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The neuropeptide Y (Y4) receptor is highly expressed in neurones of the rat dorsal vagal complex
Molecular Brain Research 48 Ž1997. 1–6
Research report
The neuropeptide Y ž Y4/ receptor is highly expressed in neurones of the rat dorsal vagal complex P.J. Larsen a
a,)
, P. Kristensen
b
Department of Medical Anatomy, UniÕersity of Copenhagen, NoÕo Nordisk A r S, BagsÕærd, Denmark b Department of Histology, Health Care DiscoÕery, NoÕo Nordisk A r S, BagsÕærd, Denmark Accepted 14 January 1997
Abstract Recently, the cDNA encoding the Y4 neuropeptide Y ŽNPY. receptor cDNA was cloned from a rat genomic library. The Y4 receptor is characterized by having a high affinity for pancreatic polypeptide ŽPP. and peptide YY ŽPYY.. By using in situ hybridization histochemistry with 35S-labelled riboprobes, we have visualized the cellular expression of mRNA encoding the Y4 receptor protein in the rat dorsal vagal complex at the light microscopical level. High densities of silver grains were observed over neurones of the dorsal vagal motor nucleus, and over neurones of a subregion of the nucleus of the solitary tract known as the subnucleus gelatinosus. Furthermore, cells within the ventral margin of the area postrema expressed high levels of Y4 mRNA. These observations indicate that circulating PP andror NPYrPYY via the blood–brain barrier-free area postrema and subpostremal area could influence neurones of the dorsal vagal complex with profound influence on numerous homeostatic mechanisms governed by this nuclear complex. Keywords: Nucleus of the solitary tract; Area postrema; Pancreatic polypeptide; Gut–brain axis; Drinking; Feeding
1. Introduction Pancreatic polypeptide ŽPP. was the first of several members to be discovered within a family of peptides recognized by their common hairpin PP-fold structure w21x. Other peptides of this 36-amino-acid peptide family are the evolutionary older members neuropeptide Y ŽNPY. and peptide YY ŽPYY. w24x. PP is exclusively synthesized and released post-prandially from the endocrine pancreas, thereby exerting its influence upon pancreatic secretion, gut motility and gall bladder contraction w16,38x. NPY is preferentially synthesized in neurones while PYY is synthesized in endocrine cells of the pancreas as well as the distal ileum and colon w6,7,28,43x. PP is released from endocrine cells of the pancreas in response to a meal via a mechanism involving vagal cholinergic neurones w38,41x whereas the post-prandial release of PYY is primarily caused by local circuits triggered by local stimulation of the intestinal mucosa w42x. The peripheral effects of PP and PYY upon gastrointestinal function are similar. In vivo, ) Corresponding author. Department of Anatomy, Section B, Panum Institute, Blegdamsvej 3, 2200 Copenhagen N, Denmark. Fax: q45 Ž3. 536-9612; E-mail: [email protected]
both peptides inhibit secretion from the exocrine pancreas, but the absence of PP receptors on rat acinar cells and the lack of inhibitory effect of PP upon CCK-stimulated secretion from isolated pancreatic acini suggests that PP exerts its effect indirectly w18,27,34x. Peripheral administration of PP and PYY inhibit centrally induced pancreatic secretion by 2-deoxy-D-glucose and this effect is greatly diminished by transection of the vagal nerves w34x, suggesting that PP and PYY interact with a site proximal to the vagal efferents. By using an in vivo radioreceptor assay, peripherally accessible binding sites for PP have been demonstrated in the dorsal vagal complex w46x, and the presence of PP-binding sites in the blood–brain barrier-free area postrema has inspired the hypothesis that gut peptides PP and PYY interacts with neurones of the dorsal vagal complex, thereby establishing a gut–brain axis w47x. This is further supported by vivo experiments demonstrating that direct application of PP to the dorsal vagal complex stimulates gastric acid secretion and motility in the rat w30x. The PP-fold peptides interact with a family of seventransmembrane NPY receptors coupled to G-binding proteins. Pharmacologically, the NPY receptors have been defined by their ability to bind NPY, PYY, PP and syn-
0169-328Xr97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 7 . 0 0 0 6 9 - 7
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P.J. Larsen, P. Kristensenr Molecular Brain Research 48 (1997) 1–6
