Interindividual variation in distribution of extramural ganglion cells in the male pelvis: a semi-quantitative and immunohistochemical study concerning nerve-sparing pelvic surgery.

OBJECTIVE: We examined distribution and numbers of extramural ganglion cells in the male pelvis, classifying them as sympathetic or parasympathetic. METHODS: Specimens were obtained from 14 formalin-fixed donated male cadavers. Semiserial sections were processed for histologic examination, and for immunohistochemistry using anti-tyrosine hydroxylase (TH) or anti-peptide histidine isoleucine (PHI). RESULTS: Like those along the sacral sympathetic trunk, most other pelvic ganglion cells were located in and along nerve components. Yet the ganglion cell clusters attached to pelvic viscera accounted for 22% to 38% of ganglion cells. These were seen at the dorsal aspect of the bladder, the bladder/prostate junction, the dorsal aspect of the seminal vesicle, and along the prostate, but not along the extrapelvic pudendal nerve, cavernous tissues including the penile hilum, the rhabdosphincter, retropubic fat or recto-urethral muscle. Two fold interindividual variation was seen for total ganglion cell number (3044 to 6522) in the pelvis. TH-positive and PHI-positive cells intermingled at various ratio in every ganglion cell cluster. Sympathetic TH-positive proportions tended to be site-specific. CONCLUSIONS: Pelvic autonomic cells exist not only in nerve components but also along viscera. Even nerve-sparing radical prostatectomy can compromise visceral ganglia. Simple classification of pelvic nerve components as sympathetic or parasympathetic would seem misleading given coexistence of both cell types in a ganglion.

Axonal neurofilament-H immunolabeling in the rabbit retina.

A monoclonal antibody directed at the multiphosphorylated epitope of axonal neurofilament-H (NF-H) was used to label axon-like fibers in the rabbit retina. NF-H-immunopositive fibers were found in the outer plexiform layer (OPL), inner plexiform layer (IPL), and optic fiber layer (OFL). The morphological characteristics of the labeled processes identified those in the OPL as horizontal cell axons and axon terminals and fibers in the OFL as axons of ganglion cells. The NF-H-positive profiles in the OPL formed a subset of horizontal cell processes labeled for calbindin. In the IPL, NF-H-immunoreactive profiles lay at all levels but were detected most often in the middle strata, 2-4. Occasionally, we observed NF-H-immuoreactive processes emerging from the IPL and entering either the GCL or the inner nuclear layer (INL). The labeled fibers in the IPL were typically very thin, less than 1 microm in diameter, and could often be followed for over 1 mm as they ran laterally across the retina. Cell bodies were never labeled by the immunoserum. To identify the NF-H-immunopositive fibers in the IPL, standard immunocytochemical double-labeling techniques were applied, using antibodies directed against several neurotransmitters or modulators thought to be expressed by axon-bearing amacrine cells. The NF-H-positive processes in the IPL were found to correspond to those labeled for tyrosine hydroxylase, somatostatin, substance P, and NADPH diaphorase activity. However, the NF-H labels did not colocalize with those against the vasoactive intestinal peptide-associated protein PHM27. Our results indicate that putative axons in the retina possess the multiphosphorylated NF-H protein found within classic axons in the central nervous system. These results thus support the idea that certain subtypes of amacrine and horizontal cells maintain true axons in the mammalian retina.

Colocalization of numerous immunoreactivities in endocrine cells of the chicken proventriculus at hatching.

The colocalization of regulatory peptide immunoreactivities in endocrine cells of the chicken proventriculus at hatching has been investigated using the avidin-biotin technique in serial sections and double immunofluorescence in the same section for light microscopy, and double immunogold staining for electron microscopy. In addition to the eight immunoreactivities previously described in this organ, cells immunoreactive for peptide histidine isoleucine (PHI), peptide gene product 9.5 (PGP), and the amidating enzyme, peptidylglycine alpha-amidating monooxygenase (PAM) were observed. All the cells immunoreactive to glucagon were also immunostained by the PHI antiserum. In addition, all the glucagon-like peptide 1, avian pancreatic polypeptide, and some of the neurotensin-like cells costored also glucagon- and PHI-immunoreactive substances. PGP- and PAM-immunoreactivities were also found in the glucagon-positive cells. A small proportion of the somatostatin-containing cells were positive for PHI but not for other regulatory peptides. These results could suggest either the existence of a very complex regulatory system or that the endocrine system of the newborn chickens is not yet fully developed.

