University of Groningen Gustatory neural processing in the brainstem of the rat Streefland, Cerien IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 1998 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Streefland, C. (1998). Gustatory neural processing in the brainstem of the rat. s.n. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne- amendment. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 04-10-2021 CHAPTER 2 AUTONOMIC BRAINSTEM PROJECTIONS TO THE PANCREAS: A RETROGRADE TRANSNEURONAL VIRAL TRACING STUDY IN THE RAT Cerien Streefland, Frans W. Maes and Béla Bohus Groningen Graduate School for Behavioral and Cognitive Neurosciences (BCN), Department of Animal Physiology, University of Groningen, The Netherlands J.Auton.Nerv.Syst. (1998) (in press) 44 Chapter 2 ABSTRACT The present study describes brainstem nuclei that participate in the autonomic innervation of the pancreas, using a retrograde viral transneuronal tracing technique. It aimed at identifying the functional architecture of the parasympathetic, gustatory-induced insulin release by the endocrine pancreas (preabsorptive insulin response, PIR). Autonomic pathways organized for reflex adjustments of the end organ, as it happens in the PIR, involve relatively simple circuits. This implies a short brainstem circuit from the rostral gustatory nucleus of the solitary tract to the dorsal motor nucleus of the vagus. The present findings confirm projections to the pancreas, originating from preganglionic neurons in the dorsal motor nucleus of the vagus. Transneuronal labeling was detected in the medial, and to a lesser extent in the lateral nucleus of the solitary tract mainly at caudal and intermediate levels. Furthermore, infected neurons were seen in the brainstem in the dorsal and ventral part of the medullary reticular formation, in the area postrema and in the raphe nuclei. Sparse labeling was found in the gustatory zone of the nucleus tractus solitarius. These results indicate that a direct connection between the rostral nucleus tractus solitarius and the medial dorsal motor nucleus of the vagus is very unlikely, so that one or more intermediate stations may be involved. Candidates to complete this pathway are the intermediate or caudal nucleus tractus solitarius, the medullary reticular formation or the parabrachial nucleus. Autonomic brainstem projections to the pancreas 45 INTRODUCTION Autonomic pathways that control the endocrine pancreas are organized for both reflex adjustments of the pancreas and integrative functions involving more complex changes that affect multiple systems. Reflex pathways involve relatively simple brainstem circuits, while integration of ascending visceral information occurs in a variety of brainstem and forebrain nuclei, that command specific autonomic and neuroendocrine responses. The cephalic phase of insulin response PIR) is mediated through such a reflex pathway. In 1967 it was shown that electrical stimulation of vagal nerves projecting to the pancreas results in release of insulin 19. Since the efferent parasympathetic influences originate from the dorsal motor nucleus of the vagus (DMnX) in the brainstem 4,5,12,24,39, it is of interest to investigate the pathways afferent to this motor nucleus. The gustatory oropharyngeal influences that play a role in the causation of insulin release, and more specifically the functional architecture of this cephalic phase is of our long term interest 6,40. After oral intake of glucose by the rat, serum insulin levels rise within one minute, before the blood glucose level starts to increase 13,43. This preabsorptive insulin response is absent after vagotomy 5,35,36, but persists in decerebrated animals 11. The local circuitry of the brainstem therefore, seems to be sufficient to elicit the PIR. This suggests a short, direct pathway within the brainstem, from the gustatory input to the endocrine pancreas. Primary afferents from gustatory receptors terminate in the rostral part of the nucleus tractus solitarius (rNTS) 9,15,30,31. The aim of the present study is to trace the medullary input to the pancreatic preganglionics in the DMnX and more specifically, to elucidate connections from the gustatory region of the NTS to the DMnX. Although descending projections from the NTS have been reported 14,21,46, no detailed information is available concerning specific descending gustatory projection pathways. To demonstrate these projections, we chose to employ the rather novel retrograde transneuronal viral tracing technique 7,8,18,38,44. The major advantage of viral tracing is the visualization of functional chains of neurons. After virus injection into the pancreatic tissue, uptake by first-order (ganglionic) neurons innervating the pancreas takes place. Here, the viruses are replicated, which leads to amplifi- cation of the tracer signal. The viruses are then released by exocytosis into the extracellular space where they may specifically infect second-order (pregangli- onic) neurons 7. This process may then be repeated, infecting neurons presynaptic to the latter. 