thetic derivatives of these peptides w11,44x. The pharmacological binding profiles and rank order of binding potencies are as follows: Y1 binds NPY, PYY and wLeu31 ,Pro 34 xNPY ) PP and COOH-terminal fragments of NPY; Y2 binds NPY, PYY and COOH-terminal fragments of NPY ) PP; Y3 binds NPY ) PYY; Y4 Žthe PP receptor. binds PP ) NPY,PYY; and the Y5 Žputative feeding receptor. binds NPY, PYY, wLeu 31 ,Pro 34 xNPY and NPY 2 – 36 ) humanPP ) COOH-terminal fragments of NPY w12x while a novel receptor Žunfortunately also designated Y5. binds NPY, PYY, wLeu31 ,Pro 34 xNPY and NPY 2 – 36 ) NPY 13 – 36 ) human PP w45x. This pharmacological classification has largely been confirmed after the recent cloning of a total of five different receptor clones each coding for either of the receptor proteins w2,10,12,13,17,25,29,35,45,48 x. A PP-preferring receptor protein designated the Y4 receptor has been cloned from both rat and human genomic DNA w2,29,48x. Until now, the in vivo expression of the mRNA encoding the Y4 receptor has only been demonstrated by Northern blot analysis, and discrepancies about the level of Y4 mRNA expression in the central nervous system ŽCNS. exist. Thus, very low levels of Y4 hybridization were detected in non-amplified human mRNA samples from various brain regions w29x while much more robust signals were obtained from various regions of the brain when a RT-PCR amplification of human Y4 cDNA was employed w2x. However, future studies await to demonstrate the detailed topographical distribution of cellular expression of the Y4 mRNA in the CNS and, subsequently, compare this distribution with that of PP-binding sites. The present study was undertaken to investigate the expression of Y4 mRNA in the rat dorsal vagal complex which has been demonstrated to be one of very few central sites possessing specific PP-binding sites w46x.
Plasmid DNA was prepared and 35 S-labelled antisense and sense RNA was transcribed using T3 or T7 polymerases as described previously w23x. Briefly, following transcription the probe preparations were precipitated repeatedly with ethanol after addition of ammonium acetate to a final concentration of 3.0 M and hydrolysed to a mean size of 100 bp. The amount of TCA-precipitable material in the final probe preparation was usually ) 90%. The two corresponding RNA probes transcribed from the opposite strands of the same plasmid template were adjusted to the same radioactivity concentration and all experiments included sections hybridized using the sense RNA probes. The final hybridization solution contained RNA probe Ž100 000 cpmrm l., deionized formamide Ž50%., dextran sulphate Ž10%., tRNA Ž1 mgrml., Ficoll 400 Ž0.02% vrw., polyvinylpyrrolidone Ž0.02% vrw., BSA fraction V Ž0.2% vrw., 10 mM DTT, 0.3 M NaCl, 0.5 mM EDTA, 10 mM Tris–Cl and 10 mM NaPO4 ŽpH 6.8.. Hybridization was performed overnight at 478C as described w23x, except that the proteinase K step was omitted and the post-hybridization washing of sections in 50% formamide was performed at 57 and 628C. After exposure to b-radiation-sensitive films ŽHyperfilm, Amersham., sections were dipped in Amersham LM-1 nuclear emulsion and exposed for 9 days before being developed in Kodak D19. Emulsion-dipped sections were Nissl-counterstained and topographic analysis of the microscopic localization of Y4 mRNA-expressing cells were facilitated by using a microscope fitted with a camera lucida device. Background labelling was evaluated over areas dominated by white matter and cells were considered
2. Materials and methods Five male Wistar rats weighing f 200 g housed under standard laboratory conditions with free access to food and water were used for this experiment. Animals were decapitated early in the light phase ŽL : D, 12 : 12 h. and the brains were rapidly frozen on dry ice and kept at y708C until use before 12-m m cryostat sections of the lower brainstem were cut. Rat Y4 receptor cDNAs were cloned by the polymerase chain reaction based on the published sequence ŽGen Bank Accession No. u35232.. Polymerase chain reactions were carried out for 30 cycles of 1 min at 948C, 1 min at 558C and 2 min at 728C, using rat genomic DNA as template and sequence-specific oligonucleotides as primers. The following cDNA fragments were isolated: Y4-2 Žbp 28– 462. and Y4-3 Žbp 442–1155. and their sequence confirmed by DNA sequencing. RNA probes were prepared from cDNA cloned into Bluescript vectors ŽStratagene..
Fig. 1. Dark-field photomicrograph demonstrating Y4 mRNA-hybridized cells in the caudal part of the dorsal vagal complex. A high density of positively hybridized cells are concentrated in the ventral marginal zone of the AP while the central and superficial parts of this circumventricular organ contain fewer Y4-expressing cells. In the dorsolateral part of the dorsal vagal complex, the subnucleus gelatinosus Žgel. contains by far the majority of Y4-expressing cells while, in the ventral portion of the dorsal vagal complex, the dorsal vagal motor nucleus ŽdmnX. contains the majority of the Y4-expressing neurones. The adjacent commissural nucleus Žncom. is almost devoid of positively labelled cells. TS, solitary tract; asterisk in central canal. Scale bar s 200 m m.
