University of Groningen

Gustatory neural processing in the 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 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 , in the and in the . 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). The effect of virus leakage from the injection area on CNS infection, was studied by dripping viral mix (2x105 pfu's) on the pancreas of 3 rats. The possibility that PrV enters the brain via the bloodstream at locations lacking a blood-brain barrier, was investigated by injecting PrV (3.3x105 pfu's) directly into the vena jugularis of 1 rat. Finally, a staining procedure was carried out without adding the first antibody, to assess nonspecific binding of the secondary antibody.

Analysis All sections were examined by light microscopy, using the brain atlas of Paxinos and Watson 34. Infected animals were included in or rejected from analysis based on the following criteria; 1) The strength of the infection. The viral labeling pattern in the DMnX should correspond closely with the DMnX labeling pattern after injection of conventional retrograde tracers into the pancreas 12,24,39 and with the localization of vagal pancreatic preganglionics determined by selective vagotomy experiments 4,5, 2) Absence of viral labeling in the nucleus ruber. In case of overinfection, lateral spread of PrV in the spinal cord may occur 18, causing significant nucleus ruber labeling, 3) Absence of viral labeling in the cerebellum. In case of overinfection, nonspecific labeling of motoneurons in the ventral horn of the spinal cord can lead to PrV labeling in the cerebellum. Infections turned out to differ between animals, ranging from very mild to severe, even when animals were compared that received the same amount of virus and had identical survival times. Cell counts were not made since they reflect infection strenght, rather than the number of cells involved in a pathway.

RESULTS

Control experiments Based on the labeling criteria, results from twelve of the sixteen rats which received a PrV injection in the 'head' of the pancreas and from nine of the fifteen rats used in control experiments, were included in the analysis. The pattern of PrV tracing results after injection of 1 µl PrV into the 'head' of the pancreas did not differ from that seen after 40 to 50 µl injections (1x104 pfu's). Pseudorabies virus injections in the 'head' or the 'tail' of the pancreas resulted in bilateral labeling within identical brainstem nuclei. However, differences were found between the amount of cells labeled in the right and the left nucleus of the and to a lesser extent in the NTS. Injections in the 'head' of the 48 Chapter 2

pancreas resulted in a higher number of labeled cells in the left DMnX and NTS and was most clear at the level where the fourth ventricle appears (ca. Bregma - 13.3). Injections in the 'tail' of the pancreas did not result in such an obvious lateralization. Additional labeling was found in the lateral columns of the DMnX in two of the four animals. Variation in the infection period showed that four days of PrV infection resulted in optimal tracing results in the brainstem. After three days the virus was visible in the spinal cord, but hardly any labeling was seen in the brainstem. Less virus and more gliosis was detected in the spinal cord after four days, probably due to the virus becoming latent. Variation in the amount of injected viral mix showed that rats receiving 1.3x102 pfu's PrV did not show any sign of viral infection. Rats that received 1.3x105 pfu's PrV showed very severe viral infection, or even died before the end of the experiment. Both 1.3x103 and 1.3x104 pfu's PrV resulted in viral infection, although 1.3x103 pfu's yielded very minor infections. Rats that received 1.3x104 pfu's showed the most usable viral infections, even though a rather wide range in infection intensity was still present. No significant viral infection occurred after dripping PrV on the pancreatic tissue. The same negative result was established after injecting PrV directly into the bloodstream. In this experiment a few virion positive cells were found in the gigantocellular reticular formation, DMnX and NTS. Virus entering the brain via the bloodstream at locations lacking a blood-brain barrier seems very unlikely, since no labeling was detected in the area postrema.