46 Chapter 2 MATERIALS AND METHODS All procedures used in this study were approved by the Committee on Animal Bioethics of the University of Groningen. Virus The virus used is the Bartha K strain of the Pseudorabies Virus (PrV) (Suid herpesvirus 1), an attenuated strain used to vaccinate pigs against Aujeszky's disease. The genome of Bartha PrV contains several mutations in genes encoding glycoproteins 27, decreasing the virulence of the virus. Plaqueing of the virus was carried out in green monkey kidney cells. Infection procedure Thirty-two male Wistar rats (190 - 210 g) were anesthetized with sodium pentobarbital (i.p., 30 mg/kg body wt.), hypnorm (i.m., 0.5 ml/kg, Janssen Pharmaceutica, NL) and atropine sulphate (i.p., 0.125 mg/kg). PrV was slowly injected (± 5 min) into the pancreatic tissue with a 30 G needle connected by a polythene tube (i.d. 0.26 mm) to a 1 ml syringe. The injections were placed in the 'head' of the pancreas, which has the highest content of insulin-secreting ß-cells 28. The peritoneum was closed and the rats were checked daily. Tissue preparation After 3 or 4 days, the rats were deeply anesthetized with sodium pentobarbital (i.p., 270 mg/kg) and perfused transcardially with 400 ml of fixative (4% paraformaldehyde in 0.1M phosphate buffer (PB), pH 7.4), preceded by a short prerinse with a 0.9 % heparinized saline solution. Brain and spinal cord were removed and postfixed for 2 hrs. The tissue was cryoprotected by overnight storage at 4 EC in 30 % sucrose in 0.1M PB. Transverse or longitudinal sections of brain and spinal cord were cut at 20-50 µm thickness on a cryostat microtome. The sections were collected in 0.1 M PB. Visualization of PrV Pseudorabies virus was visualized using an immunocytochemical peroxidase anti-peroxi- dase staining technique (PAP). Prior to the first antibody incubation, sections were rinsed in phosphate-buffered saline (PBS) and immersed for 10 minutes in 0.01 % H2O2 to exhaust endogenous peroxidase activity. The sections were rinsed again and incubated for 1 h at room temperature (RT) in 5% normal goat serum (NGS) to suppress non- specific antibody binding. Thereafter the sections were incubated overnight at RT in a primary antibody solution (1:2500, 0.1% triton X-100, 1% NGS). The rabbit-anti-PrV polyclonal antibody was kindly donated by Dr. J. Pol (Central Veterinary Institute, Lelystad, NL). After incubation the sections were thoroughly rinsed in PBS and again treated with 1% NGS for 1 h, followed by the secondary antibody incubation (goat-anti- rabbit IgG, 1:50; Zymed, USA) for 2 h at RT. The sections were rinsed again in PBS before the third incubation step (2 h at RT) in rabbit-PAP (1:500; Dakopatts, DK). Finally the sections were processed by the diaminobenzidine (DAB)-H2O2 reaction (30 mg DAB, 0.3% nickel ammoniumsulphate and 0.01 % H2O2/100 ml 0.1M Tris/HCl) After the immunocytochemical staining the sections were mounted, air dried, counterstained (with cresyl violet or neutral red/saffranine red), dehydrated, cleared in xylene and coverslipped with DPX mountant (BDH-Pool, UK). Autonomic brainstem projections to the pancreas 47 Experimental setup and control experiments Eleven rats received viral injections of 40 to 50 µl (1-1.5x104 plaque forming units (pfu's)) at 4 or 5 different locations in the ‘head’ of the pancreas. To check whether the injected volume of PrV spreads through the entire pancreas after the injection of PrV in the pancreatic ‘head’, 5 rats received a 1 µl injection at one location in the 'head' of the pancreas (with an identical amount of pfu's). Furthermore, 4 rats were given a 1 µl PrV injection in the ‘tail’ of the pancreas. In order to compare the innervation of these two pancreatic areas. To optimalize the infection (for criteria see: Analysis) 8 rats were used to vary the duration of the infection period (3 or 4 days) and the amount of pfu's injected (1.3x102 to 1.3x105 pfu's).