P.J. Larsen, P. Kristensenr Molecular Brain Research 48 (1997) 1–6
Fig. 2. Camera lucida drawing of a counterstained section of the rat dorsal vagal complex taken from a rostrocaudal level similar to that shown in Fig. 1. Cells positively hybridized for Y4 mRNA are depicted as black dots. AP, area postrema; CC, central canal; dmnX, dorsal vagal motor nucleus; dPSR, dorsal parasolitarius region; gel, subnucleus gelatinosus; Gr, nucleus gracilis; mnTS, medial nucleus of the nTS; ncom, commissural nucleus of the nTS; nI, intermediate nucleus of the nTS; TS, solitary tract; XII, hypoglossal nucleus.
positively labelled when the density of overlying photographic silver grains was ) 5 = the background labelling. At representative rostrocaudal levels of specific subnuclei, the number of positively hybridized cells were counted and compared to the number of Nissl-stained neurones. The approximate percentages of positively hybridized neurones were subsequently estimated. The nomenclature used is largely adopted from w20,31x.
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nal. Furthermore, rat brain sections hybridized with ribonucleotide probes generated from the Y4 sense strand were all devoid of a positive hybridization signal. In the lower brainstem, in situ hybridization histochemistry revealed numerous Y4 mRNA-expressing cells in the dorsal vagal complex. Except for a few positively labelled cells in the lateral reticular nucleus of the ventrolateral medulla, all other areas of the medulla oblongata were devoid of Y4 mRNA-expressing cells. In the dorsal vagal complex, high numbers of Y4 mRNA-expressing cells were present in the area postrema ŽAP., in the dorsal motor nucleus of the vagus ŽdmnX. and in the subnucleus gelatinosus of the nucleus of the solitary tract ŽnTS. ŽFigs. 1–3.. Few positively labelled cells were scattered throughout the commissural and the intermediate nuclei of the nTS. Within the AP, positively labelled cells were concentrated in the deep ventral margin of this circumventricular organ which is adjacent to the subpostremal area ŽFig. 1.. In this deep zone of the AP, ) 80% of the cells were considered positively labelled. Positively labelled cells were present throughout the rostrocaudal extent of the dmnX and the subnucleus gelatinosus of the nTS. f 40% of the rather large neurones of the dmnX were positively labelled while 70% of the neurones in the subnucleus gelatoinosus were positively hybridized with the Y4 mRNA-recognizing probe.
4. Discussion 3. Results The specificity of the in situ hybridization histochemical reaction was verified by the fact that two different probes recognizing different only partially overlapping parts of the Y4 cDNA sequence gave identical hybridization signals. All five animals examined displayed similar distribution pattern and intensity of the hybridization sig-
The rank order of binding of PP-fold proteins to the Y4 receptor is PP ) NPY, PYY, rendering PP the endogenous high-affinity ligand of this receptor w11,44x. The present observation corresponds largely to the receptor autoradiographic pattern of radioiodinated PP-binding w46x, suggestive of a somaticrperikaryal localization of the Y4 receptor protein. Compared to receptor autoradiography, the
Fig. 3. Medium-power photomicrograph showing cells positively hybridized for Y4 mRNA in the dorsal vagal motor nucleus ŽA. and the AP ŽB.. Scale bars s 50 m m
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P.J. Larsen, P. Kristensenr Molecular Brain Research 48 (1997) 1–6
currently used in situ hybridization methodology yields a far better cellular resolution of the exact distribution of Y4 mRNA expression though it is important to keep in mind that the sites of receptor protein synthesis and membrane localization are not necessarily coincident. Thus, more distant localization of the Y4 receptor protein at dendrites and axons emanating from the currently labelled cells should be considered as well. PP is a peripheral hormone not synthesized in the brain and consequently PP-mediated effects upon the CNS have to be communicated either via blood–brain barrier-free areas or via a specific uptake mechanism. PP is released from endocrine cells of the pancreas in response to a meal, sham-feeding or hypoglycaemia and these effects rely entirely upon vagal, cholinergic mechanisms w38x. Subsequently, circulating PP inhibits gastric motility, gall bladder contraction as well as exocrine pancreatic secretion w16x, suggestive of a role as a feed forward inhibitor upon digestive processes. In addition, PP and PYY have a potent emetic effect which is abolished in post-rectomized animals, suggesting that PP-fold peptides may act as endogenous emetic agents w14x. By using an in vivo radioreceptor assay, peripherally accessible binding sites for PP has been demonstrated in the rat dorsal vagal complex but it has not been possible to ascertain to what degree PP is bound by the nTS and dmnX, both areas which are considered protected by the blood–brain barrier w46x. A number of possible mechanisms via which PP