Axonal transport of neurotrophins by visceral afferent and efferent neurons of the vagus nerve of the rat

The receptor-mediated axonal transport of [¹²⁵I]-labeled neurotrophins by afferent and efferent neurons of the vagus nerve was determined to predict the responsiveness of these neurons to neurotrophins in vivo. [¹²⁵I]-labeled neurotrophins were administered to the proximal stump of the transected cervical vagus nerve of adult rats. Vagal afferent neurons retrogradely transported [¹²⁵I]neurotrophin-3 (NT-3), [¹²⁵I]nerve growth factor (NGF), and [¹²⁵I]neurotrophin-4 (NT-4) to perikarya in the ipsilateral nodose ganglion, and transganglionically transported [¹²⁵I]NT-3, [¹²⁵I]NGF, and [¹²⁵I]NT-4 to the central terminal field, the nucleus tractus solitarius (NTS). Vagal afferent neurons showed minimal accumulation of [¹²⁵I]brain-derived neurotrophic factor (BDNF). In contrast, efferent (parasympathetic and motor) neurons located in the dorsal motor nucleus of the vagus and nucleus ambiguus retrogradely transported [¹²⁵I]BDNF, [¹²⁵I]NT-3, and [¹²⁵I]NT-4, but not [¹²⁵I]NGF. The receptor specificity of neurotrophin transport was examined by applying [¹²⁵I]-labeled neurotrophins with an excess of unlabeled neurotrophins. The retrograde transport of [¹²⁵I]NT-3 to the nodose ganglion was reduced by NT-3 and by NGF, and the transport of [¹²⁵I]NGF was reduced only by NGF, whereas the transport of [¹²⁵I]NT-4 was significantly reduced by each of the neurotrophins. The competition profiles for the transport of NT-3 and NGF are consistent with the presence of TrkA and TrkC and the absence of TrkB in the nodose ganglion, whereas the profile for NT-4 suggests a p75 receptor-mediated transport mechanism. The transport profiles of neurotrophins by efferent vagal neurons in the dorsal motor nucleus of the vagus and nucleus ambiguus are consistent with the presence of TrkB and TrkC, but not TrkA, in these nuclei. These observations describe the unique receptor-mediated axonal transport of neurotrophins in adult vagal afferent and efferent neurons and thus serve as a template to discern the role of specific neurotrophins in the functions of these visceral sensory and motor neurons in vivo. J. Comp. Neurol. 393:102–117, 1998. Published 1998 Wiley-Liss, Inc.

1 This article is a US government work and, as such, is in the public domain in the United States of America.

     

Neuropeptide Y and Galanin Binding Sites in Rat and Monkev Lumbar Dorsal Root Ganalia and Spinal Cord and Effect of Peripheral Axotomy

Using monoiodinated peptide YY (PYY) and galanin as radioligands, and neuropeptide Y (NPY) fragments, the distribution of NPY binding sites and its subtypes Y1 and Y2, and of galanin binding sites, was investigated in rat and monkey lumbar (L) 4 and L5 dorsal root ganglia (DRG) and spinal cord before and after a unilateral sciatic nerve cut, ligation or crush. Receptor autoradiography revealed that [¹²⁵I]PYY bound to some DRG neurons and a few nerve fibres in normal rat DRG, and most of these neurons were small. NPY binding sites were observed in laminae I–IV and X of the rat dorsal horn and in the lateral spinal nucleus, with the highest density in laminae 1–11. [¹²⁵I]NPY binding was most strongly attenuated by NPY_13–36, a Y2 agonist, and partially inhibited by [Leu³¹,Pro³⁴]NPY, a Y1 agonist, in both rat DRG and the dorsal horn of the spinal cord. These findings suggest that Y2 receptors are the main NPY receptors in rat DRG and dorsal horn, but also that Y1 receptors exist. After sciatic nerve cut, PYY binding markedly increased in nerve fibres and neurons in DRG, especially in large neuron profiles, and in laminae III-IV of the dorsal horn, as well as in nerve fibres in dorsal roots and the sciatic nerve. Incubation with NPY_13–36 completely abolished PYY binding, which was also reduced by [Leu,³¹ Pro³⁴] NPY. However, the increase in PYY binding seen in laminae I–IV of the ipsilateral dorsal horn after axotomy was not observed after coincubation with [Leu³¹, Pro³⁴] NPY. NPY binding sites were seen in a few neurons in monkey DRG and in laminae I-II, X and IX of the monkey spinal cord. The intensity of PYY binding in laminae I-II of the dorsal horn was decreased after axotomy. Galanin receptor binding sites were not observed in rat DRG, but were observed in the superficial dorsal horn of the spinal cord, mainly in laminae I-II. Axotomy had no effect on galanin binding in rat DRG and dorsal horn. However, galanin receptor binding was observed in many neurons in monkey L4 and L5 DRG and in laminae I–IV and X of monkey L4 and L5 spinal cord, with the highest intensity in laminae I-II. No marked effect of axotomy was observed on the distribution and intensity of galanin binding in monkey DRG or spinal cord. The present results indicate that after axotomy the synthesis of NPY receptors is increased in rat DRG neurons, especially in large neurons, and is transported to the laminae I–IV of the ipsilateral dorsal horn and into the sciatic nerve. No such up-regulation of the NPY receptor occurred in monkey DRG after axotomy. The Y2 receptor seems to be the main NPY receptor in DRG and the dorsal horn of the rat and monkey spinal cord, but Y1 receptors also exist. The increase in NPY binding sites in laminae I–IV of the dorsal horn after axotomy partly represents Y1 receptors. In contrast to the rat, galanin binding sites could be identified in monkey lumbar DRG. No effect of axotomy on the distribution of galanin binding sites in rat or monkey DRG and dorsal horn was detected, suggesting their presence on local dorsal horn neurons (or central afferents).     

