PACAP and its role in primary headaches.

Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide implicated in a wide range of functions, such as nociception and in primary headaches. Regarding its localization, PACAP has been observed in the sensory trigeminal ganglion (TG), in the parasympathetic sphenopalatine (SPG) and otic ganglia (OTG), and in the brainstem trigeminocervical complex. Immunohistochemistry has shown PACAP-38 in numerous cell bodies of SPG/OTG, co-stored with vasoactive intestinal peptide (VIP), nitric oxide synthase (NOS) and, to a minor degree, with choline acetyltransferase. PACAP has in addition been found in a subpopulation of calcitonin gene-related peptide (CGRP)-immunoreactive cells in the trigeminal system. The PACAP/VIP receptors (PAC1, VPAC1, and VPAC2) are present in sensory neurons and in vascular smooth muscle related to the trigeminovascular system. It is postulated that PACAP is involved in nociception. In support, abolishment of PACAP synthesis or reception leads to diminished pain responses, whereas systemic PACAP-38 infusion triggers pain behavior in animals and delayed migraine-like attacks in migraine patients without marked vasodilatory effects. In addition, increased plasma levels have been documented in acute migraine attacks and in cluster headache, in accordance with findings in experimental models of trigeminal activation. This suggest that the activation of the trigeminal system may result in elevated venous levels of PACAP, a change that can be reduced when headache is treated. The data presented in this review indicate that PACAP and its receptors may be promising targets for migraine therapeutics.

Neurochemical characterization of zinc transporter 3-like immunoreactive (ZnT3(+)) neurons in the intramural ganglia of the porcine duodenum.

The SLC30 family of divalent cation transporters is thought to be involved in the transport of zinc in a variety of cellular pathways. Zinc transporter 3 (ZnT3) is involved in the transport of zinc into synaptic vesicles or intracellular organelles. As the presence of ZnT3 immunoreactive neurons has recently been reported in both the central and peripheral nervous systems of the rat, the present study was aimed at disclosing the presence of a zinc-enriched neuron enteric population in the porcine duodenum to establish a preliminary insight into their neurochemical coding. Double- and triple-immunofluorescence labeling of the porcine duodenum for ZnT3 with the pan-neuronal marker (PGP 9.5), substance P, somatostatin, vasoactive intestinal peptide (VIP), nitric oxide synthase (NOS), leu-enkephalin, vesicular acetylcholine transporter (VAChT), neuropeptide Y, galanin (GAL), and calcitonin gene-related peptide were performed. Immunohistochemistry revealed that approximately 35, 43, and 48 % of all PGP9.5-postive neurons in the myenteric (MP), outer submucous (OSP), and inner submucous (ISP) plexuses, respectively, of the porcine duodenum were simultaneously ZnT3(+). In the present study, ZnT3(+) neurons coexpressed a broad spectrum of active substances, but co-localization patterns unique to the plexus were studied. In the ISP, all ZnT3(+) neurons were VAChT positive, and the largest populations among these cells formed ZnT3(+)/VAChT(+)/GAL(+) and ZnT3(+)/VAChT(+)/VIP(+) cells. In the OSP and MP, the numbers of ZnT3(+)/VAChT(+) neurons were two times smaller, and substantial subpopulations of ZnT3(+) neurons in both these plexuses formed ZnT3(+)/NOS(+) cells. The large population of ZnT3(+) neurons in the porcine duodenum and a broad spectrum of active substances which co-localize with this peptide suggest that ZnT3 takes part in the regulation of various processes in the gut both in normal physiology and during pathological processes.

IP3 type 1 receptors in the heart: their predominance in atrial walls with ganglion cells.

Previously we have shown that inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are abundantly expressed in the atria of rat hearts. Since arrangement of atria is very heterogeneous, in this work we focused on the precise localization of IP3 receptors in the left atrium, where the gene expression of the type 1 IP3R was the highest. The mRNA levels of the IP3 type 1 receptors in the left atrium, left ventricle and myocytes were determined using real-time polymerase chain reaction and Taqman probe. For precise localization, immunohistochemistry with the antibody against type 1 IP3Rs was performed. The mRNA of type 1 IP3 receptor was more than three times higher in the left atrium than in the left ventricle, as determined by real-time PCR. Expression of the type 1 IP3 receptor mRNA was higher in the atria, especially in parts containing cardiac ganglion cells. The atrial auricles, which are particularly free of ganglion cells, and the ventricles (wall of the right and left ventricle and ventricular septum) contained four to five times less IP3 receptors than atrial samples with ganglia. IP3R type 1 immunoreactivity detected by a confocal microscope attributed the most condensed signal on ganglionic cells, although light immunoreactivity was also seen in cardiomyocytes. These results show that type 1IP3 receptors predominate in intrinsic neuronal ganglia of cardiac atria.

