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Nitrergic innervation of trigeminal and hypoglossal motoneurons in the cat
Brain Research 1041 (2005) 29 – 37 www.elsevier.com/locate/brainres
Research report
Nitrergic innervation of trigeminal and hypoglossal motoneurons in the cat Ines Posea,T, Simon Fungb,c, Sharon Sampognac, Michael H. Chaseb,c, Francisco R. Moralesa,b,c a
Departamento de Fisiologı´a, Facultad de Medicina, Gral. Flores 2125, Montevideo-11800, Uruguay b Websciences International 1263, Westwood Boulevard, 200, Los Angeles, CA 90024, USA c Department of Physiology, University of California, Los Angeles, CA 90024, USA Accepted 26 January 2005 Available online 8 March 2005
Abstract The present study was undertaken to determine the location of trigeminal and hypoglossal premotor neurons that express neuronal nitric oxide synthase (nNOS) in the cat. Cholera toxin subunit b (CTb) was injected into the trigeminal (mV) or the hypoglossal (mXII) motor nuclei in order to label the corresponding premotor neurons. CTb immunocytochemistry was combined with NADPH-d histochemistry or nNOS immunocytochemistry to identify premotor nitrergic (NADPH-d+/CTb+ or nNOS+/ CTb+ double-labeled) neurons. Premotor trigeminal as well as premotor hypoglossal neurons were located in the ventro-medial medullary reticular formation in a region corresponding to the nucleus magnocellularis (Mc) and the ventral aspect of the nucleus reticularis gigantocellularis (NRGc). Following the injection of CTb into the mV, this region was found to contain a total of 60 F 15 double-labeled neurons on the ipsilateral side and 33 F 14 on the contralateral side. CTb injections into the mXII resulted in 40 F 17 double-labeled neurons in this region on the ipsilateral side and 16 F 5 on the contralateral side. Thus, we conclude that premotor trigeminal and premotor hypoglossal nitrergic cells coexist in the same medullary region. They are colocalized with a larger population of nitrergic cells (7200 F 23). Premotor neurons in other locations did not express nNOS. The present data demonstrate that a population of neurons within the Mc and the NRGc are the source of the nitrergic innervation of trigeminal and hypoglossal motoneurons. Based on the characteristics of nitric oxide actions and its diffusibility, we postulate that these neurons may serve to synchronize the activity of mV and mXII motoneurons. D 2005 Elsevier B.V. All rights reserved. Theme: Motor systems and sensory motor integration Topic: Spinal cord and brainstem Keywords: Nitric oxide; Motor control; Medulla; Premotor interneurons; Neuromodulation; Reticular formation
1. Introduction Somatic motoneurons are perhaps the most completely studied cells in the central nervous system. There is a wealth of data dealing with their structure, function, electrophysiological properties, as well as their synaptic processes, including the neurotransmitters and neuromodulators that control their activity. In a comprehensive review, numerous substances including amino acids, biogenic amines, and peptides that act as neurotransmitters and neuromodulators are described in the synaptic processes that impinge on these cells [40]. * Corresponding author. Fax: +598 2 9248784. E-mail address: [email protected] (I. Pose). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.01.092
In recent years, nitric oxide (NO) has joined the list of neuromodulatory substances that act on neurons in the central nervous system [6,19]. We have previously reported that synaptic processes, containing the neuronal isoform of nitric oxide synthase (nNOS), are present in close apposition to the dendrites and cell bodies of trigeminal motoneurons in the guinea pig and that nitric oxide exerts an excitatory effect on these cells [1]. Trigeminal motoneurons participate in jaw movements associated with numerous behaviors such as mastication, deglutition, and vocalization [27,28]. During these and related motor acts, a precise coordination takes place between the jaw musculature innervated by trigeminal motoneurons and the tongue musculature that is innervated by hypoglossal motoneurons. It has been suggested that
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this coordination depends on circuits that involve overlapping or shared structures controlling both motor pools [2,13,14,24,25,47]. The present study was designed to determine the location of nitrergic premotor neurons that project to the trigeminal and hypoglossal motoneurons in the cat. An examination of the origins of the innervation of trigeminal motoneurons, prompted by our previous work [1], was combined with a study of the innervation of hypoglossal motoneurons. By means of retrograde and nNOS labeling techniques, we traced the origin of the nitrergic innervation of these brainstem motor nuclei to a population of cells within the ventro-medial medullary reticular formation. Accordingly, a novel population of premotor neurons was discovered in the present work. A portion of these data has been previously reported [32].
2. Methods Thirteen adult cats of both sexes, weighing between 3.0 and 3.5 kg, were utilized. Eight cats were employed for studies of the trigeminal nucleus and five for experiments on the hypoglossal nucleus. 2.1. Surgical procedures All experimental procedures were conducted in accord with the Guide for the Care and Use of Laboratory Animals (7th edition, National Academy Press, Washington, D.C. 1996) and approved by the Chancellor’s Animal Research Committee (ARC) of the UCLA Office for the Protection of Research Subjects (OPRS). Before anesthesia, the animals were premedicated with atropine (0.04 mg/kg, i.m.) and XylazineR (2 mg/kg, i.m.). Anesthesia was first induced with KetamineR (15 mg/kg, i.m.) and maintained with a gas mixture of isoflurane in oxygen (2–3%). The head of the cat was positioned in a heavy-duty stereotaxic frame and the calvarium was exposed. A 3.0- to 4.0-mm-diameter hole was drilled in the calvarium overlying the cerebellar cortex; after surgery, the hole was covered with bone wax. This hole provided access to the trigeminal or the hypoglossal nuclei for injection of CTb, as described below. During recovery from surgery, an analgesic (BuprenexR 0.01 mg/kg, i.m.) was administered and an antibiotic (CephazolinR) was given parenterally for 4 days. All wound margins were regularly cleaned and covered with an antibiotic ointment (FougeraR). 2.2. Micropipette and microelectrode assembly for the injection of cholera toxin subunit b (CTb) and for recording antidromic field potentials In order to inject CTb, a three-barreled micropipette assembly, consisting of a carbon fiber recording microelectrode and two side barrels containing CTb (List
Biological Laboratories, Campbell, CA), was lowered into the trigeminal (stereotaxic coordinates: P4.6, L3.5, H 4; [3]) or the hypoglossal motor nucleus (stereotaxic coordinates: P12.7, L1.2, H 6.5; [3]). The exact location of these motor nuclei in each animal was resolved by recording the antidromic field potential evoked by electrical stimulation of the masseter and hypoglosssal nerves, respectively, as described in previous communications [7,18]. CTb was injected by iontophoresis (2 AA positive current pulses, 7 s on, 7 s off, for 20 min in each barrel) at a site where the antidromic field potential was largest (4 to 5 mV, [7,18,31]). 2.3. Perfusion and immunohistochemical procedures Ten to fourteen days after the injection of CTb, the animals were sacrificed with an overdose of sodium pentobarbital and perfused with heparinized saline, followed by a solution of 4% paraformaldehyde, 15% saturated picric acid, and 0.25% glutaraldehyde in 0.1 M phosphate buffer (PB) at pH 7.4. The brainstem was removed and immersed for a 24-h post-fixation period in a solution consisting of 2% paraformaldehyde and 15% saturated picric acid in 0.1 M PB at pH 7.4. Following post-fixation, the tissue was kept in a solution of sucrose (25%) in 0.1 M PB at pH 7.4 for 2 days. The brainstem was frozen and cut into 15-Am-thick coronal sections using a cryostat. Each section was placed in one well of a 36-well tray containing a buffered solution of 0.1 M PBS, 0.3% Triton X-100 (PBST), and 0.1% sodium azide. The first section obtained was placed in the first well of the tray and consecutive sections were placed in the remaining wells in serial order. Section number 37 was placed in well 1 and the procedure was repeated until the entire brainstem was sectioned. Therefore, each well contained a sample of the entire brainstem consisting in a set of sections separated by 540 Am from one another (i.e., 15 Am 36). Using this procedure, neighboring wells contained adjacent sections. Free-floating sections were processed first for CTb immunocytochemistry and then for either nNOS immunocytochemistry or NADPH-d histochemistry. For CTb immunocytochemical detection, sections were first incubated in goat antiserum against CTb (List Biological Laboratories, CA.) at a dilution of 1:40,000 in PBS– 0.25% Triton–0.1% Na Azide (PBST-AZ), pH 7.4 at 4 8C with gentle agitation for 72 h or at 1:20,000 overnight at room temperature. The sections were rinsed over a 30-min period and placed for 90 min at room temperature in biotinylated donkey anti-goat serum (Jackson Immuno Research Laboratories, West Grove, PA) diluted 1:2000 with PBST. After rinsing for 30 min, the sections were incubated in a standard ABC complex (Vector Standard Elite kit, Vector Laboratories, Burlingame, CA) for 90 min at room temperature at a dilution of 1:400. The tissues were then rinsed for a total of 30 min and then processed with the Nickel Ammonium Sulfate enhanced DAB method consisting of immersion in 0.6% Nickel Ammonium Sulfate,
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0.02% diaminobenzidine (DAB), and 0.0015% hydrogen peroxide in 50 ml of 50 mM Tris buffer (pH = 7.6) for 15– 30 min. The reaction was terminated by rinsing the sections several times in PBST. For nNOS immunocytochemistry, free-floating sections were incubated overnight in a polyclonal antibody directed against nNOS (Accurate Chemical and Scientific Corp. Westbury, NY; dilution: 1:500). After rinsing in PBST, the tissue was incubated for 90 min in biotinylated donkey antirabbit serum (Jackson Immuno Research Laboratories) at a dilution of 1:500. The sections were then rinsed in PBST and treated with the ABC complex (Vector Standard Elite kit). Peroxidase activity was visualized with the DAB/ hydrogen peroxide method without nickel enhancement. Immunohistochemical controls for nNOS labeling included the omission of the primary antibody in the incubation buffer or the absorption test that consisted in the preincubation at 4 8C for 3 h of the antibody (1:1000) with different concentrations of the corresponding blocking peptide (50 and 100 Ag/ml; sc-648P, Santa Cruz Biotechnologies). Both procedures resulted in the absence of immunostaining. NADPH-diaphorase (NADPH-d) chemical activity was examined using nitro blue tetrazolium (NBT). To detect NADPH-d chemical activity, sections were incubated in a solution of 0.1 M PBS, pH 7.4, 0.3% Triton, 0.1 mg/ml NBT, and 1.0 mg/ml of beta-NADPH for 30–60 min at 37 8C. It has been demonstrated that after tissue fixation with aldehyde fixative solutions, the only remaining NADPH-d histochemical activity in the tissue corresponds to that of the nNOS enzyme [11,22]. The techniques for staining nNOS or for detecting NADPH-d activity are based on completely different biochemical principles [11,22], and the results obtained, which converge indicating that a group of medullary premotor neurons express nNOS, support each other. Finally, selected sections were counterstained by Pyronin Y. A number of sections were processed for choline acetyltransferase (ChAT) immunocytochemistry following the same general procedures that are described above for CTb and nNOS (see also [38]) using a goat polyclonal antibody directed against ChAT (dilution 1:2000; Chemicon, Temeluca, CA). This analysis was carried out to identify the soma and dendritic processes of brainstem motoneurons (see Fig. 1). 2.4. Data analysis The description of premotor neurons in this report corresponds to those experiments in which CTb injections were restricted to the mV or mXII, with no diffusion to other structures. The distribution of CTb-labeled neurons was first assesed using drawings of the histological sections made with a camera lucida attachment. Premotor nitrergic neurons (double-labeled, NADPH-d+/CTb+ or nNOS+/ CTb+) were found exclusively within the ventro-medial
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Fig. 1. Nitrergic fibers and terminals in the trigeminal and in the hypoglossal motor pools. (A) A trigeminal motoneuron labeled using a ChAT antibody. Blue-stained NADPH-d reactive fibers are present in close apposition to this neuron. The dashed circle encompasses NADPH-dlabeled fibers that give rise to bouton-like structures that are located near the ChAT immunostained cell body and a proximal dendritic process. The area encompassed by the circle is shown at higher magnification in AV. Small arrows point to NADPH-d-labeled fibers. (B and C) ChAT-labeled hypoglossal motoneurons and NADPH-d+ (blue) fibers.
