Somatostatin immunoreactivity in esophageal premotor neurons of the rat

Neuroscience Letters 250 (1998) 201–204

Somatostatin immunoreactivity in esophageal premotor neurons of the rat Delma L. Broussard*, Xinmin Bao, Steven M. Altschuler Division of Gastroenterology and Nutrition, The Children’s Hospital of Philadelphia, 34th and Civic Renter Boulevard, Philadelphia, PA 19104, USA Received 22 April 1998; received in revised form 3 June 1998; accepted 3 June 1998

Abstract Esophageal peristalsis is coordinated by premotor neurons localized to the central subnucleus of the nucleus of the solitary tract (NTScen). These premotor neurons project directly to motoneurons within the compact formation of the nucleus ambiguus (NAc). Somatostatin immunoreactive terminals have been previously demonstrated encircling motoneurons in the (NAc) (Cunningham, E.T., Jr. and Sawchenko, P.E., J. Neurosci., 9 (1989) 1668–1682). We combined transsynaptic tracing with pseudorabies virus and immunohistochemistry to localize somatostatin to premotor neurons within the NTScen.  1998 Published by Elsevier Science Ireland Ltd. All rights reserved

Keywords: Nucleus of the solitary tract; Somatostatin; Pseudorabies virus; Esophagus; Immunocytochemistry; Medulla oblongata

Swallowing is programmed centrally by an interneuronal network of premotor neurons (PMNs), in and around the nucleus of the solitary tract (NTS) [11,15]. Esophageal PMNs are localized exclusively to the central subnucleus of the NTS (NTScen), and have direct synaptic contact with esophageal motoneurons in the compact formation of the nucleus ambiguus (NAc) in the rat [3]. The NTScen has also been shown to be the sole site of termination of esophageal vagal afferents [2]. Therefore these PMNs are thought to be interneurons, linking esophageal sensory and motoneurons that participate in the control of esophageal motor activity. Pseudorabies virus (PRV), a swine herpes virus, has been used with increasing frequency to map central neural circuits following peripheral, or central nervous system (CNS) injection. Bartha (PRV-Ba), an attenuated strain of PRV, has been shown to be a specific transsynaptic neural tracer which is preferentially transported via motor pathways from the alimentary tract to the CNS, as a function of time [3,8].

* Corresponding author. Tel.: +1 215 5902994; fax: +1 215 5903680; e-mail: [email protected]

Demonstration of a polysynaptic circuit innervating the esophagus, utilizing PRV, provides a framework for investigating neurotransmitters that may play a role in the control of esophageal motility. Several neurotransmitters have been implicated in the central control of esophageal motility via PMNs, including somatostatin (SOM). Anterogradely labeled terminal fibers within the NAc, presumably from the NTScen, have been shown to contain SOM [9]. Additionally, neurons of the NTScen express prepro-somatostatin mRNA [10]. However, co-localization of somatostatinimmunoreactive (SOM-IR) neurons within the NTScen which project to the esophagus, have not been conclusively demonstrated. Therefore, we utilized PRV to identify esophageal PMNs in order to investigate the presence of SOM containing neurons within the NTScen. Six adult male Sprague–Dawley rats (300–450 g) were anesthetized with intramuscular ketamine (85 mg/kg) and xylazine (12 mg/kg) prior to injection of PRV-Ba, (4 m1, 5 × 108 pfu/ml, courtesy of Dr. L.W. Enquist, Department of Molecular Biology Princeton University, Princeton, NJ) into the cervical esophagus. A ventral midline incision of the neck was used to expose the cervical esophagus. The esophagus was mobilized, and gauze inserted under the

0304-3940/98/$19.00  1998 Published by Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00471- 6

