Mesopontine cholinergic projections to the hypoglossal motor nucleus

Neuroscience Letters 413 (2007) 121–125

Mesopontine cholinergic projections to the hypoglossal motor nucleus Irma Rukhadze ∗ , Leszek Kubin Department of Animal Biology 209E/VET, School of Veterinary Medicine and Center for Sleep and Respiratory Neurobiology, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA 19104-6046, USA Received 17 October 2006; received in revised form 16 November 2006; accepted 16 November 2006

Abstract Mesopontine cholinergic (ACh) neurons have increased discharge during wakefulness, rapid eye movement (REM) sleep, or both. Hypoglossal (12) motoneurons, which play an important role in the control of upper airway patency, are postsynaptically excited by stimulation of nicotinic receptors, whereas muscarinic receptors presynaptically inhibit inputs to 12 motoneurons. These data suggest that ACh contributes to sleep/wakerelated changes in the activity of 12 motoneurons by acting within the hypoglossal motor nucleus (Mo12), but the origins of ACh projections to Mo12 are not well established. We used retrograde tracers to assess the projections of ACh neurons of the mesopontine pedinculopontine tegmental (PPT) and laterodorsal tegmental (LDT) nuclei to the Mo12. In six Sprague–Dawley rats, Fluorogold or B subunit of cholera toxin, were pressure injected (5–20 nl) into the Mo12. Retrogradely labeled neurons, identified as ACh using nitric oxide synthase (NOS) immunohistochemistry, were found bilaterally in discrete subregions of both PPT and LDT nuclei. Most retrogradely labeled PPT cells (96%) were located in the PPT pars compacta region adjacent to the ventrolateral tip of the superior cerebellar peduncle. In the LDT, retrogradely labeled neurons were located exclusively in its pars alpha region. Over twice as many ACh neurons projecting to the Mo12 were located in the PPT than LDT. The results demonstrate direct mesopontine ACh projections to the Mo12. These projections may contribute to the characteristic of wakefulness and REM sleep increases, as well as REM sleep-related decrements, of 12 motoneuronal activity. Published by Elsevier Ireland Ltd. Keywords: Acetylcholine; Hypoglossal motoneurons; Laterodorsal tegmental nucleus; Pedunculopontine tegmental nucleus; REM sleep; Upper airway

Cholinergic (ACh) neurons of the laterodorsal and pedinculopontine tegmental (LDT and PPT) nuclei have increased discharge during wakefulness and/or rapid eye movement (REM) sleep [9,14,27,31]. Some of these neurons have axonal projections to the brainstem regions containing respiratory neurons. They may also project directly to the cranial motor nuclei, including the hypoglossal motor nucleus (Mo12) that innervates the genioglossus, an important upper airway dilator ([13,36]; reviewed in ref. [17]). By acting on different receptors within the Mo12, acetylcholine may increase or decrease the activity of 12 motoneurons [2,4,19]. Nicotinic receptors may mediate wake- and/or REM sleep-related excitation, and decreased ACh cell activity during slow-wave sleep (SWS) may contribute to the characteristic of the state decrements of Mo12 activity. Muscarinic (m2) receptors are also present in the Mo12 [21], and in vitro studies show that they presynaptically suppress glutamatergic inputs to 12 motoneurons [2]. However, interpretation

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0304-3940/$ – see front matter. Published by Elsevier Ireland Ltd. doi:10.1016/j.neulet.2006.11.059

of these findings in the context of sleep/wake-dependent control is difficult because little is known about the sources of ACh projections to the Mo12. In order to define the location of PPT and LDT neurons that may contribute to state-dependent modulation of 12 motoneurons, we studied the topography of mesopontine ACh projections to the Mo12. Six male Sprague–Dawley rats (body weight: 396 ± 11 g (S.E.), Charles River, MA) were used. All animal handling procedures were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania and followed the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The animals were anesthetized with isoflurane (3%) followed by Nembutal (60 mg/kg i.p.), placed in a stereotaxic head holder and the dorsal medullary surface exposed. Retrograde tracers, Fluorogold (FG; Fluorochrome, LLC; 1%/20 nl in two rats or 4%/5 nl in three rats) or cholera toxin B subunit (CTb; List Biological Lab.; 1%/5 nl in one rat) were pressure injected using glass pipettes (A-M Systems, tip diameters 20–25 ␮m), with the injection volume monitored by observing the movement of the meniscus in the pipette through a calibrated microscope. The