Quantitative autoradiographic localization of [125I]neuropeptide Y receptor binding sites in rat spinal cord and the effects of neonatal capsaicin, dorsal rhizotomy and peripheral axotomy.

Using in vitro quantitative receptor autoradiography the present study reports on the distribution and possible changes of [125I]neuropeptide Y (NPY) binding sites in the rat spinal cord following neonatal capsaicin treatment, dorsal rhizotomy and sciatic nerve section. In control spinal cord the highest density of [125I]NPY binding sites was noticed in the superficial layers of the dorsal horn whereas low-to-moderate densities of [125I]NPY binding sites were detected in the deeper dorsal horn and in the ventral horn. In comparison with control rats, neonatally treated capsaicin rats showed a significant (P less than 0.001) bilateral decrease in [125I]NPY binding sites in the superficial layers of the dorsal horn. Unilateral dorsal rhizotomy and unilateral sciatic nerve section also exhibited a significant (P less than 0.05) depletion in [125I]NPY labeling in the superficial layers of the dorsal horn ipsilateral to the surgery. These results suggest that a certain proportion of [125I]NPY receptor sites is located on the primary afferent fibers of the superficial layers of the dorsal horn. This peptide thus could play an important role in the modulation of nociceptive transmission by acting directly on primary afferent terminals.     

Association of neuropeptide Y Y1 receptors with glutamate-positive and NPY-positive neurons in rat hippocampal cultures

The hippocampus is particularly enriched with neuropeptide tyrosine (NPY) and NPY receptors including the Y1, Y2 and Y5 subtypes. We have previously reported on the enrichment of cultured rat hippocampal neurons in specific [125I][Leu31, Pro34]PYY/BIBP3226-sensitive (Y1) binding sites and Y1 receptor mRNAs [St-Pierre et al. (1998) Br. J. Pharmacol., 123, p183]. We have now identified which cell types express the Y1 receptor. The majority of Y1 receptors, visualized using either the radiolabeled probe [125I][Leu31,Pro34]PYY or two antibodies directed against distinct domains of the Y1 receptor, was expressed in neurons as revealed by neuron-specific enolase (NSE) immunostaining. One antibody was directed against the second extracelllular loop of the Y1 receptor (amino acids 185-203) whereas the second was directed against the intracellular C-terminal loop (amino acids 355-382). The labelling was evident over both perikarya and processes. Neurons labelled by the various Y1 receptor probes were mostly glutamate-positive as revealed by double immunostaining. Most interestingly, a number of NPY-positive cultured hippocampal neurons were also enriched with the Y1 receptor, suggesting that this subtype may act as an autoreceptor to regulate NPY release in the hippocampus. These results thus provide an anatomical basis for the modulation of glutamate and NPY release by the Y1 receptor in the hippocampus.     