Immunohistochemical characterization of neurons in the porcine ciliary ganglion.

The present study was aimed at disclosing the chemical coding of nerve structures in the porcine ciliary ganglion (CG) using immunohistochemical methods. The substances under investigation included markers of "classical" neurotransmitters, choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DbetaH) as well as neuropeptides, somatostatin (SOM), galanin (GAL), substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY). Immunoreactivity to ChAT and VAChT was found virtually in all the neuronal somata and in numerous intraganglionic, varicose nerve fibres which often formed basket-like formations around the nerve cell bodies. Many CG neurons contained immunoreactivity for SOM (46%) or GAL (29%). Interestingly, a small number (approx. 1%) of the cholinergic somata stained for TH but not for DbetaH; nevertheless, some extra- and intraganglionic nerve fibres displayed immunoreactivity for DbetaH or TH. The CG perikarya stained neither for vasoactive intestinal polypeptide (VIP) nor for neuropeptide Y (NPY), but some NPY- or VIP-positive nerve terminals were observed within nerve bundles distributed outside the ganglion. SP- and CGRP-immunoreactivity was found in some intraganglionic nerve fibres only. The present study revealed that the porcine CG consists of cholinergic neurons many of which contain SOM and GAL. Thus, it can be assumed that in the pig, these neuropeptides are involved, complementary to acetylocholine, in the parasympathetic postganglionic nerve pathway to structures of the eye including the ciliary and iris sphincter muscles.

Mass spectrometric analysis of single identified neurons of an insect.

The combination of retrograde labelling with dextran-tetramethylrhodamine and MALDI-TOF mass spectrometry was used to analyse for the first time the peptidome of a series of morphologically identified single neurosecretory cells of an insect. Eight postero-lateral cells of the metathoracic ganglion of the American cockroach, Periplaneta americana, were used to demonstrate that: (1) the complete dissection procedure can be documented and (2) the mass spectrometric analysis of the dissected somata results in highly reproducible mass spectra. In total, 21 FMRFamide-related peptides were detected in each of the postero-lateral cells which release their neurosecretions via thoracic perisympathetic organs. Direct analysis of these neurohemal organs confirmed the co-storage of FMRFamide-related peptides. Two additional abundant peptides from thoracic perisympathetic organs which were not detectable in the postero-lateral cells were characterized using ESI-Q-TOF MS/MS. De novo sequencing yielded two related peptides (FERL/IEamides) without any similarity with known peptide families of insects.

Evidence that urocortin is absent from neurons of the Edinger-Westphal nucleus in pigeons.

The Edinger-Westphal nucleus (EWN) is a central preganglionic parasympathetic cell group that gives rise to cholinergic input to the ciliary ganglion, thereby regulating several neurovegetative ocular functions. Recently, the supposed presence of the neuropeptide urocortin (UCN) has been reported in EWN neurons in rodent brain. The purpose of the present study was to examine the distribution of UCN in avian brain and to investigate by immunohistochemical analysis the possible use of this substance as an EWN marker in a non-mammalian class of vertebrates. Brain tissue of pigeons was incubated with a specific antibody against UCN and the results showed labeling of many small neurons, forming a double wing in the dorsal mesodiencephalic transition area. Their size and shape, however, differed from those of EWN neurons, and they were preferentially located rostral to the EWN. Double-label experiments employing an antibody against the enzyme choline acetyltransferase (ChAT) showed that UCN is not localized to the cholinergic cells of the EWN and confirmed the rostral distributionof UCN never overlapping the ChAT+ EWN cells. Taken together, these results suggest that, at least in pigeons, the UCN+ population does not belong to the traditionally defined EWN.

Cholinergic axons in the rat prostate and neurons in the pelvic ganglion.