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pipette was inserted into the Mo12 at the level of the caudal end of the area postrema, aiming at AP −13.68 mm relative to bregma [24], 0.3 mm lateral to midline and 1.1 mm below the medullary surface. Seven days after the injections, the rats were deeply anesthetized with Nembutal (80 mg/kg, i.p.) and transcardially perfused with phosphate-buffered saline (PBS, pH 7.4, with 5 USP units/ml heparin and 0.004% lidocaine) and then with 4% paraformaldehyde in PBS. The brains were post-fixed for 48 h at 4 ◦ C, cryoprotected in 30% sucrose and cut into five series of 35 ␮m coronal sections. One series was subjected to immunohistochemical procedures to visualize nitric oxide synthase (NOS) as a reliable and unique marker for ACh neurons within the mesopontine tegmentum [8,33] and the remaining series were used to provide cytoarchitectonic landmarks or for other procedures. To visualize NOS, sections were incubated for 48 h in mouse anti-NOS antibodies (1:4000; Lot #22K4838; Sigma), in PBS containing 0.3% Triton X and 5% horse serum, then for 1 h at room temperature in biotinylated anti-mouse antibodies (1:200; Vector), and then for 1.5 h in Fluorescein (FITC)-conjugated egg white-avidin (1:1,000; Jackson). The sections from the experiment with CTb were first immunohistochemically processed to visualize CTb and then subjected to NOS immunohistochemistry. They were incubated for 48 h at 4 ◦ C in goat CTb antiserum (1:20,000; Lot# 7032A2; List) in PBS containing 0.2% Triton X and 5% donkey serum. Subsequently they were incubated for 2 h in Cy3-labeled, donkey anti-goat antibodies (1:200; Jackson). Every fifth section from AP levels −9.30 to −7.04 mm caudal to bregma [24] was examined for the presence of cells containing the retrograde tracer and NOS-immunoreactivity using a Leica DML microscope and appropriate filter sets (FG, Cy3 and FITC; Chroma Technologies). Cells were regarded retrogradely labeled when they contained FG or CTb grains within a clearly identifiable cell body, the cell nucleus was present, and the grains were not visible under filters other than the one appropriate for a given fluorophore. NOS cells were identified on the basis of a uniform distribution of immunofluorescence (FITC) within the cell body and proximal dendrites. Photographs were taken with a Polaroid digital camera (DMC-2) and then enhanced using Photoshop software (Adobe). Image processing was limited to adjustments of the color balance and contrast in order to most faithfully represent the image seen under direct microscopic observation. The locations of double-labeled cells were mapped onto closest standard cross-sections from a rat brain atlas [24]. Statistical comparisons were conducted using Students’s paired t-test (Sigma Plot, Jandel) and the variability of the means characterized by the standard error (S.E.). In all six rats, tracer injections were placed in the center of the Mo12 (Fig. 1A–C). In each animal, some tracer spread was visible in the surrounding areas, such as the dorsal motor nucleus of vagus (DMV) or the reticular formation located lateral or ventral to the Mo12, but with the volumes used, the amount and intensity of such a spread represented a small fraction of that deposited within the Mo12. The pontomedullary distribution of retrogradely labeled neurons was consistent with previous reports (e.g. [5,7,10,32]), and so was the distribution of ACh neurons [1,8,13,28,29,33]. As previously defined in rats, the PPT