Regionally-selective down-regulation of NPY receptor subtypes in the obese Zucker rat. Relationship to the Y5 ‘feeding’ receptor

Experiments were performed to examine whether there are regionally and subtype selective changes in the density of neuropeptide Y (NPY) receptors in the obese Zucker rat which has an increased synthesis and release of NPY confined to the hypothalamus. Competition binding assays were employed to examine the feasibility of using [¹²⁵I]peptide YY ([¹²⁵I]PYY) to measure neuropeptide Y (NPY) Y5 ‘feeding’ receptors in the hypothalamus, hippocampal and cerebral cortex following masking of Y1 and Y2 receptors. Y5 receptors could not be discriminated from the binding to Y1 and Y2 receptors in hypothalamic, hippocampal or cerebral cortex homogenates, possibly owing to the small population of Y5 receptors expressed in the brain and the lack of selective ligands for this receptor. Quantitative receptor autoradiography was used to examine for regional changes in NPY receptor subtypes in obese versus lean Zucker rats. The non-selective Y1, Y2, Y4 and Y5 receptor ligand [¹²⁵I]PYY and the more selective Y1, Y4 and Y5 ligand [¹²⁵I][Leu³¹,Pro³⁴]PYY were employed, in conjunction with masking compounds in an attempt to measure any regional changes in the recently cloned NPY Y5 ‘feeding’ receptor. Specific [¹²⁵I]PYY and [¹²⁵I][Leu³¹,Pro³⁴]PYY binding was significantly reduced in the hypothalamic dorsomedial and arcuate nuclei as well as in the dorsal and lateral (perifornical) areas of obese Zucker rats, as compared to lean rats. In addition there were significant reductions in binding to the thalamic reuniens and centromedial nucleus, and hippocampal dentate gyrus of obese rats as compared to lean rats. Masking [¹²⁵I]PYY binding to Y1 receptors using 1 μM BIBP3226 demonstrated that the reduced NPY receptor density was due to reductions in Y2 or Y5 receptor density. The binding which was sensitive to BIBP3226, i.e. Y1 receptor density, was not different between obese and lean rats. Attempts using [¹²⁵I]PYY and the relatively selective Y2 agonist, [13–36]NPY to mask Y2 receptors and reveal Y5 receptors failed to leave any specific binding suggesting that [13–36]NPY was not selective enough to separate binding to Y2 and Y5 receptors. However, using [¹²⁵I][Leu³¹,Pro³⁴]PYY, masking binding to Y1 receptors using 1 μM BIBP3226 and masking any binding to Y4 using 1 nM rat pancreatic polypeptide left a small amount of binding remaining in the thalamus and hypothalamus, presumably to Y5 receptors which was significantly reduced in obese versus lean rat brain. These data suggest that there is a selective down-regulation in Y5 ‘feeding’ receptors in the obese Zucker rat which is known to possess a hyperactive arcuate-paraventricular NPY system.     

Trigeminal and dorsal root ganglion neurons express CCK receptor binding sites in the rat, rabbit, and monkey: possible site of opiate-CCK analgesic interactions.

125I-Bolton-Hunter sulfated cholecystokinin-8 was used to localize and characterize cholecystokinin (CCK) receptor binding sites in trigeminal and dorsal root ganglia, and in the spinal cord of the rat, rabbit, and monkey. In the rabbit and monkey, a substantial number, 90 +/- 21% and 24 +/- 8%, respectively, of trigeminal and dorsal root ganglion neurons express CCK binding sites. In the spinal cord, the highest concentration of CCK receptors is found in laminae I and II, which is the major termination site of dorsal root ganglia neurons expressing CCK receptor binding sites. Neonatal capsaicin treatment of the rat results in a 70% decline in CCK receptor binding sites in laminae I and II of the spinal cord, indicating that dorsal root ganglia neurons are a major source of CCK receptors in the spinal cord. Pharmacological experiments using selective CCK-A and CCK-B receptor antagonists demonstrate that CCK-B is the prominent CCK receptor subtype in trigeminal and dorsal root ganglia neurons in the rat, rabbit, and monkey. In the rat and rabbit spinal cord, CCK-B binding sites are the prominent subtype, whereas in the monkey cord, CCK-A is the prominent receptor subtype. These results demonstrate that CCK-B receptors are expressed by a substantial percentage of dorsal root ganglion neurons at all spinal levels, and that CCK may antagonize opiate analgesia at the level of the primary afferent neuron itself.     

Association of neurotensin binding sites with sensory and visceromotor components of the vagus nerve