medullary reticular formation. This part of the reticular formation also contained numerous nitrergic neurons that were not retrogradely labeled. Therefore, initially, the boundaries of the medullary region containing nitrergic neurons were delimited and their number was estimated. In a subsequent analysis, the subregion occupied by CTb+labeled nitrergic interneurons was delimited and their number was counted. The area of the region in the coronal plane was approximately 2 mm2. In control cats, in which the injection sites did not include neither the mV nor the mXII, the medullary NADPH-d+ or nNOS+ neurons did not display the retrograde CTb label. The stereological disector method, as described by Coggeshall [8] and Coggeshall and Lekan [9], was employed (see also Refs. [31,42]). This method required an estimation of the volume occupied by the region being studied (volume reference or Vref) and an estimation of bdensityQ (N d), in this case, of the double-labeled neurons in histological samples of the ventral medulla. An estimate of the number of double-labeled cells in the region was obtained by the formula (Vref)d (N d). To estimate numerical densities, neurons were counted in breferenceQ sections. bLook-upQ sections (i.e., those that were immediately adjacent to breferenceQ sections in a consecutive well) were also examined to determine which neurons were observed in both breference band blook-upQ sections. These neurons were not counted to avoid an overestimation of the number of cells [8,9,31]. A total of ten breferenceQ and ten blook-upQ
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sections was examined per animal. Photomicrographs (10 magnification) were obtained using an Olympus BH-2 microscope; breferenceQ and blook-upQ sections were superimposed using PhotoshopR software to determine the neurons that were observed in both sections. To determine the size of the soma profile, a 100 oil immersion objective lens was employed. Stained neurons in which the nucleolus was apparent were photographed and their major and minor soma diameters were measured. The nucleolus was clearly visible using Nomarsky optics. In order to illustrate neurons and to count them, photomicrographs were obtained by means of a digital camera, attached to an Olympus microscope, that was connected to a microcomputer. The designation of different regions of the reticular formation was based upon the nomenclature utilized by Taber [45] and Berman [3], see also [31].
3. Results Examples of nitrergic fibers within the trigeminal and hypoglossal motor pools are presented in Figs. 1A, AV and B, C, respectively. Motoneurons were labeled with the ChAT antibody; their cytoplasm is stained in brown. These sections were also processed to detect NADPH-d activity (stained fibers in blue). To determine the location of the cell bodies of the neurons that are the source of the nitrergic fibers shown above, CTb was injected into the trigeminal (mV) or into the hypoglossal (mXII) motor nucleus. Brainstem sections were later processed to detect this label together with NADPH-d activity or nNOS immunoreactivity. The extent of the CTb injection was examined in 10 sections for each cat. The deposits of CTb consisted of a circular central zone of dark reaction product (mean diameter: 1.0 mm F 0.07 SEM) surrounded by a lighter-stained zone. CTb deposits in two different animals are shown in Fig. 2. Fig. 2A is an example in which the deposit of CTb included a significant portion of the mV with little diffusion to adjacent structures. This localization of the injection to the mV was accomplished in six animals. In two other cats, the injection site was dorsal to the mV. Tissue from these latter animals was used as controls for putative CTb diffusion. Fig. 2B is an example of an injection within the mXII. In four animals, the injections were localized to this motor nucleus and in one animal, which was used as control for CTb diffusion, the injection was located ventrally. The mXII of the cat is an elongated structure which extends in the order of 5 mm in the anteroposterior direction. Therefore, the spherical CTb injection in this nucleus did not encompass the entire nucleus. In contrast, CTb injections encompassed most of the mV nucleus due to its spherical shape. The differences in shape of these nuclei likely influence the number of labeled premotor neurons for each nucleus (see Discussion). There was only one region that contained double-labeled NADPH-d+/CTb+ or nNOS+/CTb+ neurons; it was located in
Fig. 2. Cholera toxin subunit b (CTb) tracer injections in the trigeminal and hypoglossal motor pools. (A) Schematic diagram of a coronal section of the pons of a cat. The dark area represents the deposit of CTb in the mV; the gray area, circled by a dashed line, corresponds to the extracellular difusion of CTb. The filled line represents the boundaries of this nucleus, as observed in another section stained by a Nissl technique. (B) Same as in A for deposits in the mXII, in a different cat. bc, Brachium conjunctivum; mV, trigeminal motor nucleus; mXII, hypoglossal nucleus; nXII, hypoglossal nerve; p, pyramid; pV, sensory principal trigeminal nucleus; SO, superior olive; sV, spinal trigeminal complex; Vt, trigeminal tract.
the rostral part of the ventro-medial medullary reticular formation in an area corresponding to the nucleus magnocellularis (Mc) and the ventral aspect of the nucleus reticularis gigantocelularis (NRGc). Examples of these premotor nitrergic neurons are shown in Figs. 3A, B for NADPH-d/ CTb processing and in Figs. 4A–D and 5A for nNOS/CTb processing. The diagrams of the brain stem at the level of the ventral medullary reticular formation shown in Figs. 4E and 5B illustrate the location of double-labeled nNOS+/CTb+ neurons (filled circles) for one selected trigeminal and one selected hypoglossal experiment, respectively. Empty triangles depict the location of single-labeled NOS /CTb+ (nonnitrergic premotor) neurons. Empty circles illustrate the location of single-labeled NOS+/CTb (non-premotor nitrergic) neurons. These results show that the same region that contains premotor nitrergic cells also contains non-nitrergic
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Fig. 3. Premotor medullary NADPH-d containing interneurons. These sections were processed for both CTb immunostaining (dark cytoplasmic granules) and for the detection of NADPH-d activity (blue). The neurons are located in the ventro-medial medullary reticular formation. (A) Sections from a cat in which CTb was injected into the mV. Arrows point to two double-labeled NADPH-d+/Ctb+ neurons. Another NADPH-d+ neuron that did not contain the retrograde label is also shown. (B) Sections from another cat in which CTb was injected into the mXII. The arrow points to a double-labeled NADPH-d+/CTb+ neuron. A single-labeled NADPH-d+ neuron is also visible. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
premotor neurons, as well as other nitrergic cells that, because they did not display the retrograde label, did not appear to innervate either the mV or the mXII. The retrograde tracer CTb also labeled neurons belonging to the different populations of cells in the brainstem that have been described previously as containing premotor trigeminal and/or hypoglossal neurons [5,16,17,23,24, 25,30,31,39,46]. They are located in the trigeminal sensory system, in regions adjacent to the mV, in the raphe nuclei and in different subdivisions of the pontine and medullary reticular formation. Retrogradely labeled neurons in these
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Fig. 4. Examples of double-labeled (nNOS+/CTb+) trigeminal interneurons in the medullary reticular formation. The section was processed for both CTb and nNOS immunostaining. nNOS immunoreactive neurons have a golden brown cytoplasm; CTb retrogradely labeled neurons display black cytoplasmatic granules resulting from the Nickel enhancement technique employed. All photographs are from the same section. (A) Two nNOS positive neurons. The arrowhead points to one of these neurons, that does not contain CTb. The arrow points to the other cell that is a double-labeled nNOS+/CTb+ neuron. Note that its proximal dendrites (pointed out by custom/diamond arrows) display both nNOS and CTb reactivity. (B) Another example of a double-labeled cell following CTb injection into the mV. As in A, custom/diamond arrows point to the proximal dendrites containing nNOS and CTb. (C) A CTb-labeled cell that does not display nNOS reactivity. The diamond arrowheads point to the dendritic processes of this neuron. In contrast to the double-labeled neurons in A and B, it is clear that these processes do not display nNOS-like immunoreactivity. (D) A nNOS+ cell that does not contain CTb; note the absence of black granules in the soma and dendrites (arrowheads). The asterisk indicates a CTb-labeled fiber in close proximity to the cell body. (E) Schematic diagram representing the section from which the photomicrographs A, B, C, and D were taken. It is a coronal section of the medulla at a level immediately caudal to the mVII nucleus. Triangles represent CTb+ neurons; empty circles indicate nNOS+ neurons which do not contain the retrograde label; filled circles represent double-labeled (nNOS+/CTb+) pretrigeminal neurons. Each symbol represents one neuron. IO, inferior olive; p, pyramid.
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nucleus (mVII) and extends caudally to the level of the rostral portion of the inferior olivary complex. The stereotaxic boundaries are (approximately) P7.7 to P10, L1.0 to L2.5 and H 7 to H 8.5, according to Berman atlas [3]. Table 1 summarizes the results for all experiments. Ipsilateral to the CTb injection side, an average of 473 F 10 neurons displayed CTb when this label was injected into the mV (70 F 8 neurons CTb+ cells were found on the contralateral side). The number of ipsilateral double-labeled CTb+/nNOS+ pretrigeminal neurons was 60 F 15. When CTb was injected into the mXII, there was an average of 120 F 32 retrogradely labeled neurons ipsilateral to the injection side (18 F 2 cells were found on the contralateral side). The number of ipsilateral double-labeled, CTb+/ nNOS+ prehypoglossal neurons was 40 F 17. Examples of these neurons and example diagrams of their location are shown in Figs. 3–5. Contralateral to the injection side, there were 33 F 14 pretrigeminal nitrergic neurons and 16 F 5 prehypoglossal nitrergic neurons.