Fluorogold or green fluorescent pseudorabies virus labeled postganglionic neurons in the pelvic ganglion that innervate the prostate gland. Small cholinergic neurons were demonstrated by immunohistochemistry (IHC) with antiserum against vesicular acetylcholine transferase (VAChT). Large, mainly adrenergic neurons, were surrounded by preganglionic cholinergic boutons. In the prostate, M3 type muscarinic receptors were found in the outer muscle layer surrounding the prostatic acini. The antiserum against VAChT marked the inner epithelial layer. Antisera against the vesicular monoamine transporters VMAT1 and VMAT2 demonstrated staining of the inner secretory layer and adrenergic fibers in the outer muscle layer, respectively, of the prostatic acini. These results provide new evidence for the presence of neural elements that have a cholinergic influence over the rat prostate gland.

Neuropeptides in perisympathetic organs of Manduca sexta: specific composition and changes during the development.

We used a combination of matrix-assisted laser desorption-ionization time-of-flight mass spectrometry and immunocytochemistry to investigate the peptides from abdominal perisympathetic organs of Manduca sexta. Altogether three mass peaks, detected in mass spectra from single abdominal perisympathetic organs were identical with already known neuropeptides, namely CAP(2b), CCAP, and Manduca-allatotropin. Only CAP(2b) was found throughout the postembryonic development. In larvae, perisympathetic organs of the abdominal ganglia 1 and 7 do not accumulate neuropeptides. During the metamorphosis, the number of putative hormones stored in the abdominal perisympathetic organs, increases dramatically. Not a single substance, however, obtained in mass spectra of larval perisympathetic organs disappeared in the respective adult neurohemal organs. Peptides from abdominal perisympathetic organs are different from those of thoracic perisympathetic organs and the retrocerebral complex. Manduca-FLRFa-2 and -3 are enriched in thoracic perisympathetic organs; FLRFa-1, corazonin and adipokinetic hormone are abundant peptides of the retrocerebral complex. The majority of ion signals, however, represent unknown substances. An antiserum which recognized CAP(2b) allowed the morphological characterization of a median neurosecretory system in the abdominal ventral nerve cord of M. sexta, which resembles that of cockroach embryos. Double stainings confirmed that crustacean cardioactive peptide (CCAP) becomes colocalized with CAP(2b) in median neurosecretory cells during the last larval instar. This colocalization continues in adult insects.

alpha(2a) and alpha(2c) adrenoceptors on spinal neurons controlling penile erection.

The thoracolumbar and lumbosacral spinal cord contain respectively sympathetic and parasympathetic preganglionic neurons that supply the organs of the pelvis including the penis. These neurons are influenced by supraspinal information and receive aminergic projections from the brainstem. The presence of the alpha(1)- and alpha(2)-adrenoceptor subtypes has been demonstrated in the rat spinal cord. In this species, we looked for the presence of alpha(2a)- and alpha(2c)-adrenoceptor subtypes in the sympathetic and parasympathetic preganglionic neurons controlling erection. In adult male rats, transsynaptic axonal transport of pseudorabies virus injected into the penis was combined with immunohistochemistry against alpha(2a)- and alpha(2c)-adrenoceptor subtypes. At 4 days survival time, neurons infected with the pseudorabies virus were solely found in the intermediolateral cell column and dorsal gray commissure of segment T12-L2 and in the intermediolateral cell column of segment L6-S1. Neurons and fibers immunoreactive for alpha(2a)- and alpha(2c)-adrenoceptor subtypes were mainly present in the intermediolateral cell column, the dorsal gray commissure and the ventral horn of the T12-L2 and L5-S1 spinal cord, the dorsal horn displayed only immunoreactive fibers. Pseudorabies virus-infected neurons in the autonomic nuclei were both immunoreactive for alpha(2a)- and alpha(2c)-adrenoceptor subtypes and closely apposed by alpha(2a)- and alpha(2c)-immunoreactive fibers. The results suggest an intraspinal modulation of the noradrenergic and adrenergic control of the autonomic outflow to the penis by pre- and postsynaptic alpha(2) adrenoceptors.

Estrogen receptor-alpha immunoreactivity in parasympathetic preganglionic neurons innervating the bladder in the adult ovariectomized cat.