exerts its action upon the dorsal vagal complex is possible. First, PP could interact with somatic Y4 receptors upon AP neurones projecting to the adjacent nuclei of the dorsal vagal complex. The AP projects heavily to the dorsal nuclei of the dorsal vagal complex while ventrally situated nuclei, including the dmnX, are sparsely innervated w8x. Second, it is possible that Y4 receptors are present on dendrites emanating from deeper nuclei Že.g. the subnucleus gelatinosus. which via protrusion into the AP proper are situated outside the blood–brain barrier. Ultrastructural and Golgi studies confirm the presence of such antenna-like dendrites monitoring the systemic circuit w8,19,32x. Third, Y4 receptor protein synthesized within motor neurones of the dmnX could be transported to terminals located in peripheral autonomic parasympathetic ganglia, thus, acting as pre-synaptic receptors. Finally, analogous to the brain insulin uptake system w33x the presence of a specific transport mechanism conveying PP over the blood–brain barrier should be considered. The AP contained a high number of Y4 mRNA-expressing cells. The AP is a circumventricular organ which is highly vascularized by an extensive capillary plexus w9x. The absence of a blood–brain barrier in the AP allows neuronal elements in the organ and the immediate adjacent subpostremal area of the dorsal vagal complex to be directly exposed to blood-borne substances as well as the cerebrospinal fluid of the fourth ventricle w5,22x. The anatomical features of the AP makes the organ ideally
suited for chemoreception and perhaps neurosecretion w5x. In addition, the AP is also target for vagal and hypothalamic afferent fibres w20,26,39x, implying that it may participate in visceroception by means of both humorally and neurally mediated inputs. The efferent projections from the neurones of the AP are preferentially directed towards the ventrally lying dorsal vagal complex but fibres also target the A1 and C1 catecholaminergic neurones of the ventrolateral medulla as well as rostrally situated nuclei of the brainstem w3,8,36,39x. The functional role of the AP as a central player in the physiological mechanisms underlying emesis and vomiting has been continuously confirmed since the 1950s w5x whereas other roles of this organ are somewhat uncertain. The current study gives evidence that neurones of the AP are directly sensitive to circulating PP which could mediate its emetic and stimulatory actions on gastric motility and pancreatic secretion via Y4 receptors in the AP. The topographically distinct distribution of Y4 expression in the nTS is interesting because innervation of the nTS by vagal afferents from the alimentary canal is viscerotopically organized w31x. Thus, oesophageal afferents preferentially terminate within the ventral portion of the medial nTS which apparently is the only the visceral organ represented by primary terminals within this subnucleus w1x. Conversely, gastric afferents from the ventricle terminate preferentially within the subnucleus gelatinosus although some gastric sensory innervation terminate in the medial and commissural subnuclei of the nTS w40x. The high proportion of Y4 mRNA-expressing neurones in the subnucleus gelatinosus suggests that neurones involved in processing input from the gastric afferents are sensitive to PP. Whether mediated via dendritic receptors protruding into the AP or via PP concentrated in this subnucleus due to specific transport, these Y4 sites may be part of an explanatory mechanism by which PP exerts its stimulatory action upon gastric acid secretion and motility w30x. However, Y4 mRNA expression was also pronounced in neurones of the dmnX which by possessing pre-synaptic Y4 receptors may constitute another target of circulating PP influencing gastric emptying, the exocrine pancreas and gallbladder contraction. Circulating PP may, however, not be the only source of PP-fold peptides influencing Y4 receptive neurones of the AP and the adjacent dorsal vagal complex because a large majority of the catecholaminergic nor-adrenergic neurones of the dorsal vagal complex also synthesize NPY w4,15,37x. Thus, NPY-immunoreactive neurones have been demonstrated in the ventral margin of the AP and the subpostremal area and it is possible that these neurones influence local Y4 autoreceptors situated upon the very same neurones of the AP. Despite the fact that NPY has a low affinity for the Y4 receptor, it is possible that extracellular concentrations of NPY may be sufficiently high within the nTS to exert local modulatory actions of physiological relevance via Y4 receptors.
P.J. Larsen, P. Kristensenr Molecular Brain Research 48 (1997) 1–6
Acknowledgements We wish to thank Dr. J. Rasmussen ŽDepartment of Molecular Biology, NOVO-Nordisk. for cloning of Y4 cDNAs. Also, the expert photographic assistance of Mrs. G. Hahn is gratefully acknowledged. This work was supported by grants from the Danish Medical Research Council Ž12-1642-1., Danish Diabetes Association, NOVONordisk Foundation and Danish Medical Association.