Specific neurotensin (NT) binding sites were recently shown to be highly concentrated in the nucleus of the solitary tract (NTS), which receives primary vagal afferents, and in the dorsal motor nucleus of the vagus (DMN), which contains the cell bodies of origin of vagal preganglionic neurons. To investigate the relationship of these binding sites with sensory and visceromotor components of the vagus nerve, they were labeled here in vitro, using monoiodo[Tyr3]neurotensin (125I-NT) and visualized by light microscopic radioautography in the dorsomedial medulla of both intact and unilaterally vagotomized rats, in the nodose ganglia of intact animals, and in ligated vagus nerves. Unilateral vagotomy performed above the nodose ganglion resulted in a significant ipsilateral decrease in 125I-NT binding within both the NTS and the DMN, suggesting that NT binding sites were associated with both primary afferent fibers and preganglionic nerve cell bodies. The selective radioautographic labeling of a subpopulation (approximately 15%) of neuronal perikarya in the nodose ganglion confirmed that a proportion of vagal afferent neurons contained NT binding sites. Following vagus nerve ligation, a pile up of radiolabeled NT binding sites was observed on both sides of the nerve crush, indicating that NT receptor components were transported both anterogradely and retrogradely along fibers of the vagus nerve. We conclude that NT receptors are synthesized and transported within a subpopulation of afferent and efferent components of the vagus nerve and that NT may therefore act presynaptically upon vagal axon terminals in both central and peripheral nervous systems.     

Neuropeptide Y‐stimulated [35S]GTPγs functional binding is reduced in the hippocampus after kainate‐induced seizures in mice

Kainate‐induced seizures constitute a model of temporal lobe epilepsy where prominent changes are observed in the hippocampal neuropeptide Y (NPY) system. However, little is known about the functional state and signal transduction of the NPY receptor population resulting from kainate exposure. Thus, in this study, we explored functional NPY receptor activity in the mouse hippocampus and neocortex after kainate‐induced seizures using NPY‐stimulated [³⁵S]GTPγS binding. Moreover, we also studied levels of [¹²⁵I]‐peptide YY (PYY) binding and NPY, Y1, Y2, and Y5 receptor mRNA in these kainate‐treated mice. Functional NPY binding was unchanged up to 12 h post‐kainate, but decreased significantly in all hippocampal regions after 24 h and 1 week. Similarly, a decrease in [¹²⁵I]‐PYY binding was found in the dentate gyrus (DG) 1 week post‐kainate. However, at 2 h, 6 h, and 12 h, [¹²⁵I]‐PYY binding was increased in all regions, and in the CA1 also at 24 h post‐kainate. NPY mRNA levels were prominently increased in hippocampal regions, reaching maximum at 12 and 24 h. Y1 and Y5 mRNA levels were lowered in the DG at 24 and 2 h, respectively, while Y2 mRNA levels were elevated at 24 h in the DG and CA3. This study confirms rat kainate studies by showing pronounced adaptive changes in the mouse hippocampus both with regard to NPY synthesis and NPY receptor synthesis and binding, which may contribute to regulating neuronal seizure susceptibility after kainate. However, the potential seizure‐suppressant effects of increased NPY gene expression at late time points post‐kainate could be attenuated by the novel finding of reduced NPY‐receptor G‐protein activation. Synapse 68:427–436, 2014 . © 2014 Wiley Periodicals, Inc.     

Characterization of a new neuropeptide Y Y5 agonist radioligand: [125I][cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP

In order to optimally characterize a class of neuropeptide Y (NPY) receptors expressed in a tissue enriched with multiple subtypes (Y1, Y2, Y4 and Y5) and to establish its detailed distribution, it is critical to use highly selective and specific probes that possess very low non-specific binding. In that context, we recently reported on the development of [125I][hPP(1-17), Ala31, Aib32]NPY as Y5 receptor radioligand. However, the non-specific binding obtained with this radioligand was too high to allow for detailed receptor autoradiography studies [Br. J. Pharmacol. 139 (2003) 1360]. Iodinated [cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP may represent a better Y5 radioligand in that regard. Accordingly, [125I][cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP binding was investigated in rat brain membrane homogenates and its specificity and selectivity established in rat Y1, Y2, Y4 and Y5 transfected HEK293 cells. No specific binding was detected in HEK293 cells transfected with the rat Y1, Y2 or Y4 receptors, while saturable binding was observed in cells transfected with the rat Y5 receptor cDNA and in rat brain membrane homogenates (KD of 0.5-0.7 nM). Competition binding experiments performed in rat brain membrane homogenates demonstrated that specific [125I][cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP binding was competed with nanomolar affinities by Y5 agonists and antagonists such as [Leu31,Pro34]PYY, PYY(3-36), [cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP, [Ala31, Aib32]NPY, [hPP(1-17), Ala31, Aib32]NPY, CGP71683A and JCF109, but not by Y1 (BIBP3226 and BIBO3304), Y2 (BIIE0246) and Y4 (GR231118) ligands. Non-specific binding was also lower than that reported for [125I][hPP(1-17), Ala31, Aib32]NPY. Interestingly, detailed analysis of competition binding curves obtained with [Leu31, Pro34]PYY, hPP, PYY(3-36) and [cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP against specific [125I][cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP sites were best fitted to a two-site model. Additionally, receptor autoradiography studies revealed the presence of specific [125I][cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP binding sites in the lateral septum and area postrema while other brain regions contained much lower levels of specific binding. Taken together, these data suggest that [125I][cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP represents a useful tool to study the unique feature of the Y5 receptor subtype.     