Evidence from confocal fluorescence microscopy for a dense, reciprocal innervation between AVP-, somatostatin-, VIP/PHI-, GRP-, and VIP/PHI/GRP-immunoreactive neurons in the rat suprachiasmatic nucleus.

The rat suprachiasmatic nucleus (SCN) consists of several classes of neurons which can be identified by their transmitter content. Knowledge of putative interaction between these different cell types is essential in order to understand the possibilities of information processing within the SCN. The aim of the present study was therefore to obtain more information about the mutual innervation between the main cell classes in the rat SCN, viz. those containing the neuropeptides arginine vasopressin (AVP), vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI), gastrin-releasing peptide (GRP) and somatostatin respectively. For this purpose, vibratome sections were double-immunolabelled for seven different peptide combinations and subsequently analysed by high-resolution confocal laser scanning fluorescence microscopy. Attention was focused on axosomatic appositions, the occurrence and frequency of which were quantitatively estimated. Our analysis of double-immunolabelled sections demonstrated that some of the VIP- and some of the GRP-immunoreactive nerve cells and endings showed colocalization. Assuming, on the basis of literature data, that VIP and PHI are always colocalized at the cellular level, the five main cell classes in the SCN appeared to be interconnected, at least axosomatically, in the following reciprocal way: AVP <--> VIP/PHI, AVP <--> GRP, AVP <--> somatostatin, somatostatin <--> VIP/PHI, somatostatin <--> GRP, VIP/PHI <--> GRP, VIP/PHI/GRP <--> GRP, VIP/PHI/GRP <--> VIP/ PHI. In addition to this heterologous axosomatic innervation, these cell groups also showed substantial homologous innervation. Supported by electron microscope data from the literature showing the existence of axodendritic synapses for some of these peptide combinations, our findings strongly suggest that the rat SCN comprises a complex synaptic network with strong interactive capabilities, which is probably a requisite for its biological clock function.

Peptide histidine methionine in cerebrospinal fluid of patients with senile dementia of the Alzheimer type.

Immunoreactivities (IRs) of peptide histidine methionine (PHM) as well as somatostatin and vasoactive intestinal peptide (VIP) in the cerebrospinal fluid (CSF) were measured in patients with senile dementia of the Alzheimer type (SDAT) and age-matched control subjects. We found statistically significant reductions in the PHM-IR and somatostatin-IR levels in the CSF from patients with SDAT, as compared with those of the controls. However, the VIP-IR level in the CSF from SDAT was not different from that of the controls. These results suggest that selective degeneration of neurons containing somatostatin and PHM or the alteration in metabolism of PHM in the CSF might occur in SDAT.

Effects of damage to SCN neurons and efferent pathways on circadian activity rhythms of hamsters.

This experiment was designed to determine if entrainment to a light:dark (LD) schedule and the free-running rhythm in constant light are altered by partial lesions of suprachiasmatic nuclei (SCN) cells or SCN output pathways. Twenty-four male golden hamsters were housed under 12L:12D. Hamsters received either lesions (n = 16), sham surgery (n = 4), or no surgery (n = 4), and were placed into individual cages with running wheels under 14L:10D. Each time after 4 weeks, the LD schedule was phase advanced by 6 h, phase delayed by 6 h, and then the animals were exposed to constant dim light. At the end of the experiment, brain sections were processed for peptide histidine isoleucine (PHI) and gastrin releasing peptide (GRP) immunohistochemistry. Alternate sections were stained for cells and fibers. Behavioral results indicate that (a) very few SCN cells and SCN efferent fibers, as labeled by PHI and GRP immunohistochemistry, are necessary for the expression of circadian rhythmicity in wheel running, and (b) damage to pathways rostral to the SCN may be more critical for entrainment and rhythmicity than damage to caudal pathways. Targets of PHI- and GRP-immunoreactive SCN efferent fibers were also identified.

[Basic study of peptide histidine isoleucine (PHI) immunoreactivity in the rat respiratory tract].