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esophagus prior to PRV injection. Following each 1 ml circumferential injection with a Hamilton syringe (Hamilton), the injection site was swabbed with a cotton-tipped applicator followed by a saline rinse to prevent tracer spread [1– 3]. Two rats each were sacrificed 48 and 60 h postinjection. Two additional rats underwent unilateral cervical vagotomy prior to esophageal injections of PRV, followed by a survival time of 80 h to assess the specificity of PRV. After the designated survival period, rats were reanesthetized and perfused transcardially with a periodate–lysine–paraformaldehyde fixative. The brain and cervical spinal cord were removed, postfixed and cryoprotected as previously described [3]. One in four floating tissue sections was incubated for 36–48 h with rabbit antisera against PRV (RB 132, 1:6000, courtesy of Dr. L.W. Enquist), in normal goat serum, Triton X-100 and phosphate-buffered saline (PBS). Immunoreactive cells for PRV-IR cells were detected by an avidin-biotin complex immunoperoxidase reaction [3]. To localize SOM-IR cells within the NTScen, three adult male Sprague–Dawley rats (300–450 g) received PRV-Ba injections (4 ml) into the cervical esophagus. Eighty hours later the rats were reanesthetized and received a left lateral ventricle injection of colchicine (50 mg/100 g body weight) injected in 5–10 ml saline with a Hamilton syringe. The stereotaxic coordinates for the injection from the bregma were: AP: −0.8 mm; L: 1.5 mm; H: −3.5 mm from the dura. The pretreatment of animals with colchicine prior to immunocytochemical processing has been shown previously to increase levels of neuronal peptide immunoreactivity [9]. After 24 h, the rats were reanesthetized, transcardially perfused and tissue sections prepared as described above. One in four tissue sections was processed immunocytochemically for PRV and SOM using a double label indirect immunofluorescence technique. The primary antibodies were goat anti-PRVi (G282, dilution 1:400. courtesy of Dr. L.W. Enquist), and rabbit anti-SOM (1:200, Incstar). Free-floating tissue sections were incubated in normal donkey serum, Triton X-100 and PBS for 36–48 h. The secondary antibodies were donkey anti-goat antibody conjugated to Texas Red (1:200, Incstar) and donkey anti-rabbit conjugated to fluorescein isothiocyanate (FITC, 1:40, Incstar) to detect PRV and SOM, respectively. Tissue sections were mounted on slides, coverslipped and photographed using a Leitz DMR Research Microscope (Wetzlar, Germany). Each fluorochrome was photographed individually and the resulting color slide scanned into the computer program Adobe Photoshop (Adobe). Color slides were subsequently configured to behave as transparencies, and the double-labeled neurons revealed themselves by appearing a different color from either of the original fluorochromes. Retrograde vagal transport of PRV was observed at each survival time. Following injection of PRV into the esophagus, PRV-IR neurons were limited to first-order neurons (motoneurons) bilaterally within the NAc after the 48 h survival period (Fig. 1B). After 60 h, second-order neurons

within the NTS were specifically labeled within the NTScen (Fig. 1C). Additionally, an increased number of PRV-IR cells within the NAc was observed compared to the earlier survival time. At the 80 h survival time, there was unilateral labeling of the NAc and NTScen contralateral to the vagotomy. There was also an increase in the density of PRV-IR cells within the NTScen when compared to brain stem sections from the 60 h survival period. Within the NTS, contralateral to the vagotomy, there were putative third-order neurons (neurons projecting to PMNs) observed in areas not previously labeled at the earlier time periods. These included the interstitial and intermediate subnuclei of the NTS (NTSis and NTSint), as well as a few cells in the gelatinosus subnucleus of the NTS (NTSgel). Following the double label protocol, SOM-IR cell bodies were observed within the NTS (Fig. 2A,B). There was diffuse perikaryal staining for SOM throughout the NTScen. The average number of SOM-IR cells within the NTScen was 14/section. Aggregate cell counts from all animals revealed that out of the 136 PRV-IR cells counted within the NTScen (average 17 cells/section), 36% were doublelabeled (PRV-IR and SOM-IR positive). These cell counts, however, represent low estimates, due to the high density of PRV-IR cells that resulted in blurring of individual cell margins [1]. Within the NAc, there were small numbers of SOM-IR fibers. A number of properties of PRV-Ba make this virus use-

Fig. 1. (A) Diagrammatic representation of the location of the NAc and NTScen subnuclei in transverse sections at different rostro-caudal levels through the medulla. Dots indicate the locations of PRV-IR neurons shown in high power photomicrographs (E,C). (B) Light-field photomicrograph through the NA after an esophageal injection of PRV and a survival time of 48 h. PRV-IR cells are localized to the NAc. (C) Counterstained photomicrograph through the NTS following PRV injection into esophagus with a 60 h survival period. There is dense PRV labeling within the NTS restricted to the NTScen, in addition to the NAc (not shown). Scale bars, 100 mm. Both figures are at the same magnification. 4V, fourth ventricle; AP, area postrema.