Localization of AT(2) receptors in the nucleus of the solitary tract of spontaneously hypertensive and Wistar Kyoto rats using [125I] CGP42112: upregulation of a non-angiotensin II binding site following unilateral nodose ganglionectomy

We have examined the binding distribution of a selective AT(2) receptor ligand [125I] CGP42112 in the brain of adult Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). AT(2) receptor localization was also examined in the rat brainstem following unilateral nodose ganglionectomy. Specific [125I] CGP42112 binding was observed in discrete brain regions from both rat strains, including the nucleus of the solitary tract (NTS), and did not differ between WKY and SHR. [125I] CGP42112 binding in the NTS revealed an AT(2) receptor component that was displaceable by PD 123319 and Ang II (50-58%), as well as a non-angiotensin II receptor component (42-49%). Following unilateral nodose ganglionectomy, [125I] CGP42112 binding density on the denervated side of the NTS was increased approximately two-fold in both WKY and SHR. This increased [125I] CGP42112 binding density in the ipsilateral NTS was comprised of a greater non-angiotensin II component than that observed in the sham groups, since only approximately 30% was displaced by PD123319 and angiotensin II. Furthermore, [125I] CGP42112 also revealed high binding density on the denervated side in the dorsal motor nucleus and the nucleus ambiguus in both WKY and SHR. AT(2) receptor immunoreactivity was also visualised in the NTS of sham operated rats, but was not observed in the dorsal motor nucleus or the nucleus ambiguus, nor was it up-regulated following nodose ganglionectomy. These results demonstrate, for the first time, an AT(2) receptor binding site in the NTS, as well as a non-angiotensin II [125I] CGP42112 binding site. These studies also demonstrate that nodose ganglionectomy represents a useful model in which to study a non-angiotensin II [125I] CGP42112 binding site that is up-regulated following degeneration of afferent vagal nerves.     

Quantitative autoradiographic localization of neuropeptide Y receptors in the rat lower brainstem

Using quantitative autoradiography, we have characterized the binding of ¹²⁵I-Bolton-Hunter coupled neuropeptide Y ([¹²⁵I]NPY) and observed the localization of ¹²⁵I-NPY receptors in the rat lower brainstem. [¹²⁵I]NPY bound to the receptors in a specific, saturable and reversible manner with high affinity. The binding was blocked only by unlabeled NPY but not by NPY-related peptides i.e. peptide YY, pancreatic polypeptide (avian and human), nor by neurotensin. [¹²⁵I]NPY receptors were revealed to be coupled to the guanosine triphosphate (GTP)-binding protein. Regional distribution study showed that [¹²⁵I]NPY has a distinctive pattern of distribution in the rat lower brainstem, being particularly concentrated in the area postrema and the medial subnucleus of the nucleus tractus solitarii. These results suggest that such NPY receptors have an important role in cardiovascular regulation.     

Developments of α-bungarotoxin receptors in cultured chick ciliary ganglion neurons

We have maintained embryonic chick ciliary ganglion neurons in dissociated cell culture and studied the progressive appearance of surface receptors for [¹²⁵I]α-bungarotoxin. Cultures were established from 8-day-old embryos and fed a medium supplemented with 180 μg/ml of a soluble protein extract prepared from the eye, the target organ for the ciliary ganglion. Approximately 8064 neurons survived per ganglion and there was no evident loss of neurons through two weeks in culture. Binding of [¹²⁵I]α-bungarotoxin was determined at room temperature on intact cells still attached to their coverslips. Non-specific binding was less than 2% of the total. Specific binding of [¹²⁵I]α-bungarotoxin was saturable with respect to both time of incubation (20–30 min) and concentration of toxin (5–10 nM), with an apparent K_d = 1.0 nM. Binding sites for [¹²⁵I]α-bungarotoxin increased during the first week in culture from 1.8 fmol per 10⁴ neurons at 1 day in vitro (DIV) to 8.6 fmol per 10⁴ neurons at 7 DIV, after which the number of sites seemed to plateau. Light microscopic autoradiography was performed on cultures at 4 DIV and showed most of the grains associated with the surfaces of neuronal cell bodies, while scattered grains occurred over neuronal processes. When compared with previous reports on the in vivo development of α-bungarotoxin receptors in chick ciliary ganglia, the appearance of receptors in these cultured neurons followed a time course similar to, but at lower levels than, their in vivo counterparts. Nevertheless, this culture system should prove useful for the study of questions concerning the regulation, surface distribution and intracellular pathways of neuronal α-bungarotoxin receptors.     

