Phoenixin: A candidate pruritogen in the mouse.

Phoenixin (PNX) is a 14-amino acid amidated peptide (PNX-14) or an N-terminal extended 20-residue amidated peptide (PNX-20) recently identified in neural and non-neural tissue. Mass spectrometry analysis identified a major peak corresponding to PNX-14, with negligible PNX-20, in mouse spinal cord extracts. Using a previously characterized antiserum that recognized both PNX-14 and PNX-20, PNX-immunoreactivity (irPNX) was detected in a population of dorsal root ganglion (DRG) cells and in cell processes densely distributed to the superficial layers of the dorsal horn; irPNX cell processes were also detected in the skin. The retrograde tracer, Fluorogold, injected subcutaneously (s.c.) to the back of the cervical and thoracic spinal cord of mice, labeled a population of DRG, some of which were also irPNX. PNX-14 (2, 4 and 8 mg/kg) injected s.c.to the nape of the neck provoked dose-dependent repetitive scratching bouts directed to the back of the neck with the hindpaws. The number of scratching bouts varied from 16 to 95 in 30 min, commencing within 5 min post-injection and lasted 10-15 min. Pretreatment of mice at -20 min with nalfurafine (20 µg/kg, s.c.), the kappa opioid receptor agonist, significantly reduced the number of bouts induced by PNX-14 (4 mg/kg) compared with that of saline-pretreated mice. Our results suggest that the peptide, PNX-14, serves as one of the endogenous signal molecules transducing itch sensation in the mouse.

Phoenixin: a novel peptide in rodent sensory ganglia.

Phoenixin-14 amide, herein referred to as phoenixin, is a newly identified peptide from the rat brain. Using a previously characterized rabbit polyclonal antiserum against phoenixin, enzyme-immunoassay detected a high level (>4.5 ng/g tissue) of phoenixin-immunoreactivity (irPNX) in the rat spinal cords. Immunohistochemical studies revealed irPNX in networks of cell processes in the superficial dorsal horn, spinal trigeminal tract and nucleus of the solitary tract; and in a population of dorsal root, trigeminal and nodose ganglion cells. The pattern of distribution of irPNX in the superficial layers of the dorsal horn was similar to that of substance P immunoreactivity (irSP). Double-labeling the dorsal root ganglion sections showed that irPNX and irSP express in different populations of ganglion cells. In awake mice, intrathecal injection of phoenixin (1 or 5 µg) did not significantly affect the tail-flick latency as compared to that in animals injected with artificial cerebrospinal fluid (aCSF). Intrathecal administration of phoenixin (0.5, 1.25 or 2.5 µg) significantly reduced the number of writhes elicited by intraperitoneal injection of acetic acid (0.6%, 0.3 ml/30 g) as compared to that in mice injected with aCSF. While not affecting the tail-flick latency, phoenixin antiserum (1:100) injected intrathecally 10 min prior to the intraperitoneal injection of acetic acid significantly increased the number of writhes as compared to mice pre-treated with normal rabbit serum. Intrathecal injection of non-amidated phoenixin (2.5 µg) did not significantly alter the number of writhes evoked by acetic acid. Our result shows that phoenixin is expressed in sensory neurons of the dorsal root, nodose and trigeminal ganglia, the amidated peptide is bioactive, and exogenously administered phoenixin may preferentially suppress visceral as opposed to thermal pain.

Neuronostatin is co-expressed with somatostatin and mobilizes calcium in cultured rat hypothalamic neurons.