Estrogen affects autonomic functions such as micturition. The sacral cord is important in the control of micturition and contains numerous estrogen receptor-alpha immnoreactive (ER-alpha IR) neurons. Therefore, the present double labeling study examines whether sacral parasympathetic preganglionic neurons innervating the bladder are immunoreactive for ER-alpha. In the sacral cord of seven female ovariectomized cats, the distribution of ER-alpha IR neurons was studied using the H222 and 1D5 antibodies. Choleratoxin subunit b (CTb) was injected into the bladder wall to visualize its preganglionic neurons. ER-alpha IR was present in the nuclei of cells in laminae I, II, V, VII, and X, and in nuclei and cytoplasm of neurons in the sacral parasympathetic nucleus. The vast majority of CTb labeled neurons contained ER-alpha IR nuclei, indicating that preganglionic neurons innervating the bladder express ER-alpha. The results suggest that estrogen modulates micturition in the cat via ER-alpha in bladder preganglionic neurons.

Decreased neurocalcin immunoreactivity in sympathetic and parasympathetic neurons of the major pelvic ganglion in aged rats.

In the rat the majority of sympathetic and parasympathetic postganglionic neurons that innervate the pelvic viscera are located together in the major pelvic ganglia. We have ascertained that it is only the sympathetic population of this ganglion that exhibits age-associated attrition. Recent immunohistochemical investigations of the distribution of calcium binding proteins in this ganglion in young adult and aged rats have demonstrated that calbindin-D28k is only present in the sympathetic neurons and that the number of calbindin-immunoreactive sympathetic neurons of the aged ganglion was dramatically reduced. In the present study we have investigated the distribution of neurocalcin (NC) alpha isoform in the major pelvic ganglion. In young adults 98.7% of sympathetic neurons (identified by anti-tyrosine hydroxylase immunostaining) and 98% of parasympathetic neurons (identified by anti-nitric oxide synthase immunostaining) contained NC immunoreactivity and these figures are reduced to 68 and 45.5% in the aged group. Thus, unlike calbindin-D28k, NC is not confined to the sympathetic neuron population in the major pelvic ganglion and decreases significantly in old age in both neuronal populations. The likely effects are to impair intracellular calcium-dependent signalling in neurons of the major pelvic ganglion, possibly compounding the effects of the previously reported decrease in calbindin-D28k in the sympathetic population.

Calbindin-D28k in cerebrovascular extrinsic innervation system of the rat.

Calbindin-D28k, one of the calcium-binding proteins, belongs to the EF hand family and is commonly found in neurons. It serves as a representative neuronal marker for neuroanatomical investigations. The authors' knowledge of its precise function, however, is yet very limited. In this study, we examined the existence of nerve fibers with calbindin-D28k immunoreactivity in the cerebral blood vessels and ganglia that innervate the cerebral blood vessels in the rat. Numerous nerve fibers with calbindin-D28k immunoreactivity were observed on the walls of the major extracerebral arteries forming the circle of Willis and its branches. Calbindin-D28k immunoreactivity was seen in many neurons of the trigeminal, dorsal root and jugular ganglia. A small number of neurons showed calbindin-D28k immunoreactivity in the otic and superior cervical ganglia. Calbindin-D28k immunoreactivity was not detected in the sphenopalatine or internal carotid ganglia. Pericellular basket-like formations of nerve terminals with calbindin-D28k immunoreactivity were observed in the sphenopalatine, otic, internal carotid and superior cervical ganglia. The present study demonstrated calbindin-D28k immunoreactivity in the cerebrovascular nerve fibers as well as in their origins--the cranial ganglia. These findings are significant in understanding the calcium-mediated mechanism of the neural control of the cerebral blood vessels.

Pituitary adenylate cyclase-activating polypeptide innervation of the mudpuppy cardiac ganglion.

The presence and potential origin of the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) was determined in cardiac ganglia of the mudpuppy, Necturus maculosus. Although PACAP has been implicated in the regulation of cardiac function in several mammalian species, the presence of this peptide in the autonomic nervous system (ANS) of other species is unclear. Thus, this study is the first to characterize this highly conserved peptide in the ANS of a non-mammalian species. PACAP-immunoreactivity was observed in nerve fibers throughout the mudpuppy cardiac ganglia and often was co-localized with the sensory neuropeptides substance P and calcitonin gene-related peptide. Removal of all extrinsic inputs to the ganglia by organ culture eliminated PACAP-immunoreactivity in the cardiac ganglia, whereas bilateral vagotomies only partially reduced PACAP-labeling. PACAP-immunoreactive neurons were observed in both high thoracic dorsal root ganglia and in vagal sensory ganglia. While no PACAP-positive neurons were observed in caudal medulla brainstem regions, PACAP-containing nerve fibers were found in the region of the nucleus solitarius. These results suggest that, in the mudpuppy, PACAP is found primarily in visceral afferent fibers, originating from cells in either the dorsal root ganglia or vagal sensory ganglia. Based on their anatomic localization, these afferent fibers may function to transmit important sensory information to cardiovascular centers in the brain as well as serving as local reflex inputs to modulate postganglionic parasympathetic output within the cardiac ganglion itself.