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Research report
The neuropeptide Y ž Y4/ receptor is highly expressed in neurones of the rat dorsal vagal complex P.J. Larsen a
a,)
, P. Kristensen
b
Department of Medical Anatomy, UniÕersity of Copenhagen, NoÕo Nordisk A r S, BagsÕærd, Denmark b Department of Histology, Health Care DiscoÕery, NoÕo Nordisk A r S, BagsÕærd, Denmark Accepted 14 January 1997
Abstract Recently, the cDNA encoding the Y4 neuropeptide Y ŽNPY. receptor cDNA was cloned from a rat genomic library. The Y4 receptor is characterized by having a high affinity for pancreatic polypeptide ŽPP. and peptide YY ŽPYY.. By using in situ hybridization histochemistry with 35S-labelled riboprobes, we have visualized the cellular expression of mRNA encoding the Y4 receptor protein in the rat dorsal vagal complex at the light microscopical level. High densities of silver grains were observed over neurones of the dorsal vagal motor nucleus, and over neurones of a subregion of the nucleus of the solitary tract known as the subnucleus gelatinosus. Furthermore, cells within the ventral margin of the area postrema expressed high levels of Y4 mRNA. These observations indicate that circulating PP andror NPYrPYY via the blood–brain barrier-free area postrema and subpostremal area could influence neurones of the dorsal vagal complex with profound influence on numerous homeostatic mechanisms governed by this nuclear complex. Keywords: Nucleus of the solitary tract; Area postrema; Pancreatic polypeptide; Gut–brain axis; Drinking; Feeding
1. Introduction Pancreatic polypeptide ŽPP. was the first of several members to be discovered within a family of peptides recognized by their common hairpin PP-fold structure w21x. Other peptides of this 36-amino-acid peptide family are the evolutionary older members neuropeptide Y ŽNPY. and peptide YY ŽPYY. w24x. PP is exclusively synthesized and released post-prandially from the endocrine pancreas, thereby exerting its influence upon pancreatic secretion, gut motility and gall bladder contraction w16,38x. NPY is preferentially synthesized in neurones while PYY is synthesized in endocrine cells of the pancreas as well as the distal ileum and colon w6,7,28,43x. PP is released from endocrine cells of the pancreas in response to a meal via a mechanism involving vagal cholinergic neurones w38,41x whereas the post-prandial release of PYY is primarily caused by local circuits triggered by local stimulation of the intestinal mucosa w42x. The peripheral effects of PP and PYY upon gastrointestinal function are similar. In vivo, ) Corresponding author. Department of Anatomy, Section B, Panum Institute, Blegdamsvej 3, 2200 Copenhagen N, Denmark. Fax: q45 Ž3. 536-9612; E-mail: [email protected]
both peptides inhibit secretion from the exocrine pancreas, but the absence of PP receptors on rat acinar cells and the lack of inhibitory effect of PP upon CCK-stimulated secretion from isolated pancreatic acini suggests that PP exerts its effect indirectly w18,27,34x. Peripheral administration of PP and PYY inhibit centrally induced pancreatic secretion by 2-deoxy-D-glucose and this effect is greatly diminished by transection of the vagal nerves w34x, suggesting that PP and PYY interact with a site proximal to the vagal efferents. By using an in vivo radioreceptor assay, peripherally accessible binding sites for PP have been demonstrated in the dorsal vagal complex w46x, and the presence of PP-binding sites in the blood–brain barrier-free area postrema has inspired the hypothesis that gut peptides PP and PYY interacts with neurones of the dorsal vagal complex, thereby establishing a gut–brain axis w47x. This is further supported by vivo experiments demonstrating that direct application of PP to the dorsal vagal complex stimulates gastric acid secretion and motility in the rat w30x. The PP-fold peptides interact with a family of seventransmembrane NPY receptors coupled to G-binding proteins. Pharmacologically, the NPY receptors have been defined by their ability to bind NPY, PYY, PP and syn-
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P.J. Larsen, P. Kristensenr Molecular Brain Research 48 (1997) 1–6
thetic derivatives of these peptides w11,44x. The pharmacological binding profiles and rank order of binding potencies are as follows: Y1 binds NPY, PYY and wLeu31 ,Pro 34 xNPY ) PP and COOH-terminal fragments of NPY; Y2 binds NPY, PYY and COOH-terminal fragments of NPY ) PP; Y3 binds NPY ) PYY; Y4 Žthe PP receptor. binds PP ) NPY,PYY; and the Y5 Žputative feeding receptor. binds NPY, PYY, wLeu 31 ,Pro 34 xNPY and NPY 2 – 36 ) humanPP ) COOH-terminal fragments of NPY w12x while a novel receptor Žunfortunately also designated Y5. binds NPY, PYY, wLeu31 ,Pro 34 xNPY and NPY 2 – 36 ) NPY 13 – 36 ) human PP w45x. This pharmacological classification has largely been confirmed after the recent cloning of a total of five different receptor clones each coding for either of the receptor proteins w2,10,12,13,17,25,29,35,45,48 x. A PP-preferring receptor protein designated the Y4 receptor has been cloned from both rat and human genomic DNA w2,29,48x. Until now, the in vivo expression of the mRNA encoding the Y4 receptor has only been demonstrated by Northern blot analysis, and discrepancies about the level of Y4 mRNA expression in the central nervous system ŽCNS. exist. Thus, very low levels of Y4 hybridization were detected in non-amplified human mRNA samples from various brain regions w29x while much more robust signals were obtained from various regions of the brain when a RT-PCR amplification of human Y4 cDNA was employed w2x. However, future studies await to demonstrate the detailed topographical distribution of cellular expression of the Y4 mRNA in the CNS and, subsequently, compare this distribution with that of PP-binding sites. The present study was undertaken to investigate the expression of Y4 mRNA in the rat dorsal vagal complex which has been demonstrated to be one of very few central sites possessing specific PP-binding sites w46x.