Neuronostatin (NST) is a newly identified peptide of 13-amino acids encoded by the somatostatin (SST) gene. Using a rabbit polyclonal antiserum against the human NST, neuronostatin-immunoreactive (irNST) cells comparable in number and intensity to somatostatin immunoreactive (irSST) cells were detected in the hypothalamic periventricular nucleus. Fewer and/or less intensely labeled irNST cells were noted in other regions such as the hippocampus, cortex, amygdala, and cerebellum. Double-labeling hypothalamic sections with NST- and SST-antiserum revealed an extensive overlapping of irNST and irSST cells in the periventricular nucleus. Pre-absorption of the NST-antiserum with NST (1 microg/ml) but not with SST (1 microg/ml) abrogated irNST and vice versa. The activity of NST on dissociated and cultured hypothalamic neurons was assessed by the Ca(2+) imaging method. NST (10, 100, 1000 nM) concentration-dependently elevated intracellular Ca(2+) concentrations [Ca(2+)](i) in a population of hypothalamic neurons with two distinct profiles: (1) a fast and transitory increase in [Ca(2+)](i), and (2) an oscillatory response. Whereas, SST (100 nM) reduced the basal [Ca(2+)](i) in 21 of 61 hypothalamic neurons examined; an increase was not observed in any of the cells. Optical imaging with a slow-responding voltage sensitive dye DiBAC(4)(3) showed that NST (100 nM) depolarized or hyperpolarized; whereas, SST (100 nM) hyperpolarized a population of hypothalamic neurons. The result shows that NST and SST, though derived from the same precursor protein, exert different calcium mobilizing effects on cultured rat hypothalamic neurons, resulting in diverse cellular activities.

Amylin suppresses acetic acid-induced visceral pain and spinal c-fos expression in the mouse.

Amylin is a member of calcitonin or calcitonin gene-related peptide (CGRP) family. Immunohistochemical study revealed a dense network of amylin-immunoreactive (irAMY) cell processes in the superficial dorsal horn of the mice. Numerous dorsal root ganglion (DRG) and trigeminal ganglion cells expressed moderate to strong irAMY. Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed amylin receptor mRNA in the mouse spinal cord, brain stem, cortex, hypothalamus and hippocampus. The nociceptive or antinociceptive effects of amylin were evaluated in the acetic acid-induced writhing test. Amylin (0.1, 0.5 and 1 mg/kg, intraperitoneally (i.p.) or 1-10 microg, intrathecally (i.t.)) reduced the number of writhes in a dose-dependent manner. Pretreatment of the mice with the amylin receptor antagonist salmon calcitonin (8-32), either by i.p. or i.t., antagonized the effect of amylin on acetic acid-induced writhing test. Locomotor activity was not significantly modified by amylin injected either i.p. (0.01-1 mg/kg) or i.t. (1-10 microg). Measurement of c-fos mRNA by RT-PCR or proteins by Western blot showed that the levels were upregulated in the spinal cord of mice injected with acetic acid and the increase was attenuated by pretreatment with amylin (10 microg, i.t.). Collectively, our result demonstrates that irAMY is expressed in DRG neurons with their cell processes projecting to the superficial layers of the dorsal horn, and that the peptide by interacting with amylin receptors in the spinal cord may be antinociceptive.

Nalfurafine prevents 5'-guanidinonaltrindole- and compound 48/80-induced spinal c-fos expression and attenuates 5'-guanidinonaltrindole-elicited scratching behavior in mice.

The aims of the present study were to establish if nalfurafine, a kappa opioid agonist, inhibits compulsive scratching in mice elicited by the s.c. administration (behind the neck) of 5'-guanidinonaltrindole (GNTI), a kappa opioid antagonist; to assess if nalfurafine prevents c-fos expression provoked by GNTI or compound 48/80, two chemically diverse pruritogens; and to distinguish on the basis of neuroanatomy, those neurons in the brainstem activated by either GNTI-induced itch or formalin-induced pain (both compounds given s.c. to the right cheek). Pretreatment of mice with nalfurafine (0.001-0.03 mg/kg s.c.) attenuated GNTI (0.3 mg/kg)-evoked scratching dose-dependently. A standard antiscratch dose of nalfurafine (0.02 mg/kg) had no marked effect on the spontaneous locomotion of mice. Tolerance did not develop to the antiscratch activity of nalfurafine. Both GNTI and compound 48/80 provoked c-fos expression on the lateral side of the superficial layer of the dorsal horn of the cervical spinal cord and pretreating mice with nalfurafine inhibited c-fos expression induced by both pruritogens. In contrast to formalin, GNTI did not induce c-fos expression in the trigeminal nucleus suggesting that pain and itch sensations are projected differently along the sensory trigeminal pathway. Our data indicate that the kappa opioid system is involved, at least in part, in the pathogenesis of itch; and that nalfurafine attenuates excessive scratching and prevents scratch-induced neuronal activity at the spinal level. On the basis of our results, nalfurafine holds promise as a potentially useful antipruritic in human conditions involving itch.