nucleus was divided into two subdivisions: PPT pars compacta (PPTc) and PPT pars dissipata (PPTd) [26,30]. PPT neurons were clustered dorsal and ventrolateral to the superior cerebellar peduncle (scp) at AP −8.8 to −7.04 from bregma (PPTc region) and also scattered ventral and medial to the scp (PPTd region). LDT neurons were located at AP levels −9.16 to −8.3 either as a dense cluster embedded in the central gray (CG), corresponding to the core of the nucleus, or as diffusely scattered cells distributed dorsal and ventral to the border between the CG and the adjacent dorsomedial reticular formation (the LDT pars alpha region—LDT␣). Fig. 1 shows examples of tracer injections and NOS-positive neurons that were retrogradely labeled from the Mo12. In individual animals with FG injection, we found three to seven double-labeled neurons in the PPT, and one to four in the LDT, nucleus (in every fifth 35 ␮m section), and two cells were found in the animal with CTb injection. Nearly all double-labeled neurons were located in the same discrete subregions of the PPT and LDT nuclei. Of the 24 cells found in all six rats in the PPT nucleus, 23 were located in a subregion of the PPTc adjacent to the ventrolateral tip of the scp and only 1 in the PPTd region (Fig. 2). In the LDT nucleus, all 10 double-labeled cells found in the 6 rats were located in the LDT␣ region (Fig. 2). The ratios of ipsi- to contralaterally located double-labeled neurons were 11:13 for the PPT, and 5:5, for the LDT nucleus. Relative to the total number of retrogradely labeled and NOS-positive cells found bilaterally in both nuclei, the mean percentage of those located in the PPT nucleus was 69.8 ± 4.9%; this was significantly more than in the LDT nucleus (p < 0.02, t5 = 3.9). In order to assess the proportions of PPT and LDT cells projecting to the Mo12 relative to all mesopontine ACh cells, we counted NOS-positive neurons in the PPT and LDT nuclei. The number of NOS-positive neurons found bilaterally in different animals (counts obtained from every fifth section and then multiplied by 5) ranged from 1410 to 4250 for the PPT (mean: 2970 ± 500), and from 1465 to 4370 (mean: 2880 ± 380) for the LDT nucleus. These numbers were similar to those reported earlier for the PPT nucleus and slightly lower than in other studies for the LDT nucleus. For example, the numbers of ACh neurons counted bilaterally in the PPT were 2070 [13], 3655 [26] or 3252 [6]. In the LDT nucleus, previously reported bilateral counts of ACh cells were 3304 [3], 4227 [13] or 5526 [11]. Relative to our counts, the percentages of NOS cells retrogradely labeled from the Mo12 were 0.8 ± 0.2% for the PPT and significantly less, 0.30 ± 0.08%, for the LDT nucleus (p < 0.03, t5 = 3.3). Thus, a limited number of mesopontine ACh neurons located in discrete portions of the PPT and LDT nuclei have bilateral projections to the Mo12. Such neurons may directly modulate 12 motoneuronal activity in a state-dependent manner. The information about descending pontine ACh projections to the orofacial motor nuclei has been scanty and conflicting. In one study, ACh projections to the trigeminal (Mo5), facial (Mo7) or Mo12 were reported to be 5:1 ipsilateral and originate mainly in the PPT nucleus [36]. While the main location within the PPT complex of our double-labeled cells is consistent with that study, we also found a similar number of double-labeled cells in the contralateral PPT nucleus and less numerous but