mu-Opioid receptor inhibits N-type Ca2+ channels in the calyx presynaptic terminal of the embryonic chick ciliary ganglion.

A study was made on the mechanisms by which enkephalins inhibit synaptic transmission at calyx-type presynaptic terminals in the ciliary ganglion of chick embryos at stages 39-40. Excitatory postsynaptic currents (EPSCs) were recorded by nystatin-perforated patch clamp at low [Ca2+]o and high [Mg2+]o. [Leu5]enkephalin (L-ENK, 1-10 microM) reduced the quantal content (m) without changing the quantal size (q). This effect was antagonized by naloxone (1 microM). Similar results were observed under conventional whole-cell clamp of the postsynaptic neuron. A specific agonist of the mu-opioid receptor, [D-Ala2, M-Me-Phe4,Gly5]enkephalin-ol (DAMGO) reduced m without changing q. A specific agonist of the delta-opioid receptor, [d-Pen2, d-Pen5]enkephalin (DPDPE) also reduced m without changing q. Both L-ENK and [Met5]enkephalin (M-ENK) reduced the stimulus-dependent increment of the intraterminal Ca2+ concentration (Delta[Ca2+]t) without affecting the decay time constant of the intraterminal Ca2+ concentration and basal Ca2+ level. This effect was antagonized by naloxone. DAMGO reduced Delta[Ca2+]t more effectively than DPDPE. When extracellular Ca2+ was replaced by Ba2+, the stimulus-dependent increment of the intraterminal Ba2+ concentration (Delta[Ba2+]t) was also reduced by L-ENK or DAMGO. L-ENK reduced Delta[Ca2+]t even in the presence of 4-aminopyridine (4-AP), which blocks the transient K+ conductance during the falling phase of the presynaptic action potential. When N-type Ca2+ channels were blocked by omega-conotoxin GVIA (omega-CgTxGVIA), the Delta[Ca2+]t was no longer sensitive to L-ENK and DAMGO. It is suggested that enkephalins reduce the transmitter release through presynaptic opioid receptors. The mu-opioid receptor may suppress presynaptic Ca2+ influx by selectively inhibiting N-type Ca2+ channels.

The distribution of vesicular acetylcholine transporter in the human male genitourinary organs and its co-localization with neuropeptide Y and nitric oxide synthase.

Because doubt still remains concerning the distribution of nerves that are unequivocally cholinergic in the human genitourinary organs, we have used a specific marker, namely, an antibody to vesicular acetylcholine transporter (VAChT), to immunolabel cholinergic axons and cell bodies in specimens of urinary bladder, seminal vesicle, vas deferens, and prostate gland obtained from neonates and children post mortem. In addition some sections were double-immunolabeled with VAChT and either neuropeptide Y (NPY) or nitric oxide synthase (NOS). The results demonstrated a rich cholinergic innervation to the muscle coat of the bladder body with a much less prominent, but nonetheless significant, cholinergic innervation to the smooth muscle components of the seminal vesicle, vas deferens, and prostate. Small ganglia were scattered throughout the detrusor muscle of the urinary bladder, approximately 75% of the intramural neurons being VAChT immunoreactive, whereas approximately 95% contained NPY and approximately 40% contained NOS. VAChT immunoreactivity was observed in 40% of neurons in ganglia scattered throughout the pelvic plexus. Almost all these cholinergic neurons contained NPY and approximately 65% contained NOS. Almost all the cholinergic nerve fibers throughout the genitourinary organs also contained NPY. Although NOS was sparse in the cholinergic nerves of the bladder body, it occurred in the majority of cholinergic nerves at the bladder neck and was also present in a proportion of the cholinergic nerves in the other organs examined. VAChT-immunoreactive nerves were also observed in a sub-epithelial location in all the organs examined, the majority containing NPY, whereas a small proportion contained NOS. Although doubt remains about the function of sub-epithelial cholinergic nerves in the urinary bladder, the majority of similar nerves in the seminal vesicle, vas deferens, and prostate gland are considered to be secretomotor. Collectively these findings demonstrate that the cholinergic innervation of the male genitourinary system is well established in the neonate and child. Neurourol. Urodynam. 19:185-194, 2000.