Nicotinic acid adenine dinucleotide phosphate enhances quantal neurosecretion at the frog neuromuscular junction: possible action on synaptic vesicles in the releasable pool.

Inositol 1,4,5-trisphosphate (IP(3)) and cyclic adenosine diphosphate-ribose (cADPR) are second messengers that enhance neurosecretion by inducing Ca(2+) release from smooth endoplasmic reticulum (SER). The putative intracellular messenger, nicotinic acid adenine dinucleotide phosphate (NAADP), releases Ca(2+) from stores that are distinct from SER. Evidence is presented here that NAADP causes a concentration-dependent increase in quantal output that is associated with an increase in probability of transmitter release at the frog neuromuscular junction. This effect is mimicked by A23187, a Ca ionophore that promotes Ca(2+) entry at the plasmalemma. The response to NAADP is potentiated by IP(3) but antagonized by cADPR. Thapsigargin completely blocks IP(3) and cADPR responses and decreases but does not prevent the response to NAADP. We conclude that NAADP, whose receptors are widely distributed in the brain, enhances neurosecretion by releasing Ca(2+) from an internal store near the plasmalemma, possibly from synaptic vesicles in the releasable pool. These data also support the hypothesis of a two-pool model for Ca(2+) oscillations at the presynaptic site.

Orexins: a role in medullary sympathetic outflow.

Orexin A and B, also known as hypocretin 1 and 2, are two recently isolated hypothalamic peptides. As orexin-containing neurons are strategically located in the lateral hypothalamus, which has long been suspected to play an important role in feeding behaviors, initial studies were focused on the involvement of orexins in positive food intake and energy metabolism. Recent studies implicate a more diverse biological role of orexins, which can be manifested at different level of the neuraxis. For example, canine narcolepsy, a disorder with close phenotypic similarity to human narcolepsy, is caused by a mutation of hypocretin receptor 2 gene. Results from our immunohistochemical and functional studies, which will be summarized here, suggest that the peptide acting on neurons in the rostral ventrolateral medulla augment sympathoexcitatory outflow to the spinal cord. This finding is discussed in the context of increased sympathetic activity frequently associated with obesity.

G protein activation by endomorphins in the mouse periaqueductal gray matter.

The midbrain periaqueductal gray matter (PAG) is an important brain region for the coordination of mu-opioid-induced pharmacological actions. The present study was designed to determine whether newly isolated mu-opioid peptide endomorphins can activate G proteins through mu-opioid receptors in the PAG by monitoring the binding to membranes of the non-hydrolyzable analog of GTP, guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS). An autoradiographic [(35)S]GTPgammaS binding study showed that both endomorphin-1 and -2 produced similar anatomical distributions of activated G proteins in the mouse midbrain region. In the mouse PAG, endomorphin-1 and -2 at concentrations from 0.001 to 10 microM increased [(35)S]GTPgammaS binding in a concentration-dependent manner and reached a maximal stimulation of 74.6+/-3.8 and 72.3+/-4.0%, respectively, at 10 microM. In contrast, the synthetic selective mu-opioid receptor agonist [D-Ala(2),NHPhe(4), Gly-ol]enkephalin (DAMGO) had a much greater efficacy and produced a 112.6+/-5.1% increase of the maximal stimulation. The receptor specificity of endomorphin-stimulated [(35)S]GTPgammaS binding was verified by coincubating membranes with endomorphins in the presence of specific mu-, delta- or kappa-opioid receptor antagonists. Coincubation with selective mu-opioid receptor antagonists beta-funaltrexamine or D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Phe-Thr-NH(2) (CTOP) blocked both endomorphin-1 and-2-stimulated [(35)S]GTPgammaS binding. In contrast, neither delta- nor kappa-opioid receptor antagonist had any effect on the [(35)S]GTPgammaS binding stimulated by either endomorphin-1 or -2. These findings indicate that both endomorphin-1 and -2 increase [(35)S]GTPgammaS binding by selectively stimulating mu-opioid receptors with intrinsic activity less than that of DAMGO and suggest that these new endogenous ligands might be partial agonists for mu-opioid receptors in the mouse PAG.