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Fig. 1. Examples of tracer injection sites (A–C) and mesopontine cholinergic neurons retrogradely labeled from the Mo12 (D–I). (A–C) Tracer injection sites in three of the six rats of the present study; FG 4%/5 nl in (A), FG 1%/20 nl in (B), and CTb 1%/5 nl in (C). The images in (A and B) were acquired under simultaneous fluorescent and dark-field illumination to superimpose tracer distribution over anatomical landmarks; FG-labeled 12 motoneurons (light blue) surround the centers of the injection sites (arrowheads) marked by FG-induced necrosis, whereas light pink patches of tissue represent fiber tracts. (D) NOS-positive neurons in LDT␣ region at AP level −9.16 [24]; the midline is to the right, and the dashed line shows the border of the central gray. (E) Expanded image of the region framed in (D), with the filled arrow pointing to a NOS-positive neuron that was retrogradely labeled with CTb from the Mo12, as shown in (F). The empty arrow in (F) points to a cell that was also retrogradely labeled but NOS-negative. (G) NOS-positive neurons in the PPTc and LDT regions. (H) Expanded image of the region framed in (G), with the filled arrow pointing to a NOS-positive neuron that was retrogradely labeled with CTb from the Mo12, as shown in (I).

also bilateral projections from the LDT nucleus. It is possible that the relatively lesser projections from the LDT escaped detection in that earlier study in which only three rats received tracer injections into the Mo12. Our data compare favorably to the results from a larger study of descending cholinergic projections to the medullary reticular formation (MRF) and medullary nuclei [25]. In that study, in two rats with relatively small retrograde tracer injections that mainly involved the Mo12 (cases 2 and 5), six neurons per rat were found in the ipsilateral PPT and four or five in the contralateral PPT. In contrast, another study concluded that the PPT nucleus does not project to either Mo5 or Mo12 [12]. Indeed, 18 PPT neurons were retrogradely labeled bilaterally in 4 rats of that study

following tracer injections into the medial portion of the Mo7, but the labeling was interpreted as caused by tracer diffusion outside the Mo7 (LDT projections were not investigated). It is of interest to compare pontine ACh projections to the Mo12 to those to adjacent structures, such as the MRF and viscerosensory nucleus of the solitary tract (NTS). Following large tracer injections that specifically targeted the MRF, about 174 cells were labeled per rat (97 ipsi- and 77 contralaterally) in the PPT, and about 109 per rat in the LDT nucleus (74 ipsiand 35 contralaterally) [13]. In another study with injections involving to variable degrees the Mo12, NTS and adjacent portions of the MRF, 73 cells per rat were found in the PPT nucleus (45 ipsi- and 28 contralaterally; cases 6, 8 and 16 in

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Fig. 2. Distribution of NOS-positive and retrogradely labeled from the Mo12 neurons found in all six animals. Each symbol corresponds to one double-labeled cell and is placed on the closest standard levels from a rat brain atlas [24]. Shadings delineate the regions containing NOS-positive cells in the PPT and LDT nuclei obtained by superposing the areas containing NOS cells at the corresponding AP levels in the six animals used in this study. Abbreviations: CG, central gray; CnF, cuneiform nucleus; mlf, medial longitudinal fasciculus; scp, superior cerebellar peduncle; VTg, ventral tegmental nucleus; xscp, decussation of superior cerebellar peduncle.

[25]). In contrast, in the cases 10 and 11 of the same study that primarily targeted the NTS, no PPT cells were labeled. These data suggest that pontine ACh neurons project relatively densely to the MRF and minimally or not at all to the NTS. Following tracer injections centered in the Mo12, and with minimal diffusion beyond the nucleus (Fig. 1A–C), we found that nearly all retrogradely labeled cells were located within well defined subregions of the PPT and LDT nuclei. This consistency of the anatomical origin of the projections supports our interpretation that Mo12 was the main site of the effective tracer uptake in our experiments, and that a small but consistent contingent of PPT/LDT neurons have direct bilateral projections to the Mo12. Most PPT neurons and many cells of the LDT proper have rostrally projecting axons [22,28,35], whereas LDT␣ neurons often have both ascending and descending projections [13,28]. Our data suggest that the PPTc cells located around the tip of the superior cerebellar peduncle include a relatively large contingent of those that have descending projections. Therefore, it appears plausible that most neurons of the PPT and those in the central core of the LDT are key members of the ascending reticular activating system, whereas subsets of ACh neurons located in the LDT␣ and PPTc regions additionally modulate 12 motoneurons and other medullary targets. Our results provide anatomical basis supportive of such an arrangement and encourage further functional tests of this possibility. Electrophysiological studies suggest that many LDT␣ neurons have highest firing rates during REM sleep [9,14]. Through their projections to the dorsomedial pontine reticular formation,