Neuropeptide Y modulates neurotransmitter release and Ca2+ currents in rat sensory neurons

Using 125I-labeled neuropeptide Y (NPY) and peptide YY (PYY), we demonstrated the existence of specific receptors for these peptides on rat dorsal root ganglion (DRG) cells grown in primary culture. Scatchard analysis of membrane homogenates indicated that the peptides bound to 2 populations of sites, with approximate affinities of 0.08 and 6.5 nM. Only low levels of binding were detected on sympathetic neurons cultured from the same animals or on a variety of neuronal clonal cell lines. The binding of 125I-NPY and 125I-PYY to DRG cell membranes was considerably reduced by the nonhydrolyzable analog of GTP, Gpp(NH)p. The major effect of Gpp(NH)p was to reduce the number of lower-affinity NPY binding sites without altering the number of high-affinity binding sites. NPY potently inhibited Ca2+ currents recorded under voltage clamp in rat DRG cells. Both the transient and sustained portions of the Ca2+ current were inhibited. The inhibitory effects of NPY were completely blocked following treatment of the cells with pertussis toxin. Depolarization elicited a large influx of Ca2+ into DRG neurons as assessed using fura-2-based microspectrofluorimetry. This influx of Ca2+ could be partially inhibited by NPY. Furthermore, NPY effectively inhibited the depolarization-induced release of substance P from DRG cells in vitro. Thus, NPY may be an important regulator of sensory neuron function in vivo.     

Distribution of [Leu,Pro]NPY-sensitive, BIBP3226-insensitive [I]PYY(3–36) binding sites in rat brain: possible relationship to Y5 NPY receptors

Recently, using molecular cloning approaches, three new neuropeptide Y (NPY)/peptide YY (PYY) receptors have been described in rodent brain, with pharmacological profiles that differ from the three previously described Y₁, Y₂ and Y₃ NPY receptors and the Y₄ pancreatic polypeptide- (PP-) preferring receptor. Two of these new receptors are spice variants and are called Y₅ receptors, whilst a third receptor has been called Y₆ and has been suggested to be expressed only in the mouse. In the absence of a totally selective Y₅ and/or Y₆ radioligands, we have examined [¹²⁵I]PYY(3–36) binding, which binds Y₂ and Y₅/Y₆ receptors, using homogenate assays and quantitative receptor autoradiography to study the distribution of the three newly discovered Y₅/Y₆ receptors by masking binding to Y₁ receptors with high concentrations of the non-peptidergic selective Y₁ antagonist, BIBP3226, and using either [Leu³¹,Pro³⁴]NPY or human PP to mask binding to Y₅ and Y₆ receptors, leaving binding to Y₂ receptors. Using this approach, [¹²⁵I]PYY(3–36) labels a small population of Y₁ receptors and a larger population of binding sites that are insensitive to BIBP3226, human PP and [Leu³¹,Pro³⁴]NPY, presumed to be Y₂ receptors. There was also [¹²⁵I]PYY(3–36) binding to sites sensitive to NPY, human PP and [Leu³¹,Pro³⁴]NPY, but insensitive to BIBP3226, located in the hypothalamus, amygdala, hippocampus and thalamus. As one of the recently cloned Y₅ receptors is synthesized in these regions, as shown by in-situ hybridization techniques, we suggest that the small population of [¹²⁵I]PYY(3–36) binding sites which are sensitive to human PP and [Leu³¹,Pro³⁴]NPY, but insensitive to BIBP3226, may represent binding to Y₅ receptors. We have been unable, however, to visualize a smaller population of Y₆ receptors which are labelled by [¹²⁵I]PYY₃–36 and sensitive to [Leu³¹,Pro³⁴]NPY, but not to BIBP3226 and human PP, confirming that the murine Y₆ receptor does not appear to be expressed in rat brain.     