Identification of the G-protein-coupled ORL1 receptor in the mouse spinal cord by [35S]-GTPgammaS binding and immunohistochemistry.

1 Although the ORL1 receptor is clearly located within the spinal cord, the functional signalling mechanism of the ORL1 receptor in the spinal cord has not been clearly documented. The present study was then to investigate the guanine nucleotide binding protein (G-protein) activation mediated through by the ORL1 receptor in the mouse spinal cord, measuring the modulation of guanosine-5'-o-(3-[35S]-thio) triphosphate ([35S]-GTPgammaS) binding by the putative endogenous ligand nociceptin, also referred as orphanin FQ. We also studied the anatomical distribution of nociceptin-like immunoreactivity and nociceptin-stimulated [35S]-GTPgammaS autoradiography in the spinal cord. 2 Immunohistochemical staining of mouse spinal cord sections revealed a dense plexus of nociceptin-like immunoreactive fibres in the superficial layers of the dorsal horn throughout the entire length of the spinal cord. In addition, networks of fibres were seen projecting from the lateral border of the dorsal horn to the lateral grey matter and around the central canal. 3 In vitro [35S]-GTPgammaS autoradiography showed high levels of nociceptin-stimulated [35S]-GTPgammaS binding in the superficial layers of the mouse dorsal horn and around the central canal, corresponding to the areas where nociceptin-like immunoreactive fibres were concentrated. 4 In [35S]-GTPgammaS membrane assay, nociceptin increased [35S]-GTPgammaS binding of mouse spinal cord membranes in a concentration-dependent and saturable manner, affording maximal stimulation of 64.1+/-2.4%. This effect was markedly inhibited by the specific ORL1 receptor antagonist [Phe1Psi (CH2-NH) Gly2] nociceptin (1 - 13) NH2. None of the mu-, delta-, and kappa-opioid and other G-protein-coupled receptor antagonists had a significant effect on basal or nociceptin-stimulated [35S]-GTPgammaS binding. 5 These findings suggest that nociceptin-containing fibres terminate in the superficial layers of the dorsal horn and the central canal and that nociceptin released in these areas may selectively stimulate the ORL1 receptor to activate G-protein. Furthermore, the unique pattern of G-protein activation in the present study provide additional evidence that nociceptin is distinct from the mu-, delta- or kappa-opioid system.

Serotonin via 5-HT1B and 5-HT2B receptors stimulates anion secretion in the rat epididymal epithelium.

