclei, lateral reticular nucleus and the medial two-thirds of the reticular forma- tion (Fig. 1), in agreement with previous studies [5, 8, 10, 13, 14, 18]. Identical results were obtained with all three tracers. The distribution of ChAT-positive cells in the brainstem (Fig. 1) was also in accordance with previous reports [3, 11, 15]. They were located in the PPTg, LDTg, medial third of the reticular formation and vesti- bular nuclei. Many of the retrogradely labelled cells were found in areas containing ChAT-positive cells in the PPTg and LDTg, medial vestibular nuclei and the mid- line reticular formation, although no convincing exam- ples of cells double labelled for ChAT and True Blue or WGA-HRP were found. This was particularly clearly demonstrated when True Blue was used as a tracer as there is no interference between the two labelling pro- ducts (Fig. 2). The thoracic injection resulted in similar labelling with True Blue, and again, no double labelled cells were seen. This was not due to failure of the double labelling technique as other studies in this laboratory have shown its validity [7]. Interestingly, in all cases, a well-defined group of retrogradely labelled cells was found next to the LDTg and locus coeruleus (Fig. 2E,F,I,J). None of these cells stained for ChAT and only a few stained for TH.

Our results reaffirm the presence of direct projections from the brainstem to the cord but indicate that choli- nergic neurons are not involved in direct ponto-spinal

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control. They extend those of other studies [5, 19] which have shown the lack of a cholinergic projection from the PPTg to the spinal cord although Rye et al. [13] found a weak projection to the cervical cord from this region using WGA-HRP as a tracer. There is, however, a sig- nificant cholinergic influence in the spinal cord as immu- nocytochemical evidence has revealed large numbers of ChAT-positive terminals there particularly in lamina I, lamina III and onto motoneurons [1, 12]. This choliner- gic innervation of the cord is assumed to be intrinsic on the basis of a biochemical study which showed no de- crease in ChAT activity below a spinal lesion [9] and the fact that ChAT-positive cells are found in the dorsal horn, intermediate gray and around the central canal (lamina X) [1]. Some intrinsic spinal cholinergic neurons are thought to be involved in the modulation of primary sensory information [12] and some may have a role in the control of motor activity, analogous to the intrinsic cho- linergic system in the striatum [4]. The cholinergic sys- tems within the spinal cord have yet to be fully charac- terised.

This work was supported by the MRC (U.K.)

ABBREVIATIONS

Amb DTg LC LDTg lfp mcp MnR Mo5 MVe PPTg PrH

PY RMg rs

scp

sp5 Sp5 VTg

ambiguous nu dorsal tegmental nu locus coeruleus laterodorsal tegmental nu longitudinal fasciculus pons middle cerebellar peduncle median raphe nu motor trigeminal nu medial vestibular nu pedunculopontine tegmental nu prepositus hypoglossal nu pyramidal tract raphe magnus nu rubrospi~al tract superior cerebellar peduncle spinal tract of the trigeminal nerve nu spinal tract of the trigeminal nerve ventral tegrnental nucleus

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