PrV distribution in the Since we are interested in the medullary input to the pancreatic preganglionics in the DMnX and more specifically, in the projections from the gustatory region of the NTS to the DMnX the labeling detected within the NTS and DMnX will be described in detail (Fig.1). PrV positive neurons within the NTS and DMnX were always localized bilaterally, with a higher amount of labeled cells in the left NTS and DMnX. This lateralization was more obvious in the DMnX in comparison with the NTS. At caudal NTS levels (Fig.1A), viral labeling was detected in cell bodies and processes in the medial part of the NTS (mNTS), mainly in the area dorsal to the DMnX. Apart from the labeling of round, average sized cell bodies, larger ovoid bipolar cells appeared immunoreactive at a lower density along the tractus and at

Autonomic brainstem projections to the pancreas 49

Figure 1. Photomicrographs showing the distribution of PrV immunoreactive neurons within the dorsal vagal complex after PrV injection into the head of the pancreas. Transneuronal labeling in (A) caudal NTS and DMnX (B -14.3), (B) intermediate NTS, DMnX and AP (B -13.7), (C) intermediate NTS and DMnX (B -13.2), (D) rostral NTS and DMnX (B -12.72). Note PrV labeling lateral to the tractus (t) of the NTS. Scale bar = 100 µm.

the dorsal borders of the mNTS. Less numerous PrV positive cells were also found in the lateral part of the NTS (lNTS). The neurons in the lNTS were more variable in shape and size and showed relatively longer processes within the lNTS, which sometimes entered the solitary tract. Apart from immunopositive fibers, few PrV positive cells were seen in the commissural NTS. The DMnX showed the largest amount of PrV infected cells, situated mainly in its medial columns. Large round cells with intensely stained cell bodies, without clear fibers were situated in the medial part of the labeled DMnX area. Smaller, round cells with short fibers were found in the entire labeled area of the DMnX. At the intermediate level (Fig.1B,C), the NTS labeling was found in the medial and, at a lower density, in the lateral subnucei. The cell bodies in the mNTS were of average size and mainly situated alongside the solitary tract, the dorsal border of the mNTS and in the subpostremal area. At the level where the fourth ventricle appears, labeling was not found in regions adjacent to the fourth ventricle and the DMnX. All rats showed immunoreactive fibers in the mNTS. Sparse PrV positive cells were present in the lNTS without clear differences in shape or size compared to the neurons in the mNTS, but with relatively long, labeled processes. The 50 Chapter 2

DMnX and the area postrema (AP), showed a high degree of viral labeling. In the DMnX the same two cell types were labeled, as was the case at caudal NTS level, mainly within the internal medial part. Both DMnX and AP showed many PrV positive fibers; in the AP, the scattered positive cell bodies were very intensely stained. The relatively high, nonspecific background staining in the AP is probably due to the high vascularity, in which intrinsic peroxidase activity could still be present causing a higher background in the DAB reaction (see: Materials and Methods). Autonomic brainstem projections to the pancreas 51

Figure 2. Distribution of cell body labeling after pseudorabies infection of the ‘head’ of the pancreas. Each dot represents 5 to 10 PrV infected neurons (Drawings were modified from Paxinos and Watson 34. Abbreviations: A5, A5 noradrenergic cell group; Ap, area postrema; AP, anterior pretectal nucleus; Arc, arcuate hypothalamic nucleus; CeA, central ; CG, central gray; DMD, dorsomedial hypothalamic nucleus, diffuse; DR, dorsal raphe nucleus; GiA, gigantocellular reticular nucleus, alpha; KF, Kölliker-Fuse nucleus; lPBN, lateral parabrachial nucleus; LH, lateral hypothalamic area; LPGi, lateral paragigantocellular nucleus; MdD, medullary reticular nucleus, dorsal; NTS, nucleus of the solitary tract; PH, posterior hypothalamic area; PnO/V, pontine reticular nucleus, oral/ventral; PVN, paraventricular hypothalamic nucleus; RCh, retrochiasmatic area; RVL, rostroventrolateral reticular nucleus; SC, subcoeruleus nuclei; SN, substantia nigra.