1. The short-circuit current (Isc) technique was used to study the role of 5-hydroxytryptamine (5-HT) in the regulation of anion secretion in cultured rat cauda epididymal epithelia. 2. 5-HT, the 5-HT1B-selective agonist 5-nonyloxytryptamine (5-NOT) and the 5-HT2B-selective agonist alpha-methyl-5-hydroxytryptamine (alpha-methyl-5-HT) added basolaterally stimulated Isc in a dose-dependent manner with EC50 values of 0.4, 20 and 0.3 microM, respectively. No other agonists for 5-HT receptors had any effect. 3. The pattern of responses to 5-HT was biphasic. Pretreating the tissues with the 5-HT1B-selective antagonist isamoltane (200 microM) and the 5-HT2B-selective antagonist rauwolscine (200 microM) inhibited the rapid transient phase by 55 and 45 %, whereas the sustained phase could only be blocked by rauwolscine. 4. Removal of chloride or bicarbonate or both from the normal Krebs-Henseleit solution reduced the responses to 5-HT, 5-NOT and alpha-methyl-5-HT to varying degrees. The results suggest that 5-HT1B- and 5-HT2B-mediated responses were mainly due to chloride and bicarbonate secretion, respectively. 5. Manipulation of the cAMP and Ca2+ signal transduction pathways with chemical agents provided evidence that the responses to 5-HT were mediated through cAMP. 6. Piroxicam pretreatment abolished the Isc response to alpha-methyl-5-HT but not to 5-NOT, indicating that the 5-HT2B-mediated response, but not the 5-HT1B-mediated response, is dependent on prostaglandin synthesis. 7. Immunohistochemical studies showed that 5-HT-like immunoreactivity was detected in nerve fibres and in small granular cells surrounding the epididymal tubules. 8. It is suggested that the 5-HT released from serotonergic nerve endings and/or from mast cells regulates electrolyte and fluid secretion in the epididymis.

Local regulation of epididymal anion secretion by pituitary adenylate cyclase-activating polypeptide.

The present study investigated the role of pituitary adenylate cyclase-activating polypeptide (PACAP) in the regulation of electrogenic anion secretion by the rat cauda epididymal epithelium. PACAP38, which has been shown to affect secretory function in various exocrine and endocrine tissues, gave rise to a concentration-dependent increase in the short-circuit current (Isc). The PACAP38 effect was restricted to the apical aspect of the epididymal cells. The Isc response to PACAP38 was abolished in Cl-(-)free solution and completely inhibited by the Cl- channel blocker, diphenylamine-dicarboxylic acid. The Isc response to PACAP38 was also suppressed by pretreatment of the cells with the adenylate cyclase inhibitor, MDL12330A. The localization of PACAP38 was further investigated using an immunohistochemical technique. PACAP38 immunoreactivity was observed in the cauda epididymal tubules as well as in the cultured epithelium, indicating its epithelial origin. The present results suggest that Cl- secretion in the epididymis may be regulated by PACAP38, which could be locally synthesized and released by the epithelial cells, in a paracrine or autocrine fashion.

Potentiation of NMDA currents by pituitary adenylate cyclase activating polypeptide in neonatal rat sympathetic preganglionic neurons.

Whole cell patch-clamp recordings were made from sympathetic preganglionic neurons (SPNs) in the intermediolateral cell column of thoracolumbar spinal cord slices of 12- to 16-day-old rats, and the effects of pituitary adenylate cyclase activating polypeptide (PACAP)-38 on N-methyl-D-aspartate (NMDA)- and kainate (KA)-induced inward currents were examined. PACAP, in concentrations (10-30 nM) that caused no significant change of holding currents, reversibly increased NMDA-induced currents but not KA-induced currents. At higher concentrations (>30 nM), the peptide produced a sustained inward current. The potentiating effect of PACAP was nullified by prior incubation of the slices with the adenylate cyclase inhibitor MDL-12,330A (25 microM). Further, superfusing the slices with the membrane-permeable cyclic AMP analogue N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (100-300 microM) in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (700 microM) increased the NMDA currents. This result suggests that PACAP selectively increases NMDA-receptor-mediated responses in the rat SPNs, probably via a cyclic-AMP-dependent mechanism, providing evidence that the peptide may be involved in synaptic plasticity.

Excitatory action of pituitary adenylate cyclase activating polypeptide on rat sympathetic preganglionic neurons in vivo and in vitro.