Fig. 5. Examples of double-labeled (nNOS+/CTb+) hypoglossal interneurons in the medullary reticular formation. This section was processed for both CTb and nNOS immunostaining. (A) Arrows point to double-labeled, nNOS+/CTb+ interneurons following the injection of CTb into the mXII. The arrowhead points to a nitrergic neuron that does not display the retrograde label. (B) Schematic diagram representing the coronal section of the medulla at the level of the caudal portion of the mVII nucleus from which the microphotograph in A was taken. Triangles represent CTb+ neurons; empty circles indicate nNOS+ neurons which did not contain the retrograde label; filled circles represent double-labeled (nNOS+/CTb+) prehypoglossal neurons. Each symbol represents one neuron. mVII, facial motor nucleus; sV, spinal trigeminal complex; Vt, trigeminal tract.
structures did not display either NADPH-d or nNOS activity (see examples in Fig. 6). In control cats, in which the injection sites did not include neither the mV nor the mXII, the NADPH-d+ or nNOS+ neurons located in the ventro-medial medullary reticular formation did not display the retrograde CTb label. 3.1. Premotor nitrergic neurons in the ventro-medial medullary reticular formation The region occupied by premotor nitrergic neurons is located medially to the caudal half of the facial motor
Fig. 6. Examples of neurons in the parvocellularis region and the spinal trigeminal nucleus. (A) CTb-labeled neurons (containing dark granules in their cytoplasm) in the parvocellularis region immediately above the facial motor nucleus and a population of NADPH-d positive neurons in the same area. Note the close proximity of CTb and NADPH-d labeled neurons (approx. 5 Am). (B) CTb+ and NADPH-d+ neurons in the spinal trigeminal nucleus. None of the NADPH-d+ cells displayed the CTb retrograde label that had been injected into the mV. CTb-labeled neurons were not reactive to NADPH-d.
I. Pose et al. / Brain Research 1041 (2005) 29–37 Table 1 Trigeminal and hypoglossal premotor (CTb+) and premotor nitrergic (CTb+/ NOS+) cells in the ventro-medial medullary reticular formation mV +
CTb CTB+/NOS+
mXII
Ipsilateral
Contralateral
Ipsilateral
Contralateral
473 F 10 60 F 15
70 F 8 33 F 14
120 F 32 40 F 17
18 F 2 16 F 5
The table summarizes the results for all animals. The numbers are the mean F SD number of cells contained in the studied region for each category.
The populations of pretrigeminal and prehypoglossal nitrergic neurons overlapped, i.e., there was no differential distribution of these cells based on the motor nucleus to which they projected. Both trigeminal and hypoglossal nitrergic premotor neurons exhibited a variety of cell bodies: multipolar, triangular, circular, oval, or elongated. The mean soma diameter of pretrigeminal nitrergic neurons was 21.0 Am F 0.79 (SEM). The soma diameter of prehypoglossal nitrergic neurons was 19.3 Am F 0.67 (SEM). Multipolar or triangular neurons were larger than the oval or elongated cells. 3.2. Nitrergic neurons in the ventro-medial medullary reticular formation Premotor nitrergic cells were found to be part of a larger population of nitrergic cells that were located in the ventromedial medullary reticular formation and occupied the Mc and the ventral aspect of the nucleus NRGc. The region where these nitrergic cells are located is bounded by the following coordinates: P7 to P13.5, L1.0 to L2.6 and H6.4 to H8.5. Therefore, it extends more rostrally (P7 vs. P7.7) and caudally (P13.5 vs. P10) than the region occupied by the retrogradely labeled nitrergic neurons. Within this region, there were in total, an estimated 7200 F 23 nitrergic neurons.
4. Discussion In the present study, we describe a population of premotor neurons within the brainstem that are the source of the nitrergic innervation of trigeminal and hypoglossal motoneurons. The existence of neurons displaying NADPHd activity and/or nNOS immunoreactivity in the brain stem reticular formation has been demonstrated in earlier publications [4,12,41,43,48]. In the medullary reticular formation, such neurons were found laterally in the parvocellularis region and medially in the magnocellularis (Mc) and in the nucleus reticularis gigantocellularis (NRGc). The present data expand these observations by demonstrating that nitrergic neurons in the Mc and in the ventral portion of the NRGc in the rostral ventro-medial medulla innervate brainstem motor pools, whereas those in the parvocellularis reticular formation do not. As shown for
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other premotor populations of cells in the rostral portion of the ventro-medial medulla [13,16,24,25,26,31], there was a bilateral distribution with clear ipsilateral dominance. Given the size of the hypoglossal injections, it is possible that the number of premotor interneurons for this motor nucleus was underestimated. This is one possibility but not the only one. Another, is that the axons of nitrergic premotor neurons branch and present a pattern of divergence such that each interneuron would eventually innervate all hypoglossal motoneurons. This is the case, for example, of homonymous Ia afferents of many muscles, each of which branches to innervate all motoneurons of the corresponding motor nucleus [36]. If this were also the case for this premotor population, one should expect that all hypoglossal premotor neurons would have been labeled. The distinction between these possibilities requires evidence that this study was not designed to obtain. It is well established that neurons of the Mc and NRGc project to the facial motor nucleus, bilaterally and to motoneurons of the spinal cord ([10,14,15,24,25,37]; the spinal cord data are reviewed in Ref. [21]). The present evidence indicates that some of these premotor cells may be nitrergic. It should be noted that it was only recently, and after the abovementioned publications appeared, that nitrergic fibers in motor nuclei were described and that for this reason, perhaps, the expression of nNOS in premotor neurons has not been explored until the present [1]. In neuronal cell bodies, and most likely in synaptic terminals, nNOS often colocalizes with classical neurotransmitters such as glutamate or acetylcholine [29,49]. Maqbool et al. [29] presented evidence indicating that a number of ventro-medial medullary neurons that display glutamate-like immunoreactivity also exhibit NADPH-d activity. Therefore, these data suggest that nitrergic premotor interneurons utilize glutamate as a primary neurotransmittter. In a previous study [31], we described inhibitory premotor glycinergic cells (activated during active sleep) whose distribution overlaps with that of the nitrergic neurons described in the present report. Due to the fact that NO has excitatory effects on trigeminal motoneurons [1], it seems contradictory that this molecule (NO) and an inhibitory neurotransmiter (glycine) are, respectively, produced and released simultaneously by the same synaptic terminal. This reasoning notwithstanding, Spike et al. [44] have described GABAergic and glycinergic neurons in the spinal cord that also contain NADPH diaphorase. We suggest that medullary glycinergic premotor inhibitory neurons may correspond to those CTb+ neurons found in the present study that are not nitrergic. Therefore, there must be two different populations of premotor cells intermingled within the same medullary region, i.e., those which are nitrergic/glutamatergic and have excitatory functions, and those that are glycinergic (and non-nitrergic) and have inhibitory functions; however, additional research is needed in order to verify this hypothesis.
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Cholinergic neurons in the pontine tegmentum display strong nNOS immunoreactivity, which indicates that these cells, which utilize ACh as neurotransmitter, also produce NO as a neuromodulator [49]. Cholinergic premotor neurons have been described in the medulla [16], mainly in the dorsal parvocellularis region, lateral to the NRGc, which makes it unlikely that the premotor nitrergic neurons described in the present report are cholinergic. The search for other neurotransmitter/neuromodulators that are colocalized in nitrergic premotor interneurons is of further interest considering that there are a number of premotor neurons in the ventro-medial medulla that contain enkephalinergic peptides [17]. The existence of premotor interneurons of different neurochemical phenotypes in the ventral medulla explains, in part, the invariably complex motor responses that are induced when this region is stimulated in electrophysiological experiments [35,34,50]. Many neurons in the lateral reticular formation and within the sensory trigeminal nuclei displayed nNOS immuno- and NADPH-d reactivity; however, in contrast to those in the medial reticular formation, none of these are last-order neurons for these motor nuclei. Therefore, these cells cannot influence, directly, the excitability of trigeminal and hypoglossal motoneurons. Because of their location, however, nitric oxide that is produced by them could directly affect the excitability of other neighboring premotor reticular and sensory cells, and thus function to modulate their input to hypoglossal and trigeminal motor nuclei. Nitrergic modulation in premotor structures have been shown in the oculo-motor system by Moreno-Lo´pez et al. [33], who have reported that local changes in NO concentration in the prepossitus hypoglossi nucleus induces motor responses. 4.1. The putative modulatory role of premotor nitrergic neurons The present report is the first in which a population of premotor interneurons containing nNOS has been described. Consequently, physiological experiments are, in due course, required to study their function(s). In the past, other groups of premotor interneurons containing classical neurotransmitters with the capability of directly interacting with ionotropic receptors have been described in the rostral ventro-medial medullary reticular formation. In general, these neurons have been ascribed important roles in the fine control of movements of the tongue and jaw during, for example, mastication or deglutition [20,21,27,28,34,37]. In addition, a population of glycinergic cells in this region has been found, that are likely to be responsible for the postsynaptic inhibition of motoneurons during active sleep (see above and [31]). The medullary premotor neurons described so far utilize neurotransmitters that participate in fast synaptic transmission. Nitric oxide is distinct from these classical neurotransmitters, insofar as it is a highly diffusible membrane permeant gaseous molecule [6,19,43] which is not stored in synaptic vesicles, but is produced following the
entrance of Ca++ into the intracellular domain. In somatic motoneurons, NO has excitatory effects [1], which are most likely related to the activation of a soluble guanylate cyclase and the formation of cGMP [1]. In this regard, NO is a neuromodulatory substance since it appears as if its effects are directed, not to activate postsynaptic ionotropic receptor(s), but rather to set in motion a cascade of intracellular biochemical processes. Given the diffusibility of NO [6,19] and the fact that numerous nitrergic premotor fibers are in close apposition to motoneuron processes, it is conceivable that effective amounts of NO are produced if the premotor neurons that we describe in the present report are synchronously excited (see also [1]). The background level of excitation by NO in these two motor nuclei, under these hypothetical conditions, would be necessary for a variety of functions such as mastication that require synchronized motoneuron activity.
Acknowledgments We are grateful to J.K. Engelhardt for his critical review of the manuscript. This work was supported by the following grants from the US. Public Health Service: NS23426, NS09999, MH 43362, and AGO4307.