LDT and PPT neurons may trigger REM sleep which, indirectly, causes suppression of activity in upper airway motoneurons [15,16,18,20,23]. Our data suggest that, in addition, some PPT and LDT neurons may mediate REM sleep-related effects by acting directly within the Mo12. Consistent with this, muscarinic cholinergic receptors are present in the Mo12 [21] and mediate presynaptic inhibition of excitatory transmission to 12 motoneurons [2]. However, nicotinic receptor agonists infused into the Mo12 region exert an opposite, excitatory, effect on 12 motoneurons [19], and the excitation is likely to be postsynaptic [4]. Hence PPT/LDT projections to the Mo12 may both suppress and activate 12 motoneurons in a state-dependent manner. In addition, pontine ACh projections to the MRF adjacent to the Mo12 (discussed above) may indirectly contribute to state-dependent changes in 12 motoneuronal activity by acting on medullary premotor neurons that are located in the MRF ventrolateral to the Mo12 and transmit inspiratory drive to 12 motoneurons [34]. In summary, cholinergic PPT/LDT neurons have bilateral projections to the Mo12 that originate in a discrete portion of the PPTc and, to a lesser extent, in LDT␣. These projections may contribute to both excitatory and inhibitory, state-dependent modulation of 12 motoneuronal activity in relation to REM sleep and/or wakefulness. The excitatory component of these effects is of particular interest as a mechanism that may help maintain upper airway patency. Acknowledgement The study was supported by NIH grant HL-47600. References [1] D.M. Armstrong, C.B. Saper, A.I. Levey, B.H. Wainer, R.D. Terry, Distribution of cholinergic neurons in rat brain: demonstrated by the immunocytochemical localization of choline acetyltransferase, J. Comp. Neurol. 216 (1983) 53–68. [2] M.C. Bellingham, A.J. Berger, Presynaptic depression of excitatory synaptic inputs to rat hypoglossal motoneurons by muscarinic M2 receptors, J. Neurophysiol. 76 (1996) 3758–3770. [3] C.D. Blaha, L.F. Allen, S. Das, W.L. Inglis, M.P. Latimer, S.R. Vincent, P. Winn, Modulation of dopamine efflux in the nucleus accumbens after cholinergic stimulation of the ventral tegmental area in intact, pedunculopontine tegmental nucleus-lesioned, and laterodorsal tegmental nucleus-lesioned rats, J. Neurosci. 16 (1996) 714–722. [4] N.L. Chamberlin, C.M. Bocchiaro, R.W. Greene, J.L. Feldman, Nicotinic excitation of rat hypoglossal motoneurons, Neuroscience 115 (2002) 861–870. [5] E.T. Cunningham Jr., P.E. Sawchenko, Dorsal medullary pathways subserving oromotor reflexes in the rat: implications for the central neural control of swallowing, J. Comp. Neurol. 417 (2000) 448–466. [6] S. Deurveilher, E. Hennevin, Lesions of the pedunculopontine tegmental nucleus reduce paradoxical sleep (PS) propensity: evidence from a shortterm PS deprivation study in rats, Eur. J. Neurosci. 13 (2001) 1963–1976. [7] E.G. Dobbins, J.L. Feldman, Differential innervation of protruder and retractor muscles of the tongue in rat, J. Comp. Neurol. 357 (1995) 376– 394. [8] N.J. Dun, S.L. Dun, U. Forstermann, Nitric oxide synthase immunoreactivity in rat pontine medullary neurons, Neuroscience 59 (1994) 429–445. [9] M. El Mansari, K. Sakai, M. Jouvet, Unitary characteristics of presumptive cholinergic tegmental neurons during the sleep-waking cycle in freely moving cats, Exp. Brain Res. 76 (1989) 519–529.

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