In vivo and in vitro experiments were undertaken to evaluate the effects of pituitary adenylate cyclase activating polypeptide-38 (PACAP-38) on rat sympathetic preganglionic neurons (SPNs). Intrathecal injection of PACAP-38 (0.1-1 nmol) via an implanted cannula to the T2-T3 segments of urethane-anesthetized adult rats caused a dose-dependent increase of mean arterial blood pressure from minutes to over 1 h. The pressor response was not antagonized by prior injection of the PACAP type II receptor antagonist PACAP6-38 (0.5 nmol), but was significantly attenuated by prior intravenous administration of phentolamine (1 mg/kg). As a positive control, intrathecal injection of glutamate (1 micromol) and substance P (SP, 5 nmol) caused a short- and long-lasting pressor response. Vasoactive intestinal polypeptide (VIP, 1 nmol) had no significant pressor effect. In the second series of experiments, whole-cell patch recordings were made from antidromically identified SPNs of immature (12-16-day-old) rat thoracolumbar spinal cord slices. Applied to the spinal cord slices by superfusion, PACAP-38 (10-30 nM) caused intense neuronal discharges with or without a long-lasting membrane depolarization. The depolarization was not prevented by superfusing the slices with tetrodotoxin (0.3 microM) or low Ca2+ (0.25 mM) solution, indicating that PACAP-38 directly depolarized the SPNs. The depolarization was insensitive to the type II PACAP receptor antagonist PACAP6-38. Collectively, these results provide evidence that PACAP-38 exerts a potent and long-lasting excitatory effect on SPNs, leading to an increase of spinal sympathetic outflow and one of the consequences of which is an elevation of blood pressure.

Distribution of origin of nitric oxide synthase-immunoreactive nerve fibers in the rat epididymis.

Distribution of neuronal nitric oxide synthase-immunoreactive (nNOS-IR) nerve fibers and somata in the rat epididymis and major pelvic ganglia was studied by immunohistochemical methods. In the epididymis, the supply of nNOS-IR fibers was highest in the cauda and became progressively fewer toward the caput. In the cauda and corpus, nNOS-IR fibers were distributed throughout the subepithelial tissues and around the epithelial. The pattern of distribution of vasoactive intestinal polypeptide (VIP)- and tyrosine hydroxylase (TH)-immunoreactive fibers in the epididymis was similar but the latter was generally more numerous in a given region as compared to that of nNOS-IR fibers. A population of neurons in the major pelvic ganglia were nNOS-IR-, TH- or VIP-IR. Double-labeling studies revealed that few neurons in the major pelvic ganglia contained both nNOS-IR and TH-IR. Whereas nNOS-IR and VIP-IR appeared to co-localize in the same population of the pelvic ganglion cells. Similarly, nNOS-IR fibers in the epididymis were mostly VIP-positive and TH-negative. Unilateral injection of the fluorescent tracer Fluorogold into the junction between the vas deferens and the cauda labeled a population of neurons in the right and left major pelvic ganglia, some of which were also nNOS-IR. A small number of dorsal root ganglion cells contained Fluorogold and very few expressed NOS-IR. It may be concluded that nNOS-IR nerve fibers in the rat epididymis arise mainly from neurons in the major pelvic ganglia the major of which express VIP-IR but not TH-IR. The extensive supply of nNOS-immunoreactive fibers around the epithelium and throughout the subepithelial tissues suggests that NO may be closely associated with smooth muscle contraction.

Hyperalgesia induced by pituitary adenylate cyclase-activating polypeptide in the mouse spinal cord.