Plasmid DNA was prepared and 35 S-labelled antisense and sense RNA was transcribed using T3 or T7 polymerases as described previously w23x. Briefly, following transcription the probe preparations were precipitated repeatedly with ethanol after addition of ammonium acetate to a final concentration of 3.0 M and hydrolysed to a mean size of 100 bp. The amount of TCA-precipitable material in the final probe preparation was usually ) 90%. The two corresponding RNA probes transcribed from the opposite strands of the same plasmid template were adjusted to the same radioactivity concentration and all experiments included sections hybridized using the sense RNA probes. The final hybridization solution contained RNA probe Ž100 000 cpmrm l., deionized formamide Ž50%., dextran sulphate Ž10%., tRNA Ž1 mgrml., Ficoll 400 Ž0.02% vrw., polyvinylpyrrolidone Ž0.02% vrw., BSA fraction V Ž0.2% vrw., 10 mM DTT, 0.3 M NaCl, 0.5 mM EDTA, 10 mM Tris–Cl and 10 mM NaPO4 ŽpH 6.8.. Hybridization was performed overnight at 478C as described w23x, except that the proteinase K step was omitted and the post-hybridization washing of sections in 50% formamide was performed at 57 and 628C. After exposure to b-radiation-sensitive films ŽHyperfilm, Amersham., sections were dipped in Amersham LM-1 nuclear emulsion and exposed for 9 days before being developed in Kodak D19. Emulsion-dipped sections were Nissl-counterstained and topographic analysis of the microscopic localization of Y4 mRNA-expressing cells were facilitated by using a microscope fitted with a camera lucida device. Background labelling was evaluated over areas dominated by white matter and cells were considered
2. Materials and methods Five male Wistar rats weighing f 200 g housed under standard laboratory conditions with free access to food and water were used for this experiment. Animals were decapitated early in the light phase ŽL : D, 12 : 12 h. and the brains were rapidly frozen on dry ice and kept at y708C until use before 12-m m cryostat sections of the lower brainstem were cut. Rat Y4 receptor cDNAs were cloned by the polymerase chain reaction based on the published sequence ŽGen Bank Accession No. u35232.. Polymerase chain reactions were carried out for 30 cycles of 1 min at 948C, 1 min at 558C and 2 min at 728C, using rat genomic DNA as template and sequence-specific oligonucleotides as primers. The following cDNA fragments were isolated: Y4-2 Žbp 28– 462. and Y4-3 Žbp 442–1155. and their sequence confirmed by DNA sequencing. RNA probes were prepared from cDNA cloned into Bluescript vectors ŽStratagene..
Fig. 1. Dark-field photomicrograph demonstrating Y4 mRNA-hybridized cells in the caudal part of the dorsal vagal complex. A high density of positively hybridized cells are concentrated in the ventral marginal zone of the AP while the central and superficial parts of this circumventricular organ contain fewer Y4-expressing cells. In the dorsolateral part of the dorsal vagal complex, the subnucleus gelatinosus Žgel. contains by far the majority of Y4-expressing cells while, in the ventral portion of the dorsal vagal complex, the dorsal vagal motor nucleus ŽdmnX. contains the majority of the Y4-expressing neurones. The adjacent commissural nucleus Žncom. is almost devoid of positively labelled cells. TS, solitary tract; asterisk in central canal. Scale bar s 200 m m.