Peptide histidine isoleucine (PHI) as well as vasoactive intestinal peptide (VIP) is considered to be a neurotransmitter of non-adrenergic and non-cholinergic inhibitory nerves in the respiratory tract. We tried to measure PHI immunoreactivity in rat respiratory tracts to evaluate the movement of PHI. A high titer of PHI antiserum which did not react with VIP was obtained from a rabbit immunized with rat PHI, and rat PHI radioimmunoassay was established with this antiserum. Tracheas, extrapulmonary bronchi and lungs were dissected from rats sacrificed by microwave irradiation. They were homogenized with 0.5 N acetic acid and centrifuged at 35,000 X g. Lyophilized supernates were used as samples for the radioimmunoassay. PHI immunoreactivity per g wet weight tissue was 5.12 +/- 1.35 (mean +/- SD) pmol in the tracheas, 1.66 +/- 1.15 pmol in the extrapulmonary bronchi and 0.12 +/- 0.06 pmol in the lungs. PHI immunoreactivity occurred more in the central airways than in the peripheral ones. VIP immunoreactivity in rat respiratory tracts, assayed simultaneously by radioimmunoassay using commercialized VIP antiserum, was as strong as the of PHI, suggesting that PHI and VIP may be produced from the same precursor at the ratio of 1:1 in rat respiratory tracts.

Distribution of peptidergic nerves in the choroid plexus, focusing on coexistence of neuropeptide Y, vasoactive intestinal polypeptide and peptide histidine isoleucine.

Choroid plexus from rat, guinea-pig, rabbit and pig was investigated by light-microscopic immunohistochemistry and by radioimmunoassay for the presence of neuropeptides. A moderately dense supply of nerve fibers containing neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP), respectively, was found around blood vessels and in close relation to the secretory epithelium in both pig and rabbit, while lower densities of nerve fibers were found in rat and guinea-pig. Peptide concentrations ranged from 10-40 pmolequivalents/g (pmoleqv/g) for NPY and 0.5-6 pmoleqv/g for VIP in all four species. Peptide histidine isoleucine (PHI) immunoreactive nerve fibers were present in pig choroid plexus at a lower density than NPY and VIP but with a similar distribution. Low concentrations of substance P (0.3-3 pmoleqv/g) and calcitonin gene-related peptide (0.1-3 pmoleqv/g) were found to a varying degree in choroid plexus tissue from the different species, while immunohistochemical investigation was unable to detect any immunoreactive nerve fibers. NPY was often found to coexist with VIP and PHI in pig choroid plexus, while a lesser amount of nerve fibers showed coexistence of NPY and the noradrenaline synthetizing enzyme, dopamine-beta-hydroxylase. Surgical sympathetic denervation by excision of the superior cervical ganglion in the rabbit abolished NPY-containing nerve fibers, as revealed by immunohistochemistry, but only decreased NPY levels by one third, which may be due to different identity of the peptide being detected by the two techniques. It is concluded that NPY-containing nerve fibers have a dual origin in the choroid plexus and coexist with either noradrenaline or VIP/PHI.

Role of peptide histidine isoleucine in relaxation of cat lower esophageal sphincter.

Vasoactive intestinal peptide (VIP) is a candidate as an inhibitory neurotransmitter mediating relaxation of the lower esophageal sphincter (LES) because VIP antiserum reduces LES relaxation in response to neural stimulation. Vasoactive intestinal peptide antiserum, however, does not completely block LES relaxation. Thus it is possible that other neurotransmitters may be involved. Peptide histidine isoleucine has structural homologies with VIP, is synthesized with VIP from a common precursor protein, coexists in some nerve cells, and is coproduced with VIP in some tumors. In numerous organ systems VIP and peptide histidine isoleucine (PHI) produce similar effects, with PHI being less potent than VIP by approximately one log number. In the LES both VIP and PHI produce tetrodotoxin-resistant dose-dependent relaxation, with PHI being almost equipotent with VIP. We therefore tested the hypothesis that PHI may be a second neurotransmitter, partly responsible for relaxation of the cat LES, by using a highly specific rabbit PHI antiserum that exhibits minimal cross-binding with VIP, secretin, and glucagon. In 3 animals, LES and brain tissue were extracted in 0.1 N HCl and assayed with a PHI radioimmunoassay. The antiserum cross-reacted with cat brain and LES showing PHI concentrations greater than 100 ng/g, with the LES containing equal or greater concentrations of PHI than brain tissue. In other animals consecutive LES circular muscle strips were cut, mounted in 1-ml muscle chambers, and stimulated with 6-s square-wave trains of 0.1-, 0.2-, 0.4-, and 0.8-ms pulses at 1, 2, and 5 Hz. These parameters produced relaxation that was completely blocked by tetrodotoxin, and reduced by VIP antiserum, but not affected by adrenergic or cholinergic receptor antagonists. Some strips were incubated in 5% or 10% PHI antiserum, whereas others were incubated in the same concentration of preimmunization serum from the same animal. Incubation in normal serum did not significantly affect relaxation, whereas in the antiserum-treated strips, LES relaxation was reduced by a significant amount (20%-30%) at all parameters of stimulation tested. Incubation in antiserum however had no effect on relaxation induced by VIP (10(-8)-10(-6) M). These data suggest that PHI may play a role in LES relaxation induced by electrical stimulation.