Going into rostral direction (Fig.1D), labeled cell bodies gradually appeared less immunoreactive, and decreased in number in both medial and lateral NTS. It was therefore difficult to attribute labeling to medial or lateral parts, since the rNTS does not appear here as a clear and outstanding nucleus. More rostral, the density of PrV infected cells decreased and finally only immunopositive fibers were seen. The extreme rostral part of the NTS was not found to be infected by PrV. Viral NTS labeling occurred up to the level of bregma -12.3. Viral DMnX 52 Chapter 2

labeling was detected up to its outmost rostral localization at B -12.72 (Fig.1D). Other CNS nuclei besides the DMnX, NTS and AP, that show transneuronal labeling after pancreatic viral injection are shown in Figure 2, 3 and summarized in Table I. In the brainstem viral labeling was detected in the lateral reticular formation, the (Fig.3B), the dorsal and ventral part of the medullary reticular field (MdRF)(Fig.3B,C),the gigantocellular nuclei, the raphe obscurus (Fig.3B) and pallidus (Fig.3A) nuclei, the rostral ventrolateral medulla and the C3 catecholaminergic cell group. In the , bilateral labeling was seen in the pontine reticular formation, the A5 noradrenergic cell group, locus coeruleus and subcoeruleus region. Occasionally, scattered cells were observed in the parabrachial nucleus (PBN, dorsal lateral, extreme lateral and Kölliker Fuse subnuclei) in more severely infected animals. In the midbrain, PrV retrograde labeling was localized in the ventrolateral and dorsal part of the periaqueductal gray. In the forebrain, viral infection was found in the paraventricular, dorsomedial and lateral and to a lesser extend in the peoptic nuclei, the arcuate nucleus and the central amygdala.

Figure 3. PrV retrograde labeling after virus injection into the head of the pancreas in (A) the raphe pallidus nucleus, (B) the dorsal and ventral part of the medullary reticular formation, nucleus ambiguus and raphe obscurus nucleus, and in (C) the dorsal medullary reticular formation. Scale bar in (A) and (C) = 80 µm and in (B) 100 µm. Autonomic brainstem projections to the pancreas 53

Table I. Central nervous system nuclei transneuronally labeled by PrV injections into the pancreas Animal no. 1 2 3 4 5 6 7 8 9 10 11 Medulla oblongata Dorsal motor nucleus of the vagus + + + + + + + + + + + Nucleus of the solitary tract + + + + + + + + + + + Area postrema + + - + + + + + + + + Medullary reticular formation, dorsal + + - + + + + + - - - ventral + + - + + + + + + + + Lateral reticular formation + + - + + - - + - - - Rostral ventrolateral medulla + + + + + + + + - - - Raphe obscurus/pallidus nucleus + + + + + + + + - + + Gigantocellular reticular formation + + + + + + + + + + + Parvocellular reticular formation + + - - + + - - - - - C3 catecholaminergic cell group + + - - + - + + + + + Pons Pontine reticular formation + + - + + + + + + + + Raphe magnus nucleus + - - + + + + + + + + Dorsal raphe nucleus + + - - + - + + + + + Locus coeruleus + + + + + + + + + + + Subcoeruleus nuclei + + - + + + - + + + + A5 catecholaminergic cell group + + + + + + - + - + - Parabrachial nucleus, lateral + - - - + + + + + + + Parabrachial nucleus, medial + - - - + ------Kölliker Fuse nucleus - - - - + + + + + - - Mesencephalon Central gray matter + + - - + + + + + + + Diencephalon Paraventricular hypothalamic nucleus + + + + + + + + + + + Dorsomedial hypothalamic nucleus + - - + - & + + - + + Lateral hypothalamic nucleus + + - + + & + + + + + Preoptic nuclei + - - + + + + + + + + Arcuate nucleus + - - + + & + + + - + Telencephalon Central amygdaloid nucleus + + - + + & + + - + - Bed nucleus of the stria terminalis + - - - + & - + - - - +, five or more cells per nucleus; -, no cells; &, not determined 54 Chapter 2