The aim of the present study was to evaluate the distribution of pituitary adenylate cyclase-activating polypeptide (PACAP)-like immunoreactivity in the mouse spinal cord using an antibody against PACAP38 and to determine the behavioral profile, particularly with respect to hyperalgesia, of PACAP38 given intrathecally (i.t.) in the mouse. Immunoreactivity to PACAP38 was detected in numerous nerve fibers in the superficial layers of the dorsal horn of cervical, thoracic, lumbar and sacral segments and a few fibers extended into the deeper layers of the spinal cord. In addition, PACAP-like immunoreactivity were seen in the intermediolateral cell column of the thoracic and sacral segments. In behavioral studies, PACAP38 (0.05-0.5 microgram) produced a dose-dependent decrease of the tail-flick latency when given i.t. in the mouse. At higher doses (1-10 micrograms), PACAP38 given i.t. elicited biting and scratching behaviors lasting 10-20 min after the injection. PACAP at high doses (1-10 micrograms) also produced licking at tail, paw and penis and intense grooming behaviors immediately after the i.t. injection. Similar to substance P, these behaviors produced by PACAP can be considered as pain-like syndrome. These findings suggest that PACAP may be a sensory neurotransmitter involved in nociceptive signalling in the mouse spinal cord.

Pituitary adenylate cyclase activating polypeptide immunoreactivity in the rat spinal cord and medulla: implication of sensory and autonomic functions.

Immunoreactivity to pituitary adenylate cyclase activating polypeptide-38 was detected in numerous nerve fibres in layers I and II of the dorsal horn of the rat and some of these fibres extended into the deeper layers of all segments of the spinal cord. Immunoreactivity was also detected in the lateral funiculus projecting into the intermediolateral cell column of the lower cervical and thoracic segments and in the lateral pathway terminating in the intermediate gray area of the lower lumbar and sacral segments. Neurons in the lateral horn area were not immunoreactive nor were the ventral horn motoneurons. In the medulla, numerous immunoreactive fibres were observed in the spinal trigeminal tract and superficial layers of the caudal spinal trigeminal nucleus but few in the interpolar spinal trigeminal nucleus. A prominent immunoreactive nerve bundle emanated from the caudal spinal trigeminal nucleus and projected into the solitary tract. A dense network of immunoreactive neurons and fibres was present in the nucleus raphe obscurus, lateral reticular nucleus and parvocellular lateral reticular nucleus. Immunoreactive fibres could also be detected in the solitary tract and area postrema. Labelled somata were occasionally noted in various subnuclei of the nucleus of the solitary tract and nucleus raphe pallidus. In addition, a small number of positive neurons were detected in an area between the lateral reticular nucleus and inferior olive and near the ventral surface of the medulla (parapyramidal region). A few weakly-labelled cells were occasionally seen in the dorsal motor nucleus of vagus. A population of neurons in the trigeminal, nodose and dorsal root ganglia from all segments of the spinal cord displayed low to intense immunoreactivity. The presence of immunoreactivity in nodose and dorsal root ganglia, dorsal horn, spinal autonomic nuclei, solitary tract and in certain areas of the medulla suggests that this peptide may participate in a variety of sensory and autonomic functions.

The synaptic structure of PACAP immunoreactive axons in the intermediolateral nucleus of the rat.

Immuno-electronmicroscopic studies were performed to detect the presence and features of synaptic contacts between pituitary adenylate cyclase activating polypeptide immunoreactive (PACAP-ir) axons and cholera toxin B-horseradish peroxidase labeled preganglionic sympathetic neurons (PSNs) in the intermediolateral nucleus of the rat thoracic spinal cord. PACAP-ir axon varicosities, which contained small clear and large core synaptic vesicles, were found to form asymmetric type of synaptic contacts with dendrites and infrequently with somata of labeled preganglionic neurons. The present study provides ultrastructural evidence of PACAP-ir synaptic contacts with PSNs, raising the possibility that the peptide may function as a transmitter/modulator to these neurons.

Pituitary adenylate cyclase activating polypeptide-immunoreactivity in human spinal cord and dorsal root ganglia.