Species differences in the expression and distribution of the neuropeptide Y Y1, Y2, Y4, and Y5 receptors in rodents,guinea pig,and primates brains

The respective distribution of neuropeptide Y Y₁, Y₂, Y₄, and Y₅ receptor subtypes was investigated in rodents (rat and mouse), guinea pig, and primates (marmoset and vervet monkeys and human) brains, representing three orders of mammals. [¹²⁵I][Leu³¹,Pro³⁴]PYY (total Y₁-like; Y₁, Y₄, and Y₅) and [¹²⁵I]PYY_3–36 (total Y₂-like; Y₂ and possibly Y₅) binding sites were discretely but similarly distributed in the rat and mouse brain, each having its unique pattern. In contrast, surprisingly low levels of [¹²⁵I]PYY_3–36 binding sites were found in the guinea pig brain including in the hippocampal formation. [¹²⁵I][Leu³¹,Pro³⁴]PYY/BIBP3226-insensitive binding sites (Y₅-like) were found in different areas of the rat and guinea pig brains. The primate brains also revealed a different distribution binding profile for these various NPY receptor subtypes. Although the human and vervet brains contained very low amounts of [¹²⁵I][Leu³¹,Pro³⁴]PYY sites (Y₁-like) in most brain regions, except for the dentate gyrus of the hippocampus, the marmoset brain contains significant amounts of both [¹²⁵I][Leu³¹,Pro³⁴]PYY (Y₁-like) and [¹²⁵I]PYY_3–36 (Y₂-like) binding sites. Additionally, [¹²⁵I][Leu³¹,Pro³⁴]PYY/BIBP3226-insensitive binding sites were not clearly detected in the vervet and human brains. On the other hand, Y₅-like binding sites were observed in few regions of the marmoset brain. Finally, [¹²⁵I]hPP (Y₄/Y₅-like) were very discretely distributed in the rat brain, being concentrated in the paraventricular nucleus of the hypothalamus and the interpeduncular nucleus. The marmoset brain is apparently not enriched with specific [¹²⁵I]hPP sites. Taken together, these data show that significant species differences exist in the level of expression and distribution of various NPY receptor subtypes in the mammalian brain. J. Comp. Neurol. 402:372–384, 1998. © 1998 Wiley-Liss, Inc.     

Quantitative autoradiography of angiotensin II receptors in the rat solitary-vagal area: effects of nodose ganglionectomy or sinoaortic denervation

The experiments reported here were designed to examine whether angiotensin II (AII) receptors in the rat solitary-vagal area (SVA) are associated with the neuronal components of the baroreceptor reflex. AII receptors were characterized both in membrane preparations from the rat brainstem and by in vitro autoradiography using the radiolabeled AII antagonist [125I]Sar1,Ile8-AII([ 125I]SI-AII). Saturation analysis of [125I]SI-AII binding to membrane preparations from rat brainstem indicated binding to two high affinity sites (Kd1 0.32 nM and Bmax1 5.10 fmol/mg protein, Kd2 0.99 nM and Bmax2 7.94 fmol/mg protein). The rank order competition by unlabeled angiotensin peptides (SI-AII greater than AII greater than AIII greater than AI) in both membrane preparations and by quantitative autoradiography was consistent with the labeling of the brain AII receptor. Autoradiography of the [125I]SI-AII binding in sections through the SVA revealed that the nucleus tractus solitarius (NTS) and the dorsal motor nucleus of the vagus (DMV) were heavily labeled. Bilateral sinoartic denervation, which disrupts primary baroreceptor afferents, resulted in a small decrease in [125I]SI-AII binding in the rostral and intermediate NTS and DMV. Unilateral nodose ganglionectomy, which disrupts completely the vagal afferent input to the NTS and produces retrograde degeneration of the vagal efferent neurons in the DMV, resulted in a marked decrease in [125I]SI-AII binding at all levels of the ipsilateral NTS and 56% decrease within the ipsilateral DMV. These results indicate that AII receptors within the SVA are distributed heterogeneously, with a large portion associated with vagal afferent fibers in the NTS and vagal efferent neurons of the DMV, and a small but significant portion associated with baroreceptor afferents. The majority of AII receptors in the NTS, however, were not affected by these surgical interventions and therefore appear to be located on intrinsic interneurons or non-vagal afferents in the NTS.     

Regional Distribution of Putative NPY Y1 Receptors and Neurons Expressing Y1 mRNA in Forebrain Areas of the Rat Central Nervous System