DISCUSSION

Specificity of viral labeling This paper describes the efferent projection network from the brainstem to the pancreas. To trace these pathways, the retrograde transneuronal viral tracer pseudorabies was injected into the pancreas. It has been established by now that Bartha-PrV can be used as a specific retrograde transneuronal marker of central autonomic pathways 7,8,18,38,44. Card et al. 7 demonstrated, in an ultrastructural analysis of the pathways of PrV replication, assembly and regress in the DMnX (after PrV injection in the stomach), that nonneuronal elements like glia and brain macrophages restrict spread of virus from necrotic neurons. It has been suggested by Rinaman et al. 38, that glia and brain macrophages prevent viral spread by isolation or phagocytosis of the infected neurons. Furthermore, Card et al. 7 showed that the virus replicates within synaptically linked populations of neurons and does not spread through the extracellular space or by cell-to-cell fusion. Non-specific labeling might occur due to leakage of virus after injection and before neuronal uptake. To prevent this, injections were kept as small as possible. Identical areas were found to be virally infected after injecting a 1µl or a 40 µl of volume PrV into the pancreas, showing that no virus leakage took place in this experiment. Virus was dripped on and around the pancreatic tissue to assess the amount of labeling due to leakage; no significant viral infection was found in the CNS. The same result was seen after injecting PrV directly into the bloodstream. Infections turned out to differ between animals, ranging from very mild to severe, even when animals were compared that received the same amount of virus and had identical survival times. These differences are probably due to differences in genetic background, in the level of immunity to pseudorabies virus, or in the health status of the animals.

Autonomic pathways innervating the pancreas The primary criterion for the inclusion of the individual cases in the present analysis was that the distribution of PrV infected neurons within the DMnX should correspond closely with tracing results obtained with conventional retrograde tra- cers 12,24,39. Furthermore this distribution should agree with the localization of vagal pancreatic preganglionics determined by selective vagotomy experiments 4,5. In this study, PrV positive neurons within the DMnX were always localized bilaterally, with a higher density in the left DMnX, appearing mainly in the medial columns. These results reflect the current knowledge about the localization of vagal pancreatic preganglionics within the DMnX. Vagal stimulation of insulin release Autonomic brainstem projections to the pancreas 55

and more specifically of the cephalic phase insulin response are accomplished by preganglionics contained in the gastric and hepatic branches 33,35. The medial columns of the left and right DMnX are the sources of the anterior and posterior gastric branches, respectively. The posterior gastric branch (right DMnX) projects mainly to the 'tail' of the pancreas, the anterior gastric branch (left DMnX) to the 'head' of the pancreas. The cell bodies projecting through the hepatic branch occupy a sparse and somewhat diffuse column that basically coincides with the medial column of the left DMnX. Loewy and Haxhiu 22,23 injected pseudorabies virus in the left lobe of the pancreas of C8 spinal rats. In contrast to Loewy and Haxhiu, but in agreement with classical tracing reports and selective vagotomy experiments 4,5,12,24,39, a greater concentration of viral infection in the left DMnX was found in our study. The higher PrV labeling Loewy and Haxhiu found in the right DMnX, may be due to several reasons. The injection area (the left lobe of the pancreas) might show a different parasympathetic innervation compared to the ‘head’ of the pancreas, where we injected the PrV. Second, a small number of rats (4) contributed to the results of their study. Third, different rat strains were used (Wistar vs. Sprague Dawley). Although both sympathetic and parasympathetic efferents to the pancreas were transneuronally labeled in this study, former tracing results allow us to distinguish between nuclei that provide input to sympathetic and vagal preganglionics. Viral infection was detected in the rostral ventrolateral medulla, the catecholaminergic A5 region, the raphe nuclei and the paraventricular nucleus of the hypothalamus. These nuclei have been shown to innervate both sympathetic and parasympathetic preganglionic nuclei, after injecting PrV into the celiac ganglion 45 and the left lobe of C8 spinal rats 45, respectively. The central nucleus of the amygdala, bed nucleus of the stria terminalis, lateral hypothalamic area, central gray, locus coeruleus, PBN, NTS and MdRF, were virally labeled in this study and provide input to parasympathetic preganglionics in the DMnX 3,25,26,47,48. Although the head of the pancreas contains the highest level of insulin secreting ß-cells 28, the injected virus is not taken up solely by neurons innervating these ß-cells. Other neurons, which innervate for example "-cells in the islets of Langerhans or even blood vessels in the pancreatic tissue, could also be infected by PrV. This means that the results of this tracing experiment cannot be coupled to pancreatic insulin release specifically, but to pancreatic exo- and endocrine secretion in general.