P.J. Larsen, P. Kristensenr Molecular Brain Research 48 (1997) 1–6
Fig. 2. Camera lucida drawing of a counterstained section of the rat dorsal vagal complex taken from a rostrocaudal level similar to that shown in Fig. 1. Cells positively hybridized for Y4 mRNA are depicted as black dots. AP, area postrema; CC, central canal; dmnX, dorsal vagal motor nucleus; dPSR, dorsal parasolitarius region; gel, subnucleus gelatinosus; Gr, nucleus gracilis; mnTS, medial nucleus of the nTS; ncom, commissural nucleus of the nTS; nI, intermediate nucleus of the nTS; TS, solitary tract; XII, hypoglossal nucleus.
positively labelled when the density of overlying photographic silver grains was ) 5 = the background labelling. At representative rostrocaudal levels of specific subnuclei, the number of positively hybridized cells were counted and compared to the number of Nissl-stained neurones. The approximate percentages of positively hybridized neurones were subsequently estimated. The nomenclature used is largely adopted from w20,31x.
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nal. Furthermore, rat brain sections hybridized with ribonucleotide probes generated from the Y4 sense strand were all devoid of a positive hybridization signal. In the lower brainstem, in situ hybridization histochemistry revealed numerous Y4 mRNA-expressing cells in the dorsal vagal complex. Except for a few positively labelled cells in the lateral reticular nucleus of the ventrolateral medulla, all other areas of the medulla oblongata were devoid of Y4 mRNA-expressing cells. In the dorsal vagal complex, high numbers of Y4 mRNA-expressing cells were present in the area postrema ŽAP., in the dorsal motor nucleus of the vagus ŽdmnX. and in the subnucleus gelatinosus of the nucleus of the solitary tract ŽnTS. ŽFigs. 1–3.. Few positively labelled cells were scattered throughout the commissural and the intermediate nuclei of the nTS. Within the AP, positively labelled cells were concentrated in the deep ventral margin of this circumventricular organ which is adjacent to the subpostremal area ŽFig. 1.. In this deep zone of the AP, ) 80% of the cells were considered positively labelled. Positively labelled cells were present throughout the rostrocaudal extent of the dmnX and the subnucleus gelatinosus of the nTS. f 40% of the rather large neurones of the dmnX were positively labelled while 70% of the neurones in the subnucleus gelatoinosus were positively hybridized with the Y4 mRNA-recognizing probe.
4. Discussion 3. Results The specificity of the in situ hybridization histochemical reaction was verified by the fact that two different probes recognizing different only partially overlapping parts of the Y4 cDNA sequence gave identical hybridization signals. All five animals examined displayed similar distribution pattern and intensity of the hybridization sig-
The rank order of binding of PP-fold proteins to the Y4 receptor is PP ) NPY, PYY, rendering PP the endogenous high-affinity ligand of this receptor w11,44x. The present observation corresponds largely to the receptor autoradiographic pattern of radioiodinated PP-binding w46x, suggestive of a somaticrperikaryal localization of the Y4 receptor protein. Compared to receptor autoradiography, the
Fig. 3. Medium-power photomicrograph showing cells positively hybridized for Y4 mRNA in the dorsal vagal motor nucleus ŽA. and the AP ŽB.. Scale bars s 50 m m
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P.J. Larsen, P. Kristensenr Molecular Brain Research 48 (1997) 1–6
currently used in situ hybridization methodology yields a far better cellular resolution of the exact distribution of Y4 mRNA expression though it is important to keep in mind that the sites of receptor protein synthesis and membrane localization are not necessarily coincident. Thus, more distant localization of the Y4 receptor protein at dendrites and axons emanating from the currently labelled cells should be considered as well. PP is a peripheral hormone not synthesized in the brain and consequently PP-mediated effects upon the CNS have to be communicated either via blood–brain barrier-free areas or via a specific uptake mechanism. PP is released from endocrine cells of the pancreas in response to a meal, sham-feeding or hypoglycaemia and these effects rely entirely upon vagal, cholinergic mechanisms w38x. Subsequently, circulating PP inhibits gastric motility, gall bladder contraction as well as exocrine pancreatic secretion w16x, suggestive of a role as a feed forward inhibitor upon digestive processes. In addition, PP and PYY have a potent emetic effect which is abolished in post-rectomized animals, suggesting that PP-fold peptides may act as endogenous emetic agents w14x. By using an in vivo radioreceptor assay, peripherally accessible binding sites for PP has been demonstrated in the rat dorsal vagal complex but it has not been possible to ascertain to what degree PP is bound by the nTS and dmnX, both areas which are considered protected by the blood–brain barrier w46x. A number of possible mechanisms via which PP exerts its action upon the