References [1] V. Abudara, A. Ferna´ndez Alvarez, M.H. Chase, Nitric oxide as an anterograde neurotransmitter in the trigeminal motor pool, J. Neurophysiol. 88 (2002) 497 – 506. [2] M. Amri, A. Car, C. Roman, Axonal branching of medullary swallowing neurons projecting to the trigeminal and hypoglossal motor nuclei: demonstration by electrophysiological and fluorescent double labeling techniques, Exp. Brain Res. 81 (1990) 383 – 390. [3] A.L. Berman, The brainstem of the cat. A Cytoarchitectonic Atlas with Stereotaxic Coordinates, Univ. of Wisconsin Press, Madison, 1968. [4] D. Blottner, Z. Grozdanovic, R. Gossrau, Histochemistry of nitric oxide synthase in the nervous system, Histochem. J. 27 (10) (1995) 785 – 811. [5] R.C. Borke, M.E. Nau, R.L. Ringler, Brain stem afferents of hypoglossal neurons in the rat, Brain Res. 269 (1983) 47 – 55. [6] J.E. Brenman, D.S. Bredt, Synaptic signaling by nitric oxide, Curr. Opin. Neurobiol. 7 (1997) 374 – 378. [7] P. Castillo, C. Pedroarena, M. Chase, F.R. Morales, A medullary inhibitory region for trigeminal motoneurons in the cat, Brain Res. 549 (1991) 346 – 349. [8] R.E. Coggeshall, A consideration of neural counting methods, TINS 15 (1992) 9 – 13. [9] R.E. Coggeshall, H.A. Lekan, Methods for determining numbers of cells and synapses: a case for more uniform standards of review, J. Comp. Neurol. 364 (1996) 6 – 15. [10] C. Dauvergne, G. Pinganaud, P. Buisseret, C. Buisseret-Delmas, F. Zerari-Mailly, Reticular premotor neurons projecting to both facial and hypoglossal nuclei receive trigeminal afferents in rats, Neurosci. Lett. 311 (2) (2001) 109 – 112. [11] T. Dawson, D.S. Bredt, M. Fotuhi, P.M. Hwang, S. Snyder, Nitric oxide synthase and neuronal NADPH-diaphorase are identical in brain and peripheral tissues, Proc. Natl. Acad. Sci. 88 (1991) 7797 – 7801.
I. Pose et al. / Brain Research 1041 (2005) 29–37 [12] N.J. Dun, S.L. Dun, U. Forstermann, Nitric oxide synthase immunoreactivity in rat pontine medullary neurons, Neuroscience 59 (2) (1994) 429 – 445. [13] R.A. Fay, R. Norgren, Identification of rat brainstem multisynaptic connections to the oral motor nuclei using pseudorabies virus: I. Masticatory muscle motor systems, Brain Res. Rev. 25 (1997) 255 – 275. [14] R.A. Fay, R. Norgren, Identification of rat brainstem multisynaptic connections to the oral motor nuclei using pseudorabies virus: III. Lingual muscle motor systems, Brain Res. Rev. 25 (1997) 291 – 311. [15] P. Fort, K. Sakai, P. Luppi, D. Salvert, M. Jouvet, Monoaminergic, peptidergic, and cholinergic afferents to the cat facial nucleus as evidence by a double immunostaining method with unconjugated cholera toxin as a retrograde tracer, J. Comp. Neurol. 302 (1989) 283 – 285. [16] P. Fort, P.H. Luppi, K. Sakai, D. Salvert, M. Jouvet, Nuclei of origin of monoaminergic, peptidergic, and cholinergic afferents to the cat trigeminal motor nucleus: a double-labeling study with cholera-toxin as a retrograde tracer, J. Comp. Neurol. 301 (1990) 262 – 275. [17] P. Fort, C. Rampon, D. Gervasoni, C. Peyron, P.H. Luppi, Anatomical demonstration of a medullary enkephalinergic pathway potentially implicated in the orofacial muscle atonia of paradoxical sleep in the cat, Sleep Res., Online 1 (1998) 102 – 108. [18] S.J. Fung, J. Yamuy, S. Sampogna, F.R. Morales, M.H. Chase, Hypocretin (orexin) input to trigeminal and hypoglossal motoneurons in the cat: a double-labeling immuno-histochemical study, Brain Res. 903 (2001) 257 – 262. [19] J. Garthwaite, C.L. Boulton, Nitric oxide signaling in the central nervous system, Annu. Rev. Physiol. 57 (1995) 683 – 706. [20] K. Hiraba, M. Taira, Y. Sahara, N. Nakamura, Single-unit activity in bulbar reticular formation during food ingestion in chronic cats, J. Neurophysiol. 60 (4) (1988) 1333 – 1349. [21] J.C. Holstege, The ventro-medial medullary projections to spinal motoneurons: ultrastructure, transmitters and functional aspects, Prog. Brain Res. 107 (1996) 159 – 181. [22] B.T. Hope, G.J. Michael, K.M. Knifge, S.R. Vincent, Neuronal NADPH diaphorase is a nitric oxide synthase, Proc. Natl. Acad. Sci. 88 (1991) 2811 – 2814. [23] S. Landgren, K.A. Olsson, K.G. Wetberg, Bulbar neurons with axonal projections to the trigeminal motor nucleus in the cat, Exp. Brain Res. 65 (1986) 98 – 111. [24] Y.-Q. Li, M. Takada, N. Mizuno, Identification of premotor interneurons which project bilaterally to the trigeminalmotor, facial or hypoglossal nuclei: a fluorescent retrograde double-labeling study in the rat, Brain Res. 611 (1993) 160 – 164. [25] Y.-Q. Li, M. Takada, N. Mizuno, Premotor neurons projecting simultaneously to two orofacial motor nuclei by sending their branched axons: a study with a fluorescent retrograde double-labeling technique, Neurosci. Lett. 152 (1993) 29 – 32. [26] Y.-Q. Li, M. Takada, T. Kaneko, N. Mizuno, Premotor neurons for trigeminal motor nucleus neurons innervating the jaw-closing and jaw-opening muscles: differential distribution in the lower brainstem of the rat, J. Comp. Neurol. 356 (1995) 563 – 579. [27] J.P. Lund, K.A. Olsson, The importance of reflexes and their control during jaw movements, Trends Neurosci. 6 (1983) 458 – 463. [28] E.S. Luschei, L.J. Goldberg, Neural mechanisms of mandibular control: mastication and voluntary biting, in: Handbook of Physiology. The Nervous System, vol. II, Part 2, Am. Phys. Soc., Bethesda, MD, 1981, pp. 1237 – 1274. [29] A. Maqbool, T.F.C. Batten, P.N. McWilliam, Co-localization of neurotransmitter immunoreactivities in putative nitric oxide synthesizing neurones of the cat brain stem, J. Chem. Neuroanat. 8 (1995) 191 – 206.
37
[30] N. Mizuno, Y. Yasui, S. Nomura, K. Itoh, A. Konishi, M. Takada, M. Kudo, A light and electron microscopic study of premotor neurons for the trigeminal motor nucleus, J. Comp. Neurol. 215 (1983) 290 – 298. [31] F.R. Morales, S. Sampogna, J. Yamuy, M.H. Chase, c-fos Expression in brainstem premotor interneurons during cholinergically-induced active sleep in the cat, J. Neurosci. 19 (1999) 9508 – 9518. [32] F.R. Morales, S. Sampogna, I. Pose, Nitric oxide producing premotor trigeminal interneurons, Program No.856.1, 2002 Abstract viewer/ Itinerary planner, Society for Neuroscience, Washington, DC, 2002 (CD-ROM). [33] B. Moreno-Lo´pez, M. Escudero, J.M. Delgado-Garcı´a, C. Estrada, Nitric oxide production by brain stem neurons is required for normal performance of eye movements in alert animals, Neuron 17 (1996) 739 – 745. [34] Y. Nakamura, M. Takatori, S. Nozaki, M. Kikuchi, Monosynaptic reciprocal control of trigeminal motoneurons from the medial bulbar reticular formation, Brain Res. 89 (1975) 144 – 148. [35] Y. Nakamura, K. Hiraba, M. Taira, Y. Sahara, S. Enomoto, M. Katoh, A. Iriki, Activity during active sleep of bulbar reticular neurons firing rhythmically during mastication in cats, Exp. Neurol. 85 (1) (1984) 178 – 186. [36] S.G. Nelson, L.M. Mendell, Projection of single knee flexor Ia fibers to homonymous and heteronymous motoneurons, J. Neurophysiol. 41 (1978) 778 – 787. [37] A.S. Popratiloff, M. Streppel, A. Gruart, O. Guntinas-Lichius, D.N. Angelov, E. Stennert, J.M. Delgado-Garcia, W.F. Neiss, Hypoglossal and reticular interneurons involved in oro-facial coordination in the rat, J. Comp. Neurol. 433 (2001) 364 – 379. [38] I. Pose, S. Sampogna, M.H. Chase, F.R. Morales, Cuneiform neurons activated during cholinergically-induced active sleep in the cat, J. Neurosci. 20 (2000) 3319 – 3327. [39] C. Rampon, C. Peyron, J.M. Petit, P. Fort, D. Gervasoni, P.H. Luppi, Origin of the glycinergic innervation of the rat trigeminal motor nucleus, NeuroReport 25 (1996) 3081 – 3085. [40] J.C. Rekling, G.D. Funk, D.A. Bayliss, X.-W. Dong, J.L. Feldman, Synaptic control of motoneuronal excitability, Physiol. Rev. 82 (2) (2000) 767 – 852. [41] J. Rodrigo, D.R. Springall, O. Uttenthal, M.L. Bentura, F. AbadiaMolina, V. Riveros-Moreno, R. Martinez-Murillo, J.M. Polak, S. Moncada, Localization of nitric oxide synthase in the adult rat brain, Philos. Trans. R. Soc. Lond., Ser. B Biol. Sci. 345 (1312) (1994) 175 – 221. [42] C.B. Saper, Any way you cut it: a new journal policy for the use of unbiased counting methods, J. Comp. Neurol. 364 (1996) 5. [43] E. Southam, J. Garthwaite, The nitric oxide-cyclic GMP signaling pathway in rat brain, Neuropharmacology 11 (1993) 1267 – 1277. [44] R.C. Spike, A.J. Todd, H.M. Johnston, Coexistence of NADPH diaphorase with GABA, glycine, and acetylcholine in rat spinal cord, J. Comp. Neurol. 335 (1993) 320 – 333. [45] E. Taber, The cytoarchitecture of the brain stem of the cat: I. Brain stem nuclei of the cat, J. Comp. Neurol. 116 (1961) 27 – 69. [46] J.B. Travers, R. Norgren, Afferent projections to the oral motor nuclei in the rat, J. Comp. Neurol. 220 (1983) 280 – 298. [47] J.B. Travers, L.A. Dinardo, H. Karimnamazi, Motor and premotor mechanisms of licking, Neurosci. Biobehav. Rev. 21 (1997) 631 – 647. [48] S.R. Vincent, H. Kimura, Histochemical mapping of nitric oxide synthase in the rat brain, Neuroscience 46 (1992) 755 – 784. [49] S.R. Vincent, K. Satoh, D.M. Armstrong, P. Panula, W. Vale, H.C. Fibiger, Neuropeptides and NADPH-diaphorase activity in the ascending cholinergic reticular system of the rat, Neuroscience 17 (1986) 167 – 182. [50] C.Ch. Yang, J.Y.H. Chan, S.S.H. Chan, Excitatory innervation of caudal hypoglossal nucleus from nucleus reticularis gigantocellularis in the rat, Neuroscience 65 (1995) 365 – 374.