Neuroanatomical brainstem circuitry of the cephalic phase insulin response 56 Chapter 2

The reflex pathway that mediates the cephalic phase insulin response should involve the rNTS, where primary gustatory fibers synapse and the medial columns of the DMnX (mDMnX), where the vagal pancreatic preganglionics are localized. Experiments showed that PrV can travel two or three synapses 8. After injection into the pancreas PrV is taken up by the postganglionic neuron (first synapse), then it passes the intramural ganglion within the pancreas (second synapse), and from there it ends up in the DMnX. After crossing the third synapse within the DMnX we expected to find viral labeling in the rNTS. However, very little labeling was found in the rostral gustatory part of the NTS. This implies that there are almost no direct connections between the rNTS and the mDMnX. Accordingly, one or more intermediate stations should be involved. Figure 4 summarizes some putative pathways that match the current findings. Our tracing results suggest the following possible intermediate stations: a) the medullary reticular formation (MdRF); b) the area postrema (AP); c) the parabrachial nucleus (PBN); d) the intermediate or caudal NTS. Another possibility is, that the first relay for the information which induces the PIR, is not the rNTS but resides in intermediate or caudal NTS regions, which in turn have a direct connection with the DMnX. As for the MdRF is concerned, our results show clear PrV labeling in the medullary and pontine reticular formation and in the region of the nucleus ambi- guus, all of which could link the rNTS to the mDMnX (Fig.4). Autoradiographic studies in rat and hamster demonstrated anterograde labeling in medullary and pontine reticular areas after injecting tritiated amino acids into sites in the rostral NTS, where electrophysiological responses were recorded during taste stimulation of the anterior 29,49. Anterograde 1,2 and retrograde 14,17,47 studies in rat and hamster, confirmed rNTS projections to the reticular formation. Furthermore, the existence of direct projections from the MdRF to the DMnX has been established 47. With respect to the AP, our results always showed intense labeling of cell bodies and fibers in the AP. The AP is a major target for sensory fibers and it has been implicated in integration of visceral sensation, neurally as well as humorally 42. However, since the afferent input from the NTS to the AP is relatively sparse 42, and only a limited number of fibers penetrate the DMnX 10,42,50 (Fig.4), we consider it rather unlikely that the AP serves as an intermediary station between the rNTS and the mDMnX. The so-called pontine taste area 32 within the parabrachial nucleus may also be a relay between rNTS and mDMnX. Projections from the gustatory NTS to various subdivisions of the PBN have been found 2,16,37,46,49. Although there is no evidence for direct contacts with the DMnX 16,20,41, PBN neurons are known to Autonomic brainstem projections to the pancreas 57 project to the MdRF, and thus provide another option for the link between taste re ceptors and th e pancreas ( Fig. 4).

Figure 4. Diagram summarizing possible pathways in the brainstem to connect oral taste receptors to DMnX areas innervating the pancreas. Dashed lines represent sparse projections. PBN, parabrachial nucleus; MdRF, medullary reticular formation; AP, area postrema; rNTS and i/cNTS, rostral and intermediate/caudal nucleus of the solitary tract; mDMnX, medial part of the dorsal motor nucleus of the vagus; X, vagal nerve; V/VII/IX/X, trigeminal, facial, glossopharyngeal and vagal gustatory afferents.

Regarding the intra-NTS neurones, the rNTS might also project to intermediate or caudal levels of the NTS by means of interneurones (Fig. 4). Halsell et al. 14, showed that neurons within the rNTS project not only to the PBN and medullary reticular formation, but also to the cNTS. Furthermore, Beckman and Whitehead 2 frequently observed HRP labeled axons, projecting from rostral NTS to more caudal levels, in hamster. Intranuclear NTS connections would permit the integration of taste with general viscerosensory information. It is possible that the first relay for taste fibers conveying information to initiate the PIR is not situated in the rNTS, but in the intermediate or caudal regions of the NTS where we found PrV labeling. It is known that taste fibers ter- minate mainly in the rostrolateral NTS 15, but terminations in the lateral and medial parts of the commissural, intermediate and interstitial NTS, which overlap the PrV infected areas we found, are present 15. Hence, one option is that taste information 58 Chapter 2