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Fig. 2. Indirect immunofluorescent labeling of the NTScen. (A) Highpower photomicrograph of Texas Red labeled PRV-IR neurons within the NTScen, transsynaptically labeled after PRV injection into the esophagus and an 80 h survival period. (B) Numerous fluorescein labeled SOM-IR neurons within the NTScen from the same field as shown in (A). The majority of PRV-IR cells express SOM-IR. Arrows indicate representative double-labeled cells for SOM and PRV. Scale bar, 50 mm.

ful in studying swallowing-related neurons within the CNS. PRV-Ba possesses two prominent alterations in the viral genome that reduce virulence. This genetic alteration results in a longer postinfection survival period, in addition to controlled viral transport in a hierarchical manner between synaptically linked neurons [7,8]. Our results are consistent with previous groups who have demonstrated preferential retrograde transport of PRV from the gastrointestinal tract at similar survival periods [8]. Because colchicine can inhibit the replication of PRV, we chose a long survival period to ensure that PRV-IR cells were observed within esophageal PMNs. Additionally, the longer survival period helped to maximize the number of PRV-IR cells within the NTScen. In this study we have shown SOM-IR neurons within the NTScen which are transsynapatically connected to the esophagus via the NAc. In contrast to Cunningham and Sawchenko [9], our studies show significantly more SOM-IR cells within the NTScen. One possible explanation for this difference is that we utilized a larger dose of intraventricular colchicine than the other investigators. We also used a different antiserum to somatostatin. In another experiment. prepro-somatostatin mRNA was co-localized within NTScen neurons [10] projecting to the NAc. Both of these studies indirectly suggest somatostatinergic projections from the NTScen to the NAc. The localization of SOM-IR in the NTScen suggests a role for this neurotransmitter in the coordination of swallowing activity. Somatostatin is a tetradecapeptide that may regulate many different aspects of CNS function, but primarily neuroendocrine control. However, localization of SOM within the medulla [4,12] implicates its role in autonomic functions. Pharmacological studies have described cardiovascular and respiratory responses following infusions of SOM into the medulla [16,19]. However, there are no functional studies that evaluate the effect of SOM on swallowing. There are other neuroanatomical investigations that suggest

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that SOM in the NTS may play a role in relaying viscerosensory information within the CNS [13]. Specific mechanisms of action of SOM have been investigated within vagal motoneurons within the NA. Utilizing brainstem slice preparations, Wang et al. observed inhibition of acetylcholine induced depolarization of single ambigual neurons in the presence of SOM [17]. In contrast, SOM enhances glutamate-evoked depolarization of ambigual motoneurons. Subsequent studies by this group suggest that SOM excites ambigual compact formation neurons by permitting the expression of N-methyl-D-aspartate (NMDA) receptor mediated postsynaptic response [18]. The expression of the NMDA receptor within compact formation neurons that project to the esophagus has been previously reported [5,6]. In vivo stimulation of esophageal swallowing via the NMDA receptor within the NA further supports a role for NMDA receptors in the control of esophageal motoneurons [14]. The present study supports the hypothesis that SOM is expressed within esophageal PMNs. Further studies are needed to clarify the role of SOM in motor functions of the esophagus, as well as its interactions with NMDA receptors. This research was supported by NIH grants DK-44487 (S.M.A.), Heinz Nutrition Center (D.L.B.) and The Children’s Hospital of Philadelphia (D.L.B.). Special thanks to Mr. Richard Le for technical assistance with computer graphics. [1] Altschuler, S.M., Bao, X. and Miselis, R.R., Dendritic architecture of nucleus ambiguus motoneurons projecting to the upper alimentary tract in the rat, J. Comp. Neurol., 309 (1991) 402– 414. [2] Altschuler, S.M., Bao, X., Bieger, D., Hopkins, D.A. and Miselis, R.R., Viscerotopic representation of the upper alimentary tract in the rat: sensory ganglia and nuclei of the solitary and spinal trigeminal tracts, J. Comp. Neurol., 283 (1989) 248–268. [3] Barrett, R.T., Bao, X., Miselis, R.R. and Altschuler, S.M., Brain stem localization of rodent esophageal premotor neurons revealed by transneuronal passage of pseudorabies virus, Gastroenterology, 107 (1994) 728–737. [4] Breder, C.D., Yamada, Y., Yasuda, K., Seino, S., Saper, C.B. and Bell, G.I., Differential expression of somatostatin receptor subtypes in brain, J. Neurosci., 12 (1992) 3920–3934. [5] Broussard, D.L., Bao, X., Li, X. and Altschuler, S.M., Co-localization of NOS and NMDA receptor in esophageal premotor neurons of the rat, NeuroReport, 6 (1995) 2073–2076. [6] Broussard, D.L., Weidner, E., Li, X., Pritchett, D. and Altschuler, S.M., Expression of N-Methyl-D-Aspartate (NMDA) receptor mRNA in the brainstem circuit controlling esophageal peristalsis, Mol. Brain Res., 27 (1994) 329–332. [7] Card, J.P., Whealy, M.E., Robbins, A.K. and Enquist, L.W., Pseudorabies virus envelope glycoprotein gI influences both neurotropism and virulence during infection of the rat visual system, J. Virol., 66 (1992) 3032–3041. [8] Card, J.P., Rinaman, L., Schwaber, J.S., Miselis, R.R., Whealy, M.E., Robbins, A.K. and Enquist, L.W., Neurotropic properties of pseudorabies virus: uptake and transneuronal passage in the rat central nervous system, J. Neurosci., 10 (1990) 1974– 1994. [9] Cunningham, E.T. Jr. and Sawchenko, P.E., A circumscribed