dorsal vagal complex is possible. First, PP could interact with somatic Y4 receptors upon AP neurones projecting to the adjacent nuclei of the dorsal vagal complex. The AP projects heavily to the dorsal nuclei of the dorsal vagal complex while ventrally situated nuclei, including the dmnX, are sparsely innervated w8x. Second, it is possible that Y4 receptors are present on dendrites emanating from deeper nuclei Že.g. the subnucleus gelatinosus. which via protrusion into the AP proper are situated outside the blood–brain barrier. Ultrastructural and Golgi studies confirm the presence of such antenna-like dendrites monitoring the systemic circuit w8,19,32x. Third, Y4 receptor protein synthesized within motor neurones of the dmnX could be transported to terminals located in peripheral autonomic parasympathetic ganglia, thus, acting as pre-synaptic receptors. Finally, analogous to the brain insulin uptake system w33x the presence of a specific transport mechanism conveying PP over the blood–brain barrier should be considered. The AP contained a high number of Y4 mRNA-expressing cells. The AP is a circumventricular organ which is highly vascularized by an extensive capillary plexus w9x. The absence of a blood–brain barrier in the AP allows neuronal elements in the organ and the immediate adjacent subpostremal area of the dorsal vagal complex to be directly exposed to blood-borne substances as well as the cerebrospinal fluid of the fourth ventricle w5,22x. The anatomical features of the AP makes the organ ideally
suited for chemoreception and perhaps neurosecretion w5x. In addition, the AP is also target for vagal and hypothalamic afferent fibres w20,26,39x, implying that it may participate in visceroception by means of both humorally and neurally mediated inputs. The efferent projections from the neurones of the AP are preferentially directed towards the ventrally lying dorsal vagal complex but fibres also target the A1 and C1 catecholaminergic neurones of the ventrolateral medulla as well as rostrally situated nuclei of the brainstem w3,8,36,39x. The functional role of the AP as a central player in the physiological mechanisms underlying emesis and vomiting has been continuously confirmed since the 1950s w5x whereas other roles of this organ are somewhat uncertain. The current study gives evidence that neurones of the AP are directly sensitive to circulating PP which could mediate its emetic and stimulatory actions on gastric motility and pancreatic secretion via Y4 receptors in the AP. The topographically distinct distribution of Y4 expression in the nTS is interesting because innervation of the nTS by vagal afferents from the alimentary canal is viscerotopically organized w31x. Thus, oesophageal afferents preferentially terminate within the ventral portion of the medial nTS which apparently is the only the visceral organ represented by primary terminals within this subnucleus w1x. Conversely, gastric afferents from the ventricle terminate preferentially within the subnucleus gelatinosus although some gastric sensory innervation terminate in the medial and commissural subnuclei of the nTS w40x. The high proportion of Y4 mRNA-expressing neurones in the subnucleus gelatinosus suggests that neurones involved in processing input from the gastric afferents are sensitive to PP. Whether mediated via dendritic receptors protruding into the AP or via PP concentrated in this subnucleus due to specific transport, these Y4 sites may be part of an explanatory mechanism by which PP exerts its stimulatory action upon gastric acid secretion and motility w30x. However, Y4 mRNA expression was also pronounced in neurones of the dmnX which by possessing pre-synaptic Y4 receptors may constitute another target of circulating PP influencing gastric emptying, the exocrine pancreas and gallbladder contraction. Circulating PP may, however, not be the only source of PP-fold peptides influencing Y4 receptive neurones of the AP and the adjacent dorsal vagal complex because a large majority of the catecholaminergic nor-adrenergic neurones of the dorsal vagal complex also synthesize NPY w4,15,37x. Thus, NPY-immunoreactive neurones have been demonstrated in the ventral margin of the AP and the subpostremal area and it is possible that these neurones influence local Y4 autoreceptors situated upon the very same neurones of the AP. Despite the fact that NPY has a low affinity for the Y4 receptor, it is possible that extracellular concentrations of NPY may be sufficiently high within the nTS to exert local modulatory actions of physiological relevance via Y4 receptors.
P.J. Larsen, P. Kristensenr Molecular Brain Research 48 (1997) 1–6
Acknowledgements We wish to thank Dr. J. Rasmussen ŽDepartment of Molecular Biology, NOVO-Nordisk. for cloning of Y4 cDNAs. Also, the expert photographic assistance of Mrs. G. Hahn is gratefully acknowledged. This work was supported by grants from the Danish Medical Research Council Ž12-1642-1., Danish Diabetes Association, NOVONordisk Foundation and Danish Medical Association.
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