that can induce the PIR, terminates at more caudal levels in the NTS, which in turn project directly to the DMnX (Fig. 4). In sum, the present report describes brainstem nuclei that participate in the autonomic innervation of the pancreas. Projections to the pancreas, originating from preganglionic neurons in the dorsal motor nucleus of the vagus were confirmed. Autonomic input to pancreatic vagal preganglionics was demonstrated by transneuronal labeling observed in the brainstem within the dorsal and ventral part of the medullary reticular formation, in the area postrema and in the raphe nuclei. Viral infection was detected in the medial, and to a lesser extent in the lateral nucleus of the solitary tract mainly at caudal and intermediate levels. Sparse labeling was found in the gustatory zone of the nucleus tractus solitarius, suggesting that a direct connection between the rostral nucleus tractus solitarius and the medial dorsal motor nucleus of the vagus is very unlikely. The intermediate or caudal nucleus tractus solitarius, the medullary reticular formation or the parabrachial nucleus are likely candidates to complete the brainstem circuit that mediates gustatory induced insulin release.

ACKNOWLEDGEMENTS

The authors wish to thank Dr. A.S.P. Jansen and Dr. G.J. ter Horst for helpful discussions and Prof. Dr. P.G.M. Luiten for critically reading the manuscript. J. Tiemersma, R. Poelman, R.L. Freund and J. van Wijk are acknowledged for their technical assistance. Dr. T.C. Mettenleiter and Dr. J.Pol are acknowledged for their kind donation of the Pseudorabies virus and the anti- PrV antibody, respectively.

REFERENCES

1Becker, D. and Travers, S. Projections of electrophysiologically identified loci in the orally-responsive , Soc.Neurosci.Abstr., 16 (1990) 404. 2 Beckman, M.E. and Whitehead, M.C. Intramedullary connections of the rostral nucleus of the solitary tract in the hamster, Brain Res., 557 (1991) 265-279. 3 Berk, M.L. and Finkelstein, J.A. Efferent connections of the lateral hypothalamic area of the rat: An autoradiographic investigation, Brain Res.Bull., 8 (1982) 511-526. 4 Berthoud, H.R., Fox, E.A. and Powley, T.L. Localization of vagal preganglionics that stimulate insulin and glucagon stimulation, Am.J.Physiol., 258 (1990) R160-R168. 5 Berthoud, H.R. and Powley, T.L. Identification of vagal preganglionics that mediate cephalic phase insulin response, Am.J.Physiol., 258 (1990) R523-R530. 6 Buwalda, B., Strubbe, J.H., Hoes, M.W.N. and Bohus, B. Reduced preabsorptive insulin response in aged rats: Differential effects of amphetamine and arginine-vasopressin, J.Auton.Nerv.Syst., 36 (1991) 123-128. 7 Card, J.P., Rinaman, L., Lynn, R.B., Lee, B.H., Meade, R.P., Miselis, R.R. and Enquist, L.W. Pseudorabies virus infection of the rat central nervous system: Ultrastructural Autonomic brainstem projections to the pancreas 59