Research report
Nitrergic innervation of trigeminal and hypoglossal motoneurons in the cat Ines Posea,T, Simon Fungb,c, Sharon Sampognac, Michael H. Chaseb,c, Francisco R. Moralesa,b,c a
Departamento de Fisiologı´a, Facultad de Medicina, Gral. Flores 2125, Montevideo-11800, Uruguay b Websciences International 1263, Westwood Boulevard, 200, Los Angeles, CA 90024, USA c Department of Physiology, University of California, Los Angeles, CA 90024, USA Accepted 26 January 2005 Available online 8 March 2005
Abstract The present study was undertaken to determine the location of trigeminal and hypoglossal premotor neurons that express neuronal nitric oxide synthase (nNOS) in the cat. Cholera toxin subunit b (CTb) was injected into the trigeminal (mV) or the hypoglossal (mXII) motor nuclei in order to label the corresponding premotor neurons. CTb immunocytochemistry was combined with NADPH-d histochemistry or nNOS immunocytochemistry to identify premotor nitrergic (NADPH-d+/CTb+ or nNOS+/ CTb+ double-labeled) neurons. Premotor trigeminal as well as premotor hypoglossal neurons were located in the ventro-medial medullary reticular formation in a region corresponding to the nucleus magnocellularis (Mc) and the ventral aspect of the nucleus reticularis gigantocellularis (NRGc). Following the injection of CTb into the mV, this region was found to contain a total of 60 F 15 double-labeled neurons on the ipsilateral side and 33 F 14 on the contralateral side. CTb injections into the mXII resulted in 40 F 17 double-labeled neurons in this region on the ipsilateral side and 16 F 5 on the contralateral side. Thus, we conclude that premotor trigeminal and premotor hypoglossal nitrergic cells coexist in the same medullary region. They are colocalized with a larger population of nitrergic cells (7200 F 23). Premotor neurons in other locations did not express nNOS. The present data demonstrate that a population of neurons within the Mc and the NRGc are the source of the nitrergic innervation of trigeminal and hypoglossal motoneurons. Based on the characteristics of nitric oxide actions and its diffusibility, we postulate that these neurons may serve to synchronize the activity of mV and mXII motoneurons. D 2005 Elsevier B.V. All rights reserved. Theme: Motor systems and sensory motor integration Topic: Spinal cord and brainstem Keywords: Nitric oxide; Motor control; Medulla; Premotor interneurons; Neuromodulation; Reticular formation
1. Introduction Somatic motoneurons are perhaps the most completely studied cells in the central nervous system. There is a wealth of data dealing with their structure, function, electrophysiological properties, as well as their synaptic processes, including the neurotransmitters and neuromodulators that control their activity. In a comprehensive review, numerous substances including amino acids, biogenic amines, and peptides that act as neurotransmitters and neuromodulators are described in the synaptic processes that impinge on these cells [40]. * Corresponding author. Fax: +598 2 9248784. E-mail address: [email protected] (I. Pose). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.01.092
In recent years, nitric oxide (NO) has joined the list of neuromodulatory substances that act on neurons in the central nervous system [6,19]. We have previously reported that synaptic processes, containing the neuronal isoform of nitric oxide synthase (nNOS), are present in close apposition to the dendrites and cell bodies of trigeminal motoneurons in the guinea pig and that nitric oxide exerts an excitatory effect on these cells [1]. Trigeminal motoneurons participate in jaw movements associated with numerous behaviors such as mastication, deglutition, and vocalization [27,28]. During these and related motor acts, a precise coordination takes place between the jaw musculature innervated by trigeminal motoneurons and the tongue musculature that is innervated by hypoglossal motoneurons. It has been suggested that
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this coordination depends on circuits that involve overlapping or shared structures controlling both motor pools [2,13,14,24,25,47]. The present study was designed to determine the location of nitrergic premotor neurons that project to the trigeminal and hypoglossal motoneurons in the cat. An examination of the origins of the innervation of trigeminal motoneurons, prompted by our previous work [1], was combined with a study of the innervation of hypoglossal motoneurons. By means of retrograde and nNOS labeling techniques, we traced the origin of the nitrergic innervation of these brainstem motor nuclei to a population of cells within the ventro-medial medullary reticular formation. Accordingly, a novel population of premotor neurons was discovered in the present work. A portion of these data has been previously reported [32].
2. Methods Thirteen adult cats of both sexes, weighing between 3.0 and 3.5 kg, were utilized. Eight cats were employed for studies of the trigeminal nucleus and five for experiments on the hypoglossal nucleus. 2.1. Surgical procedures All experimental procedures were conducted in accord with the Guide for the Care and Use of Laboratory Animals (7th edition, National Academy Press, Washington, D.C. 1996) and approved by the Chancellor’s Animal Research Committee (ARC) of the UCLA Office for the Protection of Research Subjects (OPRS). Before anesthesia, the animals were premedicated with atropine (0.04 mg/kg, i.m.) and XylazineR (2 mg/kg, i.m.). Anesthesia was first induced with KetamineR (15 mg/kg, i.m.) and maintained with a gas mixture of isoflurane in oxygen (2–3%). The head of the cat was positioned in a heavy-duty stereotaxic frame and the calvarium was exposed. A 3.0- to 4.0-mm-diameter hole was drilled in the calvarium overlying the cerebellar cortex; after surgery, the hole was covered with bone wax. This hole provided access to the trigeminal or the hypoglossal nuclei for injection of CTb, as described below. During recovery from surgery, an analgesic (BuprenexR 0.01 mg/kg, i.m.) was administered and an antibiotic (CephazolinR) was given parenterally for 4 days. All wound margins were regularly cleaned and covered with an antibiotic ointment (FougeraR). 2.2. Micropipette and microelectrode assembly for the injection of cholera toxin subunit b (CTb) and for recording antidromic field potentials In order to inject CTb, a three-barreled micropipette assembly, consisting of a carbon fiber recording microelectrode and two side barrels containing CTb (List
Biological Laboratories, Campbell, CA), was lowered into the trigeminal (stereotaxic coordinates: P4.6, L3.5, H 4; [3]) or the hypoglossal motor nucleus (stereotaxic coordinates: P12.7, L1.2, H 6.5; [3]). The exact location of these motor nuclei in each animal was resolved by recording the antidromic field potential evoked by electrical stimulation of the masseter and hypoglosssal nerves, respectively, as described in previous communications [7,18]. CTb was injected by iontophoresis (2 AA positive current pulses, 7 s on, 7 s off, for 20 min in each barrel) at a site where the antidromic field potential was largest (4 to 5 mV, [7,18,31]). 2.3. Perfusion and immunohistochemical procedures Ten to fourteen days after the injection of CTb, the animals were sacrificed with an overdose of sodium pentobarbital and perfused with heparinized saline, followed by a solution of 4% paraformaldehyde, 15% saturated picric acid, and 0.25% glutaraldehyde in 0.1 M phosphate buffer (PB) at pH 7.4. The brainstem was removed and immersed for a 24-h post-fixation period in a solution consisting of 2% paraformaldehyde and 15% saturated picric acid in 0.1 M PB at pH 7.4. Following post-fixation, the tissue was kept in a solution of sucrose (25%) in 0.1 M PB at pH 7.4 for 2 days. The brainstem was frozen and cut into 15-Am-thick coronal sections using a cryostat. Each section was placed in one well of a 36-well tray containing a buffered solution of 0.1 M PBS, 0.3% Triton X-100 (PBST), and 0.1% sodium azide. The first section obtained was placed in the first well of the tray and consecutive sections were placed in the remaining wells in serial order. Section number 37 was placed in well 1 and the procedure was repeated until the entire brainstem was sectioned. Therefore, each well contained a sample of the entire brainstem consisting in a set of sections separated by 540 Am from one another (i.e., 15 Am 36). Using this procedure, neighboring wells contained adjacent sections. Free-floating sections were processed first for CTb immunocytochemistry and then for either nNOS immunocytochemistry or NADPH-d histochemistry. For CTb immunocytochemical detection, sections were first incubated in goat antiserum against CTb (List Biological Laboratories, CA.) at a dilution of 1:40,000 in PBS– 0.25% Triton–0.1% Na Azide (PBST-AZ), pH 7.4 at 4 8C with gentle agitation for 72 h or at 1:20,000 overnight at room temperature. The sections were rinsed over a 30-min period and placed for 90 min at room temperature in biotinylated donkey anti-goat serum (Jackson Immuno Research Laboratories, West Grove, PA) diluted 1:2000 with PBST. After rinsing for 30 min, the sections were incubated in a standard ABC complex (Vector Standard Elite kit, Vector Laboratories, Burlingame, CA) for 90 min at room temperature at a dilution of 1:400. The tissues were then rinsed for a total of 30 min and then processed with the Nickel Ammonium Sulfate enhanced DAB method consisting of immersion in 0.6% Nickel Ammonium Sulfate,
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0.02% diaminobenzidine (DAB), and 0.0015% hydrogen peroxide in 50 ml of 50 mM Tris buffer (pH = 7.6) for 15– 30 min. The reaction was terminated by rinsing the sections several times in PBST. For nNOS immunocytochemistry, free-floating sections were incubated overnight in a polyclonal antibody directed against nNOS (Accurate Chemical and Scientific Corp. Westbury, NY; dilution: 1:500). After rinsing in PBST, the tissue was incubated for 90 min in biotinylated donkey antirabbit serum (Jackson Immuno Research Laboratories) at a dilution of 1:500. The sections were then rinsed in PBST and treated with the ABC complex (Vector Standard Elite kit). Peroxidase activity was visualized with the DAB/ hydrogen peroxide method without nickel enhancement. Immunohistochemical controls for nNOS labeling included the omission of the primary antibody in the incubation buffer or the absorption test that consisted in the preincubation at 4 8C for 3 h of the antibody (1:1000) with different concentrations of the corresponding blocking peptide (50 and 100 Ag/ml; sc-648P, Santa Cruz Biotechnologies). Both procedures resulted in the absence of immunostaining. NADPH-diaphorase (NADPH-d) chemical activity was examined using nitro blue tetrazolium (NBT). To detect NADPH-d chemical activity, sections were incubated in a solution of 0.1 M PBS, pH 7.4, 0.3% Triton, 0.1 mg/ml NBT, and 1.0 mg/ml of beta-NADPH for 30–60 min at 37 8C. It has been demonstrated that after tissue fixation with aldehyde fixative solutions, the only remaining NADPH-d histochemical activity in the tissue corresponds to that of the nNOS enzyme [11,22]. The techniques for staining nNOS or for detecting NADPH-d activity are based on completely different biochemical principles [11,22], and the results obtained, which converge indicating that a group of medullary premotor neurons express nNOS, support each other. Finally, selected sections were counterstained by Pyronin Y. A number of sections were processed for choline acetyltransferase (ChAT) immunocytochemistry following the same general procedures that are described above for CTb and nNOS (see also [38]) using a goat polyclonal antibody directed against ChAT (dilution 1:2000; Chemicon, Temeluca, CA). This analysis was carried out to identify the soma and dendritic processes of brainstem motoneurons (see Fig. 1). 2.4. Data analysis The description of premotor neurons in this report corresponds to those experiments in which CTb injections were restricted to the mV or mXII, with no diffusion to other structures. The distribution of CTb-labeled neurons was first assesed using drawings of the histological sections made with a camera lucida attachment. Premotor nitrergic neurons (double-labeled, NADPH-d+/CTb+ or nNOS+/ CTb+) were found exclusively within the ventro-medial
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Fig. 1. Nitrergic fibers and terminals in the trigeminal and in the hypoglossal motor pools. (A) A trigeminal motoneuron labeled using a ChAT antibody. Blue-stained NADPH-d reactive fibers are present in close apposition to this neuron. The dashed circle encompasses NADPH-dlabeled fibers that give rise to bouton-like structures that are located near the ChAT immunostained cell body and a proximal dendritic process. The area encompassed by the circle is shown at higher magnification in AV. Small arrows point to NADPH-d-labeled fibers. (B and C) ChAT-labeled hypoglossal motoneurons and NADPH-d+ (blue) fibers.