Immunohistochemical studies using an antibody against pituitary adenylate cyclase activating polypeptide-38 (PACAP) were performed on spinal cords and dorsal root ganglia harvested from two human cadavers. PACAP-like immunoreactivity (PACAP-LI) was detected in nerve fibers of the superficial layers of the dorsal horn, a few of which extended into the deeper laminae and as far as the ventral horn. At the thoracic segments, additional PACAP-LI nerve fibers were seen in the lateral funiculus projecting into the intermediolateral cell column. Dorsal root ganglia contained numerous PACAP-LI cell bodies of varying intensity. As a control, immunoreactivity to calcitonin gene-related peptide (CGRP) and substance P(SP) was also studied and found to be in nerve fibers of the substantia gelatinosa of the dorsal horn and in dorsal root ganglion cells. These results show that the pattern of distribution of PACAP-LI in the human spinal cord and dorsal root ganglia is similar to that of rodents and further suggest that PACAP may participate in sensory and autonomic functions.

Pituitary adenylate cyclase activating polypeptide-immunoreactive sensory neurons innervate rat adrenal medulla.

Rat adrenal chromaffin cells were invested by a dense network of nerve fibers immunoreactive to pituitary adenylate cyclase activating polypeptide-38 (PACAP-IR). Immunohistochemical studies demonstrated the presence of PACAP-IR in nodose and dorsal root ganglion cells, but not in neurons of the intermediolateral cell column and other autonomic nuclei of the thoracic and upper lumbar spinal cord. Somata of the T7 to T12 paravertebral ganglia were PACAP-negative. A few lightly labeled neurons were occasionally noted in the dorsal motor nucleus of the vagus. Injection of the retrograde tracer Fluorogold into the left adrenal medulla 3 days prior to sacrifice resulted in the labeling of a population of neurons in the ipsilateral spinal cord intermediolateral cell column (T1 to L1), ipsilateral and contralateral nodose ganglia and ipsilateral dorsal root ganglia from T7 to T10 inclusive. A small number of lightly labeled somata was occasionally noted in the dorsal motor nucleus of the vagus. Combined retrograde tracing and PACAP immunohistochemistry showed that a population of Fluorogold-containing nodose and dorsal root ganglion cells were also PACAP-positive. Pre-treatment of the rats with capsaicin caused a marked reduction of the PACAP-IR in the adrenal gland as well as in the superficial layers of the dorsal horn and caudal spinal trigeminal nucleus. These findings, in conjunction with the apparent absence of PACAP-IR in spinal sympathetic preganglionic neurons, sympathetic postganglionic neurons, and dorsal motor nucleus of the vagus, raise the possibility that PACAP-IR fibers observed in the adrenal medulla are primarily sensory in origin. As a corollary, catecholamine secretion from chromaffin cells may be modulated by the peptidergic sensory afferents in addition to the cholinergic sympathetic preganglionic nerve fibers.

c-fos expression as a marker of central cardiovascular neurons.

Immunohistochemical detection of Fos, the protein product of the immediate-early gene c-fos, was evaluated as a functional marker of central neurons sensitive to a change of blood pressure/blood volume. Controlled hemorrhage and infusion of the hypotensive agent nitroprusside or hydralazine induced the appearance of Fos-immunoreactivity (Fos-IR) in several prominent groups of central neurons: the piriform cortex, bed nucleus of the stria terminalis, islands of Calleja, subfornical organ, central nucleus of the amygdala, parabrachial nucleus, supraoptic and paraventricular nuclei, pontine A5, locus ceruleus, ventrolateral medulla, the nucleus of the solitary tract, area postrema, and intermediolateral cell column in the spinal cord. Elevation of blood pressure by infusion of phenylephrine caused the appearance of Fos-IR in fewer groups of neurons: the bed nucleus of the stria terminalis, central nucleus of the amygdala, parabrachial nucleus, the nucleus of the solitary tract and area postrema. The differential distribution of Fos neurons in hypotensive versus hypertensive animals underscores the potential application of Fos as a metabolic marker in identifying a network of neurons responding to a specific cardiovascular challenge. Further, simultaneous characterization of the transmitter phenotype of Fos-containing neurons offers an additional advantage of this method over other conventional tract-tracing techniques.