characterization of viral replication, transport, and pathogenesis, J.Neurosci., 13(6) (1993) 2515-2539. 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(6) (1990) 1974-1994. 9 Contreras, R.J., Beckstead, R.M. and Norgren, R. The central projections of the trigeminal, facial, glossopharyngeal and vagus nerves: An autoradiographic study, J.Auton.Nerv.Syst., 6 (1982) 303-322. 10 Cunningham, E.T., Miselis, R.R. and Sawchenko, P.E. The relationship of efferent projections from the area postrema to vagal motor and brain stem catecholamine- containing cell groups - An axonal transport and immunohistochemical study in the rat, Neurosci., 58 (1994) 635-648. 11 Flynn, F.W., Berridge, K.C. and Grill, H.J. Pre- and postabsorptive insulin secretion in chronic decerebrate rats, Am.J.Physiol., 250 (1986) R539-R548. 12 Fox, E.A. and Powley, T.L. Longitudinal organization within the dorsal motor nucleus represents separate branches of the abdominal vagus, Brain Res., 341 (1985) 269-282. 13 Grill, H.J., Berridge, K.C. and Ganster, D.J. Oral glucose is the prime elicitor of preabsorptive insulin secretion, Am.J.Physiol., 246 (1984) R88-R95. 14 Halsell, C.B., Travers, S.P. and Travers, J.B. Ascending and descending projections from the rostral nucleus of the solitary tract originate from separate neuronal populations, Neurosci., 72 (1996) 185-197. 15 Hamilton, R. and Norgren, R. Central projections of gustatory nerves in the rat, J.Comp.Neurol., 222 (1984) 560-577. 16 Herbert, H., Moga, M.M. and Saper, C.B. Connections of the parabrachial nucleus with the nucleus of the solitary tract and the medullary reticular formation in the rat, J.Comp.Neurol., 293 (1990) 540-580. 17 Hermann, G.E., Kohlerman, N.J. and Rogers, R.C. Hepatic-vagal and gustatory afferent interactions in the brainstem of the rat, J.Auton.Nerv.Syst., 9 (1983) 477-495. 18 Jansen, A.S.P., Farwell, D.G. and Loewy, A.D. Specificity of pseudorabies virus as a retrograde marker of sympathetic preganglionic neurons: Implications for transneuronal labeling studies, Brain Res., 617 (1993) 103-112. 19 Kaneto, A., Kosaka, K. and Nakao, K. Effects of stimulation of the vagus nerve on insulin secretion, Endocrinol., 80 (1967) 530-536. 20 Krukoff, T.L., Harris, K.H. and Jhamandas, J.H. Efferent projections from the parabrachial nucleus demonstrated with the anterograde tracer phaseolus vulgaris leucoagglutinin, Brain Res.Bull., 30 (1993) 163-172. 21 Loewy, A.D. and Burton, H. Nuclei of the solitary tract: Efferent projections to the lower brain stem and spinal cord of the cat, J.Comp.Neurol., 181 (1978) 421-450. 22 Loewy, A.D., Franklin, M.F. and Haxhiu, M.A. CNS monoamine cell groups projecting to pancreatic vagal motor neurons - A transneuronal labeling study using pseudorabies virus, Brain Res., 638 (1994) 248-260. 23 Loewy, A.D. and Haxhiu, M.A. CNS cell groups projecting to pancreas parasympathetic preganglionic neurons, Brain Res., 620 (1993) 323-330. 24 Luiten, P.G.M., Ter Horst, G.J., Koopmans, S.J., Rietberg, M. and Steffens, A.B. Preganglionic innervation of the pancreas islet cells in the rat, J.Auton.Nerv.Syst., 10 (1984) 27-42. 25 Luiten, P.G.M., Ter Horst, G.J., Karst, H. and Steffens, A.B. The course of paraventricular hypothalamic efferents to autonomic structures in medulla and spinal cord, Brain Res., 329 (1985) 374-378. 26 Luiten, P.G.M., Ter Horst, G.J. and Steffens, A.B. The hypothalamus, intrinsic connections and outflow pathways to the endocrine system in relation to the control of feeding and 60 Chapter 2