medullary reticular formation. This part of the reticular formation also contained numerous nitrergic neurons that were not retrogradely labeled. Therefore, initially, the boundaries of the medullary region containing nitrergic neurons were delimited and their number was estimated. In a subsequent analysis, the subregion occupied by CTb+labeled nitrergic interneurons was delimited and their number was counted. The area of the region in the coronal plane was approximately 2 mm2. In control cats, in which the injection sites did not include neither the mV nor the mXII, the medullary NADPH-d+ or nNOS+ neurons did not display the retrograde CTb label. The stereological disector method, as described by Coggeshall [8] and Coggeshall and Lekan [9], was employed (see also Refs. [31,42]). This method required an estimation of the volume occupied by the region being studied (volume reference or Vref) and an estimation of bdensityQ (N d), in this case, of the double-labeled neurons in histological samples of the ventral medulla. An estimate of the number of double-labeled cells in the region was obtained by the formula (Vref)d (N d). To estimate numerical densities, neurons were counted in breferenceQ sections. bLook-upQ sections (i.e., those that were immediately adjacent to breferenceQ sections in a consecutive well) were also examined to determine which neurons were observed in both breference band blook-upQ sections. These neurons were not counted to avoid an overestimation of the number of cells [8,9,31]. A total of ten breferenceQ and ten blook-upQ
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sections was examined per animal. Photomicrographs (10 magnification) were obtained using an Olympus BH-2 microscope; breferenceQ and blook-upQ sections were superimposed using PhotoshopR software to determine the neurons that were observed in both sections. To determine the size of the soma profile, a 100 oil immersion objective lens was employed. Stained neurons in which the nucleolus was apparent were photographed and their major and minor soma diameters were measured. The nucleolus was clearly visible using Nomarsky optics. In order to illustrate neurons and to count them, photomicrographs were obtained by means of a digital camera, attached to an Olympus microscope, that was connected to a microcomputer. The designation of different regions of the reticular formation was based upon the nomenclature utilized by Taber [45] and Berman [3], see also [31].
3. Results Examples of nitrergic fibers within the trigeminal and hypoglossal motor pools are presented in Figs. 1A, AV and B, C, respectively. Motoneurons were labeled with the ChAT antibody; their cytoplasm is stained in brown. These sections were also processed to detect NADPH-d activity (stained fibers in blue). To determine the location of the cell bodies of the neurons that are the source of the nitrergic fibers shown above, CTb was injected into the trigeminal (mV) or into the hypoglossal (mXII) motor nucleus. Brainstem sections were later processed to detect this label together with NADPH-d activity or nNOS immunoreactivity. The extent of the CTb injection was examined in 10 sections for each cat. The deposits of CTb consisted of a circular central zone of dark reaction product (mean diameter: 1.0 mm F 0.07 SEM) surrounded by a lighter-stained zone. CTb deposits in two different animals are shown in Fig. 2. Fig. 2A is an example in which the deposit of CTb included a significant portion of the mV with little diffusion to adjacent structures. This localization of the injection to the mV was accomplished in six animals. In two other cats, the injection site was dorsal to the mV. Tissue from these latter animals was used as controls for putative CTb diffusion. Fig. 2B is an example of an injection within the mXII. In four animals, the injections were localized to this motor nucleus and in one animal, which was used as control for CTb diffusion, the injection was located ventrally. The mXII of the cat is an elongated structure which extends in the order of 5 mm in the anteroposterior direction. Therefore, the spherical CTb injection in this nucleus did not encompass the entire nucleus. In contrast, CTb injections encompassed most of the mV nucleus due to its spherical shape. The differences in shape of these nuclei likely influence the number of labeled premotor neurons for each nucleus (see Discussion). There was only one region that contained double-labeled NADPH-d+/CTb+ or nNOS+/CTb+ neurons; it was located in
Fig. 2. Cholera toxin subunit b (CTb) tracer injections in the trigeminal and hypoglossal motor pools. (A) Schematic diagram of a coronal section of the pons of a cat. The dark area represents the deposit of CTb in the mV; the gray area, circled by a dashed line, corresponds to the extracellular difusion of CTb. The filled line represents the boundaries of this nucleus, as observed in another section stained by a Nissl technique. (B) Same as in A for deposits in the mXII, in a different cat. bc, Brachium conjunctivum; mV, trigeminal motor nucleus; mXII, hypoglossal nucleus; nXII, hypoglossal nerve; p, pyramid; pV, sensory principal trigeminal nucleus; SO, superior olive; sV, spinal trigeminal complex; Vt, trigeminal tract.
the rostral part of the ventro-medial medullary reticular formation in an area corresponding to the nucleus magnocellularis (Mc) and the ventral aspect of the nucleus reticularis gigantocelularis (NRGc). Examples of these premotor nitrergic neurons are shown in Figs. 3A, B for NADPH-d/ CTb processing and in Figs. 4A–D and 5A for nNOS/CTb processing. The diagrams of the brain stem at the level of the ventral medullary reticular formation shown in Figs. 4E and 5B illustrate the location of double-labeled nNOS+/CTb+ neurons (filled circles) for one selected trigeminal and one selected hypoglossal experiment, respectively. Empty triangles depict the location of single-labeled NOS /CTb+ (nonnitrergic premotor) neurons. Empty circles illustrate the location of single-labeled NOS+/CTb (non-premotor nitrergic) neurons. These results show that the same region that contains premotor nitrergic cells also contains non-nitrergic
I. Pose et al. / Brain Research 1041 (2005) 29–37
Fig. 3. Premotor medullary NADPH-d containing interneurons. These sections were processed for both CTb immunostaining (dark cytoplasmic granules) and for the detection of NADPH-d activity (blue). The neurons are located in the ventro-medial medullary reticular formation. (A) Sections from a cat in which CTb was injected into the mV. Arrows point to two double-labeled NADPH-d+/Ctb+ neurons. Another NADPH-d+ neuron that did not contain the retrograde label is also shown. (B) Sections from another cat in which CTb was injected into the mXII. The arrow points to a double-labeled NADPH-d+/CTb+ neuron. A single-labeled NADPH-d+ neuron is also visible. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
premotor neurons, as well as other nitrergic cells that, because they did not display the retrograde label, did not appear to innervate either the mV or the mXII. The retrograde tracer CTb also labeled neurons belonging to the different populations of cells in the brainstem that have been described previously as containing premotor trigeminal and/or hypoglossal neurons [5,16,17,23,24, 25,30,31,39,46]. They are located in the trigeminal sensory system, in regions adjacent to the mV, in the raphe nuclei and in different subdivisions of the pontine and medullary reticular formation. Retrogradely labeled neurons in these
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Fig. 4. Examples of double-labeled (nNOS+/CTb+) trigeminal interneurons in the medullary reticular formation. The section was processed for both CTb and nNOS immunostaining. nNOS immunoreactive neurons have a golden brown cytoplasm; CTb retrogradely labeled neurons display black cytoplasmatic granules resulting from the Nickel enhancement technique employed. All photographs are from the same section. (A) Two nNOS positive neurons. The arrowhead points to one of these neurons, that does not contain CTb. The arrow points to the other cell that is a double-labeled nNOS+/CTb+ neuron. Note that its proximal dendrites (pointed out by custom/diamond arrows) display both nNOS and CTb reactivity. (B) Another example of a double-labeled cell following CTb injection into the mV. As in A, custom/diamond arrows point to the proximal dendrites containing nNOS and CTb. (C) A CTb-labeled cell that does not display nNOS reactivity. The diamond arrowheads point to the dendritic processes of this neuron. In contrast to the double-labeled neurons in A and B, it is clear that these processes do not display nNOS-like immunoreactivity. (D) A nNOS+ cell that does not contain CTb; note the absence of black granules in the soma and dendrites (arrowheads). The asterisk indicates a CTb-labeled fiber in close proximity to the cell body. (E) Schematic diagram representing the section from which the photomicrographs A, B, C, and D were taken. It is a coronal section of the medulla at a level immediately caudal to the mVII nucleus. Triangles represent CTb+ neurons; empty circles indicate nNOS+ neurons which do not contain the retrograde label; filled circles represent double-labeled (nNOS+/CTb+) pretrigeminal neurons. Each symbol represents one neuron. IO, inferior olive; p, pyramid.
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nucleus (mVII) and extends caudally to the level of the rostral portion of the inferior olivary complex. The stereotaxic boundaries are (approximately) P7.7 to P10, L1.0 to L2.5 and H 7 to H 8.5, according to Berman atlas [3]. Table 1 summarizes the results for all experiments. Ipsilateral to the CTb injection side, an average of 473 F 10 neurons displayed CTb when this label was injected into the mV (70 F 8 neurons CTb+ cells were found on the contralateral side). The number of ipsilateral double-labeled CTb+/nNOS+ pretrigeminal neurons was 60 F 15. When CTb was injected into the mXII, there was an average of 120 F 32 retrogradely labeled neurons ipsilateral to the injection side (18 F 2 cells were found on the contralateral side). The number of ipsilateral double-labeled, CTb+/ nNOS+ prehypoglossal neurons was 40 F 17. Examples of these neurons and example diagrams of their location are shown in Figs. 3–5. Contralateral to the injection side, there were 33 F 14 pretrigeminal nitrergic neurons and 16 F 5 prehypoglossal nitrergic neurons.