Using monoiodinated radioligands of peptide YY (PYY), and the recently introduced neuropeptide Y (NPY) analogue [Leu³¹,Pro³⁴]NPY, receptor binding sites of the Y₁ and Y₂ type were localized in the rat brain by quantitative in vitro autoradiography. The binding specificity and affinity of both radiolabeled ligands were analysed by ligand binding studies employing rat brain membrane homogenates from cerebral cortex and hippocampus. Using in situ hybridization histochemistry, the regional distribution and cellular localization of mRNA encoding the Y₁ receptor were investigated in rat brain sections and compared to the distribution of Y₁-specific binding sites. PYY binds to both Y₁ and Y₂ receptors, while long C-terminal fragments such as NPY^13–36 and NPY^16–36 bind preferentially to Y₂ receptors. [Leu³¹, Pro³⁴]NPY is a specific agonist for Y₁ receptors. Highest densities of [¹²⁵I]PYY binding sites were found in the cerebral cortex, the thalamus, the lateral septum, the hippocampus and the mesencephalic dopaminergic areas. In order to block putative Y₂ receptors, a series of [¹²⁵I]PYY binding experiments was performed in the presence of NPY^13–36 (1 μM), a Y₂ preferring C-terminal fragment. High densities of binding sites remained present in the cerebral cortex, the thalamus and the medial mammillary nucleus when NPY^13–36 was present in the incubation medium. Furthermore, these areas were highly enriched with [¹²⁵l][Leu³¹, Pro³⁴]NPY binding sites. In contrast, the hippocampal complex had its binding capacity reduced by -50%, while the lateral septum and mesencephalic dopaminergic areas had their binding capacities reduced even further. Linear regression analysis showed a high degree of correspondence between [¹²⁵l][Leu³¹, Pro³⁴]NPY binding and that obtained with [¹²⁵I]PYY in the presence of 1 μM NPY^13–36, suggesting that the two independent approaches to visualizing Y₁ binding sites are comparable. In situ hybridization histochemistry revealed high levels of Y₁ mRNA in the granular cell layer of the hippocampal dentate gyrus, several thalamic nuclei and the hypothalamic arcuate nucleus. Moderate levels of Y₁ mRNA were seen in the frontoparietal cortex, several thalamic nuclei, the hippocampal pyramidal layers, the subiculum, the olfactory tubercle, the claustrum and a number of hypothalamic nuclei. The mesencephalon, the amygdala and most basal ganglia showed very low levels of hybridization. The present study further clarifies the anatomical distribution of multiple NPY binding sites within the central nervous system of the rat, and extends earlier suggestions that Y₁ and Y₂ receptor types are present within the central nervous system.     

Region- and stage-specific patterns of melanocortin receptor ontogeny in rat central nervous system,cranial nerve ganglia and sympathetic ganglia

Observations on developmental actions of melanotropic peptides in nervous system have been difficult to interpret in the absence of data on receptor ontogeny. We investigated binding of [¹²⁵I]Nle⁴,d-Phe⁷-α-MSH ([¹²⁵I]NDP) in developing Long Evans rats from gestational day (E) 13 by quantitative autoradiography. Regional [¹²⁵I]NDP binding characteristics were assessed by competition experiments in early postnatal brain. The study revealed region- and stage-specific, often transient ontogenetic patterns. Sympathetic ganglia exhibit high [¹²⁵I]NDP binding from E13, with a peak in superior cervical ganglion at E16–E18. The first central [¹²⁵I]NDP binding sites transiently appear in parts of thalamus between E13 and E15. The early fetal period is characterized by prominent peaks of receptor density in somatosensory and viscerosensory nuclei (trigeminal sensory nuclei, solitary tract nucleus), paralleled by receptor expression in 5th, 7th, 9th and 10th cranial nerve ganglia. During late fetal life, receptor density peaks in dorsal motor nucleus of vagus and inferior olive; binding sites transiently appear in cerebellum. Caudate-putamen, nucleus accumbens, olfactory tubercle and septohippocampal nucleus show a high perinatal maximum. Starting with late fetal piriform cortex, [¹²⁵I]NDP binding peaks sequentially in cerebral cortical areas, with highest levels in entorhinal cortex. Preoptic, septal, hypothalamic and amygdaloid areas known for elevated receptor densities in adulthood, exhibit a slow, peri- and postnatal receptor ontogeny. Temporal relations to regional developmental processes support the idea of a role of melanocortins during ontogeny.     

Central and peripheral vagal transport of cholecystokinin binding sites occurs in afferent fibers

The effects of various vagal lesions on cholecystokinin (CCK) binding sites in the nucleus tractus solitarii (NTS) and area postrema (AP) and the peripheral transport of CCK binding sites in the cervical vagus were examined in rats by in vitro autoradiography with [125I]CCK-8. Unilateral supraganglionic, but not subdiaphragmatic vagotomy significantly reduced CCK binding in the ipsilateral NTS. Specific unilateral afferent, but not efferent, vagal rootlet transections also significantly reduced NTS CCK binding ipsilateral to the transections. None of the vagal lesions altered CCK binding in the AP. Infraganglionic but not supraganglionic vagotomy eliminated the peripheral transport of vagal CCK binding sites. Together these results demonstrate that CCK receptors in the NTS are located on vagal afferent terminals, that CCK receptors in the AP are likely postsynaptic to a vagal afferent input and that the peripheral and central transport of vagal CCK binding sites occurs in afferent fibers.