204

[10]

[11]

[12]

[13]

[14]

D.L. Broussard et al. / Neuroscience Letters 250 (1998) 201–204 projection from the nucleus of the solitary tract to the nucleus ambiguus in the rat: anatomical evidence for somatostatin-28immunoreactive interneurons subserving reflex control of esophageal motility, J. Neurosci., 9 (1989) 1668–1682. Cunningham, E.T. Jr., Simmons, D.M., Swanson, L.W. and Sawchenko, P.E., Enkephalin immunoreactivity and messenger RNA in a discrete projection from the nucleus of the solitary tract to the nucleus ambiguous in the rat, J. Comp. Neurol., 307 (1991) 1–16. Doty, R.W., Richmond, W.H. and Storey, A.T., Effect of medullary lesions on coordination of deglutition, Exp. Neurol., 17 (1967) 91–106. Fodor, M., Slama, A., Guillaume, V., Videau, C., Csaba, Z., Oliver, C. and Epelbaum, J., Distribution and pharmacological characterization of somatostatin receptor binding sites in the sheep brain, J. Chem. Neuroanat., 12 (1997) 175–182. Giehl, K. and Mestres, P., Somatostatin-mRNA expression in brainstem projections into the medial preoptic nucleus, Exp. Brain Res., 103 (1995) 344–354. Kessler, J.P., Involvement of excitatory amino acids in the activ-

[15]

[16]

[17]

[18]

[19]

ity of swallowing related neurons of the ventro-lateral medulla, Brain Res., 603 (1993) 353–357. Kessler, J.P. and Jean, A., Identification of the medullary swallowing regions in the rat, Exp. Brain Res., 57 (1985) 256– 263. Koda, L.Y., Ling, N., Benoit, R., Madamba, S.G. and Bakhit, C., Blood pressure following microinjection of somatostatin related peptides into the rat nucleus tractus solitarii, Eur. J. Pharm., 113 (1985) 425–430. Wang, Y.T., Neuman, R.S. and Bieger, D., Somatostatin inhibits nicotinic cholinoceptor mediated-excitation in rat ambigual motoneurons in vitro, Neurosci Lett., 123 (1991) 236–239. Wang, Y.T., Zhang, M., Neuman, R.S. and Bieger, D., Somatostatin regulates excitatory amino acid receptor-mediated fast excitatory postsynaptic potential components in vagal motoneurons, Neurosci., 53 (1993) 7–9. Yamamoto, Y., Runold, M., Prabhakar, N., Pantaleo, T. and Lagercrantz, H., Somatostatin in the control of respiration, Acta Physiol. Scand., 134 (1988) 529.