Fig. 5. Examples of double-labeled (nNOS+/CTb+) hypoglossal interneurons in the medullary reticular formation. This section was processed for both CTb and nNOS immunostaining. (A) Arrows point to double-labeled, nNOS+/CTb+ interneurons following the injection of CTb into the mXII. The arrowhead points to a nitrergic neuron that does not display the retrograde label. (B) Schematic diagram representing the coronal section of the medulla at the level of the caudal portion of the mVII nucleus from which the microphotograph in A was taken. Triangles represent CTb+ neurons; empty circles indicate nNOS+ neurons which did not contain the retrograde label; filled circles represent double-labeled (nNOS+/CTb+) prehypoglossal neurons. Each symbol represents one neuron. mVII, facial motor nucleus; sV, spinal trigeminal complex; Vt, trigeminal tract.
structures did not display either NADPH-d or nNOS activity (see examples in Fig. 6). In control cats, in which the injection sites did not include neither the mV nor the mXII, the NADPH-d+ or nNOS+ neurons located in the ventro-medial medullary reticular formation did not display the retrograde CTb label. 3.1. Premotor nitrergic neurons in the ventro-medial medullary reticular formation The region occupied by premotor nitrergic neurons is located medially to the caudal half of the facial motor
Fig. 6. Examples of neurons in the parvocellularis region and the spinal trigeminal nucleus. (A) CTb-labeled neurons (containing dark granules in their cytoplasm) in the parvocellularis region immediately above the facial motor nucleus and a population of NADPH-d positive neurons in the same area. Note the close proximity of CTb and NADPH-d labeled neurons (approx. 5 Am). (B) CTb+ and NADPH-d+ neurons in the spinal trigeminal nucleus. None of the NADPH-d+ cells displayed the CTb retrograde label that had been injected into the mV. CTb-labeled neurons were not reactive to NADPH-d.
I. Pose et al. / Brain Research 1041 (2005) 29–37 Table 1 Trigeminal and hypoglossal premotor (CTb+) and premotor nitrergic (CTb+/ NOS+) cells in the ventro-medial medullary reticular formation mV +
CTb CTB+/NOS+
mXII
Ipsilateral
Contralateral
Ipsilateral
Contralateral
473 F 10 60 F 15
70 F 8 33 F 14
120 F 32 40 F 17
18 F 2 16 F 5
The table summarizes the results for all animals. The numbers are the mean F SD number of cells contained in the studied region for each category.
The populations of pretrigeminal and prehypoglossal nitrergic neurons overlapped, i.e., there was no differential distribution of these cells based on the motor nucleus to which they projected. Both trigeminal and hypoglossal nitrergic premotor neurons exhibited a variety of cell bodies: multipolar, triangular, circular, oval, or elongated. The mean soma diameter of pretrigeminal nitrergic neurons was 21.0 Am F 0.79 (SEM). The soma diameter of prehypoglossal nitrergic neurons was 19.3 Am F 0.67 (SEM). Multipolar or triangular neurons were larger than the oval or elongated cells. 3.2. Nitrergic neurons in the ventro-medial medullary reticular formation Premotor nitrergic cells were found to be part of a larger population of nitrergic cells that were located in the ventromedial medullary reticular formation and occupied the Mc and the ventral aspect of the nucleus NRGc. The region where these nitrergic cells are located is bounded by the following coordinates: P7 to P13.5, L1.0 to L2.6 and H6.4 to H8.5. Therefore, it extends more rostrally (P7 vs. P7.7) and caudally (P13.5 vs. P10) than the region occupied by the retrogradely labeled nitrergic neurons. Within this region, there were in total, an estimated 7200 F 23 nitrergic neurons.
4. Discussion In the present study, we describe a population of premotor neurons within the brainstem that are the source of the nitrergic innervation of trigeminal and hypoglossal motoneurons. The existence of neurons displaying NADPHd activity and/or nNOS immunoreactivity in the brain stem reticular formation has been demonstrated in earlier publications [4,12,41,43,48]. In the medullary reticular formation, such neurons were found laterally in the parvocellularis region and medially in the magnocellularis (Mc) and in the nucleus reticularis gigantocellularis (NRGc). The present data expand these observations by demonstrating that nitrergic neurons in the Mc and in the ventral portion of the NRGc in the rostral ventro-medial medulla innervate brainstem motor pools, whereas those in the parvocellularis reticular formation do not. As shown for
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other premotor populations of cells in the rostral portion of the ventro-medial medulla [13,16,24,25,26,31], there was a bilateral distribution with clear ipsilateral dominance. Given the size of the hypoglossal injections, it is possible that the number of premotor interneurons for this motor nucleus was underestimated. This is one possibility but not the only one. Another, is that the axons of nitrergic premotor neurons branch and present a pattern of divergence such that each interneuron would eventually innervate all hypoglossal motoneurons. This is the case, for example, of homonymous Ia afferents of many muscles, each of which branches to innervate all motoneurons of the corresponding motor nucleus [36]. If this were also the case for this premotor population, one should expect that all hypoglossal premotor neurons would have been labeled. The distinction between these possibilities requires evidence that this study was not designed to obtain. It is well established that neurons of the Mc and NRGc project to the facial motor nucleus, bilaterally and to motoneurons of the spinal cord ([10,14,15,24,25,37]; the spinal cord data are reviewed in Ref. [21]). The present evidence indicates that some of these premotor cells may be nitrergic. It should be noted that it was only recently, and after the abovementioned publications appeared, that nitrergic fibers in motor nuclei were described and that for this reason, perhaps, the expression of nNOS in premotor neurons has not been explored until the present [1]. In neuronal cell bodies, and most likely in synaptic terminals, nNOS often colocalizes with classical neurotransmitters such as glutamate or acetylcholine [29,49]. Maqbool et al. [29] presented evidence indicating that a number of ventro-medial medullary neurons that display glutamate-like immunoreactivity also exhibit NADPH-d activity. Therefore, these data suggest that nitrergic premotor interneurons utilize glutamate as a primary neurotransmittter. In a previous study [31], we described inhibitory premotor glycinergic cells (activated during active sleep) whose distribution overlaps with that of the nitrergic neurons described in the present report. Due to the fact that NO has excitatory effects on trigeminal motoneurons [1], it seems contradictory that this molecule (NO) and an inhibitory neurotransmiter (glycine) are, respectively, produced and released simultaneously by the same synaptic terminal. This reasoning notwithstanding, Spike et al. [44] have described GABAergic and glycinergic neurons in the spinal cord that also contain NADPH diaphorase. We suggest that medullary glycinergic premotor inhibitory neurons may correspond to those CTb+ neurons found in the present study that are not nitrergic. Therefore, there must be two different populations of premotor cells intermingled within the same medullary region, i.e., those which are nitrergic/glutamatergic and have excitatory functions, and those that are glycinergic (and non-nitrergic) and have inhibitory functions; however, additional research is needed in order to verify this hypothesis.
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Cholinergic neurons in the pontine tegmentum display strong nNOS immunoreactivity, which indicates that these cells, which utilize ACh as neurotransmitter, also produce NO as a neuromodulator [49]. Cholinergic premotor neurons have been described in the medulla [16], mainly in the dorsal parvocellularis region, lateral to the NRGc, which makes it unlikely that the premotor nitrergic neurons described in the present report are cholinergic. The search for other neurotransmitter/neuromodulators that are colocalized in nitrergic premotor interneurons is of further interest considering that there are a number of premotor neurons in the ventro-medial medulla that contain enkephalinergic peptides [17]. The existence of premotor interneurons of different neurochemical phenotypes in the ventral medulla explains, in part, the invariably complex motor responses that are induced when this region is stimulated in electrophysiological experiments [35,34,50]. Many neurons in the lateral reticular formation and within the sensory trigeminal nuclei displayed nNOS immuno- and NADPH-d reactivity; however, in contrast to those in the medial reticular formation, none of these are last-order neurons for these motor nuclei. Therefore, these cells cannot influence, directly, the excitability of trigeminal and hypoglossal motoneurons. Because of their location, however, nitric oxide that is produced by them could directly affect the excitability of other neighboring premotor reticular and sensory cells, and thus function to modulate their input to hypoglossal and trigeminal motor nuclei. Nitrergic modulation in premotor structures have been shown in the oculo-motor system by Moreno-Lo´pez et al. [33], who have reported that local changes in NO concentration in the prepossitus hypoglossi nucleus induces motor responses. 4.1. The putative modulatory role of premotor nitrergic neurons The present report is the first in which a population of premotor interneurons containing nNOS has been described. Consequently, physiological experiments are, in due course, required to study their function(s). In the past, other groups of premotor interneurons containing classical neurotransmitters with the capability of directly interacting with ionotropic receptors have been described in the rostral ventro-medial medullary reticular formation. In general, these neurons have been ascribed important roles in the fine control of movements of the tongue and jaw during, for example, mastication or deglutition [20,21,27,28,34,37]. In addition, a population of glycinergic cells in this region has been found, that are likely to be responsible for the postsynaptic inhibition of motoneurons during active sleep (see above and [31]). The medullary premotor neurons described so far utilize neurotransmitters that participate in fast synaptic transmission. Nitric oxide is distinct from these classical neurotransmitters, insofar as it is a highly diffusible membrane permeant gaseous molecule [6,19,43] which is not stored in synaptic vesicles, but is produced following the
entrance of Ca++ into the intracellular domain. In somatic motoneurons, NO has excitatory effects [1], which are most likely related to the activation of a soluble guanylate cyclase and the formation of cGMP [1]. In this regard, NO is a neuromodulatory substance since it appears as if its effects are directed, not to activate postsynaptic ionotropic receptor(s), but rather to set in motion a cascade of intracellular biochemical processes. Given the diffusibility of NO [6,19] and the fact that numerous nitrergic premotor fibers are in close apposition to motoneuron processes, it is conceivable that effective amounts of NO are produced if the premotor neurons that we describe in the present report are synchronously excited (see also [1]). The background level of excitation by NO in these two motor nuclei, under these hypothetical conditions, would be necessary for a variety of functions such as mastication that require synchronized motoneuron activity.
Acknowledgments We are grateful to J.K. Engelhardt for his critical review of the manuscript. This work was supported by the following grants from the US. Public Health Service: NS23426, NS09999, MH 43362, and AGO4307.