Alpha subunit composition of nicotinic acetylcholine receptors in the rat autonomic ganglia neurons as determined with subunit-specific anti-alpha(181-192) peptide antibodies.

The subunit composition of nicotinic acetylcholine receptors of rat autonomic ganglia neurons was studied by means of antibodies, which differentiated between different alpha subunits and specifically blocked acetylcholine-induced membrane currents. Polyclonal rabbit antibodies and mouse monoclonal antibodies were raised against synthetic peptides matching in sequence the alpha(181-192) region of alpha3, alpha4, alpha5, and alpha7 subunits of rat neuronal nicotinic acetylcholine receptors. The antibodies discriminated among alpha3, alpha4, alpha5, and alpha7 peptides in enzyme-linked immunosorbent assay and bound to native acetylcholine receptors expressed in PC-12 cells. By means of immunoperoxidase staining of cultured rat autonomic neurons followed by transmission, dark-field and phase-contrast microscopy, it was found that all cells of the superior cervical ganglia expressed the alpha3, alpha5, and alpha7 nicotinic acetylcholine receptors, whereas approximately half of the cells were clearly alpha4-positive. In contrast, only about one-third of the intracardiac neurons were alpha3-positive, about 50% were alpha4-positive, one-seventh were alpha5-positive, and one-fifth were alpha7-positive. All antibodies tested blocked acetylcholine-induced currents in the neurons of the superior cervical ganglia as was demonstrated by whole-cell patch-clamp studies. Although each antibody could block up to 80% of the current, the degree of inhibition varied considerably from cell to cell. It is concluded that alpha3, alpha5, and alpha7 subunits are expressed in all neurons of the superior cervical ganglion and in some intracardiac neurons, whereas alpha4 subunits are expressed in some but not all neurons of both tissues. The neurons of the superior cervical ganglion express heterogeneous acetylcholine receptors and differ in relative amounts of acetylcholine receptor subtypes expressed.

A parasympathetic ganglion innervating the harderian gland and lacrimal gland of the musk shrew (Suncus murinus): fluorescent tracing and immunohistochemical studies.

A small ganglion, named the peri-trigeminal ganglion (PTG), was found in the ventromedial border of the rostral half of the trigeminal ganglion (TG) in the musk shrew (Suncus murinus). In frontal sections, the PTG was semicircular or elliptical in shape. Most of the neurons constituting this ganglion were round in shape and much smaller than those of the TG. The retrograde fluorescent tracer fluoro-gold was injected into various regions of the face in order to investigate innervation by the PTG neurons. When the tracer was injected subcutaneously around the external acoustic meatus and around the circumference of the orbit, a number of labeled neurons were seen not only in the TG but also in the PTG. After applying the tracer to the lacrimal gland (LG) and the harderian gland (HG), numerous labeled neurons were detected only in the PTG. A few labeled neurons were found in the PTG after injection into the palatoglossal arch. Immunohistochemically, most of the neurons constituting the PTG were positive for vasoactive intestinal polypeptide (VIP) antiserum. And a moderate number of somatostatin (SOM)-immunoreactive neurons and a small number of leucine-enkephalin (L-ENK)-immunoreactive neurons were detected. Numerous substance P-immunoreactive nerve fibers and varicosities were found in the PTG, and fewer L-ENK-, SOM- and VIP-immunoreactive fibers were observed. The present results suggest that the PTG is an autonomic ganglion that resembles in part the pterygopalatine ganglion in other species, and mainly innervates the HG and LG.

Occurrence and distribution of substance P receptors in the cerebral blood vessels of the rat.