metabolism, Prog.Neurobiol., 28 (1987) 1-54. 27 Mettenleiter, T.C. Molecular biology of pseudorabies (aujeszky's disease) virus, Comp.Immun.Microbiol.Infect.Dis., 14(2) (1991) 151-163. 28 Miller, R.E. Pancreatic neuroendocrinology: Peripheral neural mechanisms in the regulation of the islets of Langerhans, Endocrine Rev., 2(4) (1981) 471-494. 29 Norgren, R. Projections from the nucleus of the solitary tract in the rat, Neurosci., 3 (1978) 207-218. 30 Norgren, R. The central organization of the gustatory and visceral afferent systems in the nucleus of the solitary tract. In: Katsuki, Y., Norgren, R. and Sato, M. (Eds.), Brain mechanisms of sensation, 1981, pp. 143-160. 31 Norgren, R. Central neural mechanisms of taste. In: I.Darien Smith (Ed.), Handbook of physiology - The nervous system III, Section II: Sensory processes, Amer.Physiol.Soc. Bethesda, 1984, pp. 1087-1128. 32 Norgren, R. and Leonard, C.M. Ascending central gustatory pathways, J.Comp.Neurol., 150 (1972) 217-238. 33 Norgren, R. and Smith, G.P. Central distribution of subdiaphragmatic vagal branches in the rat, J.Comp.Neurol., 273 (1988) 207-223. 34 Paxinos, G. and Watson, C. The rat brain in stereotaxic coordinates, Academic Press, New York, 1986. 35 Powley, T.L..and Berthoud, H.-R. Neuroanatomical bases of cephalic phase reflexes. In: Friedman, M.I., Tordoff, M.G. and Kare, M.R. (Eds.), Chemical Senses: Appetite and Nutrition, M. Dekker Inc. New York, 1991, pp. 391-404. 36 Powley, T.L. and Berthoud, H.R. Neuroanatomical bases of cephalic phase reflexes, Appetite., 12 (1989) 78. 37 Ricardo, J.A. and Koh, E.T. Anatomical evidence of direct projections from the nucleus of the solitary tract to the hypothalamus, amygdala, and other forebrain stuctures in the rat, Brain Res., 153 (1978) 1-26. 38 Rinaman, L., Card, J.P. and Enquist, L.W. Spatiotemporal responses of astrocytes, ramified microglia, and brain macrophages to central neuronal infection with pseudorabies virus, J.Neurosci., 13(2) (1993) 685-702. 39 Rinaman, L. and Miselis, R.R. The organization of vagal innervation of rat pancreas using cholera toxin-horse radish peroxidase conjugate, J.Auton.Nerv.Syst., 21 (1987) 109-125. 40 Roozendaal, B., Oldenburger, W.P., Strubbe, J.H., Koolhaas, J.M. and Bohus, B. The centralamygdala is involved in conditioned but not in the meal-induced cephalic insulin response in the rat, Neurosci.Lett., 116 (1990) 210-215. 41 Saper, C.B. and Loewy, A.D. Efferent connections of the parabrachial nucleus in the rat, Brain Res., 197 (1980) 291-317. 42 Shapiro, R.E. and Miselis, R.R. The central connections of the area postrema of the rat, J.Comp.Neurol., 234 (1985) 344-364. 43 Steffens, A.B. Influence of the oral cavity on insulin release in the rat, Am.J.Physiol., 230 (1976) 1411-1415. 44 Strack, A.M. and Loewy, A.D. Pseudorabies virus: A highly specific transneuronal cell body marker in the sympathetic nervous system, J.Neurosci., 10(7) (1990) 2139-2147. 45 Strack, A.M., Sawyer, W.B., Hughes, J.H., Platt, K.B. and Loewy, A.D. A general pattern of CNS innervation of the sympathetic outflow demonstrated by transneuronal pseudorabies viral infections, Brain Res., 491 (1989) 156-162. 46 Ter Horst, G.J., De Boer, P., Luiten, P.G.M. and Van Willigen, J.D. Ascending projections from the solitary tract nucleus to the hypothalamus. A Phaseolus Vulgaris Lectin tracing study in the rat, Neurosci., 31 (1989) 785-797. 47 Ter Horst, G.J., Luiten, P.G.M. and Kuipers, F. Descending pathways from hypothalamus to dorsal motor vagus and ambiguus nuclei in the rat, J.Auton.Nerv.Syst., 11 (1984) Autonomic brainstem projections to the pancreas 61

59-75. 48 Ter Horst, G.J., Toes, G.J. and Van Willigen, J.D. Locus coeruleus projections to the dorsal motor vagus nucleus in the rat, Neurosci., 45(1) (1991) 153-160. 49 Travers, J.B. Efferent projections from the anterior nucleus of the solitary tract of the hamster, Brain Res., 457 (1988) 1-11. 50 Van Der Kooy, D. and Koda, L.Y. Organization of the projection of a circumventricular organ:The area postrema in the rat, J.Comp.Neurol., 219 (1983) 328-338.