References [1] V. Abudara, A. Ferna´ndez Alvarez, M.H. Chase, Nitric oxide as an anterograde neurotransmitter in the trigeminal motor pool, J. Neurophysiol. 88 (2002) 497 – 506. [2] M. Amri, A. Car, C. Roman, Axonal branching of medullary swallowing neurons projecting to the trigeminal and hypoglossal motor nuclei: demonstration by electrophysiological and fluorescent double labeling techniques, Exp. Brain Res. 81 (1990) 383 – 390. [3] A.L. Berman, The brainstem of the cat. A Cytoarchitectonic Atlas with Stereotaxic Coordinates, Univ. of Wisconsin Press, Madison, 1968. [4] D. Blottner, Z. Grozdanovic, R. Gossrau, Histochemistry of nitric oxide synthase in the nervous system, Histochem. J. 27 (10) (1995) 785 – 811. [5] R.C. Borke, M.E. Nau, R.L. Ringler, Brain stem afferents of hypoglossal neurons in the rat, Brain Res. 269 (1983) 47 – 55. [6] J.E. Brenman, D.S. Bredt, Synaptic signaling by nitric oxide, Curr. Opin. Neurobiol. 7 (1997) 374 – 378. [7] P. Castillo, C. Pedroarena, M. Chase, F.R. Morales, A medullary inhibitory region for trigeminal motoneurons in the cat, Brain Res. 549 (1991) 346 – 349. [8] R.E. Coggeshall, A consideration of neural counting methods, TINS 15 (1992) 9 – 13. [9] R.E. Coggeshall, H.A. Lekan, Methods for determining numbers of cells and synapses: a case for more uniform standards of review, J. Comp. Neurol. 364 (1996) 6 – 15. [10] C. Dauvergne, G. Pinganaud, P. Buisseret, C. Buisseret-Delmas, F. Zerari-Mailly, Reticular premotor neurons projecting to both facial and hypoglossal nuclei receive trigeminal afferents in rats, Neurosci. Lett. 311 (2) (2001) 109 – 112. [11] T. Dawson, D.S. Bredt, M. Fotuhi, P.M. Hwang, S. Snyder, Nitric oxide synthase and neuronal NADPH-diaphorase are identical in brain and peripheral tissues, Proc. Natl. Acad. Sci. 88 (1991) 7797 – 7801.
I. Pose et al. / Brain Research 1041 (2005) 29–37 [12] N.J. Dun, S.L. Dun, U. Forstermann, Nitric oxide synthase immunoreactivity in rat pontine medullary neurons, Neuroscience 59 (2) (1994) 429 – 445. [13] R.A. Fay, R. Norgren, Identification of rat brainstem multisynaptic connections to the oral motor nuclei using pseudorabies virus: I. Masticatory muscle motor systems, Brain Res. Rev. 25 (1997) 255 – 275. [14] R.A. Fay, R. Norgren, Identification of rat brainstem multisynaptic connections to the oral motor nuclei using pseudorabies virus: III. Lingual muscle motor systems, Brain Res. Rev. 25 (1997) 291 – 311. [15] P. Fort, K. Sakai, P. Luppi, D. Salvert, M. Jouvet, Monoaminergic, peptidergic, and cholinergic afferents to the cat facial nucleus as evidence by a double immunostaining method with unconjugated cholera toxin as a retrograde tracer, J. Comp. Neurol. 302 (1989) 283 – 285. [16] P. Fort, P.H. Luppi, K. Sakai, D. Salvert, M. Jouvet, Nuclei of origin of monoaminergic, peptidergic, and cholinergic afferents to the cat trigeminal motor nucleus: a double-labeling study with cholera-toxin as a retrograde tracer, J. Comp. Neurol. 301 (1990) 262 – 275. [17] P. Fort, C. Rampon, D. Gervasoni, C. Peyron, P.H. Luppi, Anatomical demonstration of a medullary enkephalinergic pathway potentially implicated in the orofacial muscle atonia of paradoxical sleep in the cat, Sleep Res., Online 1 (1998) 102 – 108. [18] S.J. Fung, J. Yamuy, S. Sampogna, F.R. Morales, M.H. Chase, Hypocretin (orexin) input to trigeminal and hypoglossal motoneurons in the cat: a double-labeling immuno-histochemical study, Brain Res. 903 (2001) 257 – 262. [19] J. Garthwaite, C.L. Boulton, Nitric oxide signaling in the central nervous system, Annu. Rev. Physiol. 57 (1995) 683 – 706. [20] K. Hiraba, M. Taira, Y. Sahara, N. Nakamura, Single-unit activity in bulbar reticular formation during food ingestion in chronic cats, J. Neurophysiol. 60 (4) (1988) 1333 – 1349. [21] J.C. Holstege, The ventro-medial medullary projections to spinal motoneurons: ultrastructure, transmitters and functional aspects, Prog. Brain Res. 107 (1996) 159 – 181. [22] B.T. Hope, G.J. Michael, K.M. Knifge, S.R. Vincent, Neuronal NADPH diaphorase is a nitric oxide synthase, Proc. Natl. Acad. Sci. 88 (1991) 2811 – 2814. [23] S. Landgren, K.A. Olsson, K.G. Wetberg, Bulbar neurons with axonal projections to the trigeminal motor nucleus in the cat, Exp. Brain Res. 65 (1986) 98 – 111. [24] Y.-Q. Li, M. Takada, N. Mizuno, Identification of premotor interneurons which project bilaterally to the trigeminalmotor, facial or hypoglossal nuclei: a fluorescent retrograde double-labeling study in the rat, Brain Res. 611 (1993) 160 – 164. [25] Y.-Q. Li, M. Takada, N. Mizuno, Premotor neurons projecting simultaneously to two orofacial motor nuclei by sending their branched axons: a study with a fluorescent retrograde double-labeling technique, Neurosci. Lett. 152 (1993) 29 – 32. [26] Y.-Q. Li, M. Takada, T. Kaneko, N. Mizuno, Premotor neurons for trigeminal motor nucleus neurons innervating the jaw-closing and jaw-opening muscles: differential distribution in the lower brainstem of the rat, J. Comp. Neurol. 356 (1995) 563 – 579. [27] J.P. Lund, K.A. Olsson, The importance of reflexes and their control during jaw movements, Trends Neurosci. 6 (1983) 458 – 463. [28] E.S. Luschei, L.J. Goldberg, Neural mechanisms of mandibular control: mastication and voluntary biting, in: Handbook of Physiology. The Nervous System, vol. II, Part 2, Am. Phys. Soc., Bethesda, MD, 1981, pp. 1237 – 1274. [29] A. Maqbool, T.F.C. Batten, P.N. McWilliam, Co-localization of neurotransmitter immunoreactivities in putative nitric oxide synthesizing neurones of the cat brain stem, J. Chem. Neuroanat. 8 (1995) 191 – 206.
37
[30] N. Mizuno, Y. Yasui, S. Nomura, K. Itoh, A. Konishi, M. Takada, M. Kudo, A light and electron microscopic study of premotor neurons for the trigeminal motor nucleus, J. Comp. Neurol. 215 (1983) 290 – 298. [31] F.R. Morales, S. Sampogna, J. Yamuy, M.H. Chase, c-fos Expression in brainstem premotor interneurons during cholinergically-induced active sleep in the cat, J. Neurosci. 19 (1999) 9508 – 9518. [32] F.R. Morales, S. Sampogna, I. Pose, Nitric oxide producing premotor trigeminal interneurons, Program No.856.1, 2002 Abstract viewer/ Itinerary planner, Society for Neuroscience, Washington, DC, 2002 (CD-ROM). [33] B. Moreno-Lo´pez, M. Escudero, J.M. Delgado-Garcı´a, C. Estrada, Nitric oxide production by brain stem neurons is required for normal performance of eye movements in alert animals, Neuron 17 (1996) 739 – 745. [34] Y. Nakamura, M. Takatori, S. Nozaki, M. Kikuchi, Monosynaptic reciprocal control of trigeminal motoneurons from the medial bulbar reticular formation, Brain Res. 89 (1975) 144 – 148. [35] Y. Nakamura, K. Hiraba, M. Taira, Y. Sahara, S. Enomoto, M. Katoh, A. Iriki, Activity during active sleep of bulbar reticular neurons firing rhythmically during mastication in cats, Exp. Neurol. 85 (1) (1984) 178 – 186. [36] S.G. Nelson, L.M. Mendell, Projection of single knee flexor Ia fibers to homonymous and heteronymous motoneurons, J. Neurophysiol. 41 (1978) 778 – 787. [37] A.S. Popratiloff, M. Streppel, A. Gruart, O. Guntinas-Lichius, D.N. Angelov, E. Stennert, J.M. Delgado-Garcia, W.F. Neiss, Hypoglossal and reticular interneurons involved in oro-facial coordination in the rat, J. Comp. Neurol. 433 (2001) 364 – 379. [38] I. Pose, S. Sampogna, M.H. Chase, F.R. Morales, Cuneiform neurons activated during cholinergically-induced active sleep in the cat, J. Neurosci. 20 (2000) 3319 – 3327. [39] C. Rampon, C. Peyron, J.M. Petit, P. Fort, D. Gervasoni, P.H. Luppi, Origin of the glycinergic innervation of the rat trigeminal motor nucleus, NeuroReport 25 (1996) 3081 – 3085. [40] J.C. Rekling, G.D. Funk, D.A. Bayliss, X.-W. Dong, J.L. Feldman, Synaptic control of motoneuronal excitability, Physiol. Rev. 82 (2) (2000) 767 – 852. [41] J. Rodrigo, D.R. Springall, O. Uttenthal, M.L. Bentura, F. AbadiaMolina, V. Riveros-Moreno, R. Martinez-Murillo, J.M. Polak, S. Moncada, Localization of nitric oxide synthase in the adult rat brain, Philos. Trans. R. Soc. Lond., Ser. B Biol. Sci. 345 (1312) (1994) 175 – 221. [42] C.B. Saper, Any way you cut it: a new journal policy for the use of unbiased counting methods, J. Comp. Neurol. 364 (1996) 5. [43] E. Southam, J. Garthwaite, The nitric oxide-cyclic GMP signaling pathway in rat brain, Neuropharmacology 11 (1993) 1267 – 1277. [44] R.C. Spike, A.J. Todd, H.M. Johnston, Coexistence of NADPH diaphorase with GABA, glycine, and acetylcholine in rat spinal cord, J. Comp. Neurol. 335 (1993) 320 – 333. [45] E. Taber, The cytoarchitecture of the brain stem of the cat: I. Brain stem nuclei of the cat, J. Comp. Neurol. 116 (1961) 27 – 69. [46] J.B. Travers, R. Norgren, Afferent projections to the oral motor nuclei in the rat, J. Comp. Neurol. 220 (1983) 280 – 298. [47] J.B. Travers, L.A. Dinardo, H. Karimnamazi, Motor and premotor mechanisms of licking, Neurosci. Biobehav. Rev. 21 (1997) 631 – 647. [48] S.R. Vincent, H. Kimura, Histochemical mapping of nitric oxide synthase in the rat brain, Neuroscience 46 (1992) 755 – 784. [49] S.R. Vincent, K. Satoh, D.M. Armstrong, P. Panula, W. Vale, H.C. Fibiger, Neuropeptides and NADPH-diaphorase activity in the ascending cholinergic reticular system of the rat, Neuroscience 17 (1986) 167 – 182. [50] C.Ch. Yang, J.Y.H. Chan, S.S.H. Chan, Excitatory innervation of caudal hypoglossal nucleus from nucleus reticularis gigantocellularis in the rat, Neuroscience 65 (1995) 365 – 374.