The distribution of immunoreactivity to the receptor for substance P was examined in the cerebral blood vessels of the rat. Substance P immunoreactivity has been demonstrated in the nerve fibers of the cerebral blood vessels. Recently, the production of substance P receptor specific antibody has enabled the detection of localization of the substance P receptor in the central nervous system. In this study, we examined the existence of nerve fibers with substance P receptor immunoreactivity in the cerebral blood vessels and the cranial ganglia innervating the cerebral blood vessels. Sprague-Dawley rats were perfused with fixative and the pial arteries and the cranial ganglia known to innervate the cerebral blood vessels, i.e., trigeminal, sphenopalatine, internal carotid, otic and superior cervical ganglia, were dissected. All specimens were incubated with anti-substance P receptor IgG, then stained by the avidin-biotin-peroxidase complex method. Numerous nerve fibers with varicosities forming plexuses, with substance P receptor immunoreactivity were observed on the walls of the major extracerebral arteries forming the circle of Willis and its branches. Substance P receptor immunoreactivity was also detected in the endothelium of the cerebral arteries. Substance P receptor immunoreactivity was positive in many neurons of the sphenopalatine ganglion, otic ganglion, trigeminal ganglion, superior cervical ganglion and internal carotid ganglion. The present study demonstrated the existence of nerve fibers with substance P receptor immunoreactivity in the cerebral blood vessels and the cranial ganglia that innervate the cerebral blood vessels. These findings are important in understanding the responsiveness of the cerebral blood vessels to substance P.

Neuropeptide Y (NPY) expression is increased in explanted guinea pig parasympathetic cardiac ganglia neurons.

While expression of neuropeptides by sympathetic neurons is altered by decentralization and axotomy, it is not known whether similar experimental paradigms also modulate the chemical phenotype of parasympathetic cardiac ganglia neurons. The present study tested whether guinea pig parasympathetic neuron neuropeptide Y (NPY) expression was altered when cardiac ganglia preparations were maintained as organ explants in the presence or absence of colchicine. Two experimental approaches were used to examine NPY expression. First, immunocytochemical techniques were used to quantitate numbers of neurons within the cardiac ganglia exhibiting NPY-immunoreactivity; second, reverse transcription PCR was used to examine proNPY mRNA expression. In control cardiac ganglia preparations, approximately 4% of ganglia neurons exhibited NPY-immunoreactivity. The percentage of NPY-immunopositive neurons in 30- and 72-h explanted cardiac ganglia preparations, maintained in the absence of colchicine, increased to 11 and 16%, respectively. Colchicine treatment of explanted preparations further increased the percentage of NPY-positive ganglia cells 24% (30 h) and 32% (72 h). All NPY-immunoreactive neurons from control ganglia and explanted ganglia were choline acetyltransferase(ChAT)-immunoreactive, indicating retention of the cholinergic phenotype. ProNPY mRNA also was increased following ganglia explantation, consistent with the increase in the numbers of NPY-immunoreactive neurons. NPY transcripts were further increased after 30 h, but not after 72 h in colchicine-treated, explanted cardiac ganglia preparations. These results demonstrate that NPY expression is altered in explanted cardiac ganglia preparations, providing evidence that the chemical phenotype of parasympathetic cardiac neurons can be modulated.

Neuronal messengers and peptide receptors in the human sphenopalatine and otic ganglia.

A majority of the parasympathetic nerve fibers to cranial structures derive from the sphenopalatine and otic ganglia. In particular, blood vessels are invested with a rich supply of dilator fibers of parasympathetic origin. In the present study, we have examined the occurrence of noncholinergic neuromessengers and neuropeptide receptors in the human sphenopalatine and otic ganglia. Vasoactive intestinal peptide (VIP)-immunoreactive (ir) nerve cell bodies occurred in high numbers in the sphenopalatine and otic ganglia. Likewise, high numbers of NOS- and PACAP-containing nerve cell bodies were seen in both ganglia. Autofluorescent lipofuscin, characteristic of adult human nervous tissue, was present within many nerve cell bodies in both ganglia. Receptor mRNA was studied with reverse transcriptase-polymerase chain reaction (RT-PCR). Total RNA from the sphenopalatine and otic ganglia was successfully extracted. By using appropriate sense and antisense primers, oligonucleotides were designed from the human sequences derived from GenBank, corresponding to human NPY Y1, CGRP1 and VIP1 receptors. In the sphenopalatine ganglion, we revealed the presence of mRNA for the human NPY Y1 and VIP1 receptors but not the CGRP1 receptor. The otic ganglion was found to react positively only for primers to mRNA for VIP1 but not for CGRP1 or NPY Y1 receptors.