The effects of vasoactive intestinal peptide (VIP) antagonists, and VIP and peptide histidine isoleucine antisera on non-adrenergic, non-cholinergic relaxations of tracheal smooth muscle.

1. The effects of several drugs, including antagonists of vasoactive intestinal peptide (VIP), and antisera to VIP or peptide histidine isoleucine (PHI), on relaxation responses of guinea-pig isolated trachea to electrical field stimulation (EFS) have been examined. 2. beta-Adrenoceptor blockade with propranolol only partially blocked the inhibitory response to EFS, but had no effect in tissues from animals pretreated with 6-hydroxydopamine or reserpine. 3. Neither adenosine deaminase, in the presence of dipyridamole, nor the potent adenosine antagonist NPC205 (1,3-n-dipropyl-8-(4-hydroxyphenyl)-xanthine) had any effect on the inhibitory response to EFS. 4. The VIP antagonists, [Ac-Tyr1, D-Phe2]-GRF(1-29)-NH2 and [4-Cl-D-Phe6, Leu17]-VIP had no effect on the inhibitory response to EFS. Moreover, they were without effect on responses to exogenous VIP or PHI. 5. Overnight incubation with VIP antisera markedly reduced the inhibitory response to EFS. PHI antisera had a similar, but smaller effect. 6. In the presence of a concentration of VIP that is maximal for its relaxant effect, inhibitory responses to electrical stimulation were greatly inhibited. 7. Naloxone and reactive blue 2 each had no effect on inhibitory responses indicating that endogenous opioids and adenosine 5'-triphosphate (ATP) respectively are not involved. 8. The results suggest that VIP and PHI, but not adenosine, contribute to non-adrenergic, noncholinergic inhibitory nerve responses of guinea-pig trachea. Moreover, the surprising lack of effect of both VIP antagonists on these responses, and in particular, on responses to exogenous VIP, suggests that the receptors mediating VIP-induced tracheal relaxation are different from those that mediate pancreatic secretion.

Localization of vasopressin-, vasoactive intestinal polypeptide-, peptide histidine isoleucine- and somatostatin-mRNA in rat suprachiasmatic nucleus.

Messenger RNAs (mRNA) coding for vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), somatostatin and vasopressin were localized in the suprachiasmatic nucleus (SCN) of the rat hypothalamus using in situ hybridization histochemistry. Specific mRNA coding for each of these peptides was distributed in areas coextensive with the immunohistochemical localization of the appropriate peptide. The autoradiographic signal produced with probes to VIP and PHI created dense concentrations of silver grains over neuronal perikarya in the ventrolateral SCN, and the coextensive distribution of both VIP- and PHI-mRNAs suggests that both peptides are synthesized within the same neurons. The distribution of somatostatin-mRNA was distinct from the of VIP and PHI. Labeled neurons are observed at the interface of the two SCN subdivisions and the distribution of these neurons is identical to those shown to contain somatostatin immunoreactivity. Vasopressin-mRNA is also differentially concentrated within neurons in the dorsomedial subdivision of the SCN in an area that is coextensive with vasopressin-immunoreactive perikarya. The discrete pattern of hybridization for each of these mRNAs indicates that each of these peptides are synthesized in SCN neurons and reaffirms the differential distribution of each of these chemically defined cell populations within cytoarchitecturally distinct subdivisions of the nucleus.

Some blood vessels in the rat median eminence are surrounded by a dense plexus of vasoactive intestinal polypeptide/peptide histidine isoleucine (VIP/PHI) immunoreactive nerves.

Using immunohistochemistry at the light and electron microscopic level some blood vessels along the median eminence were shown to be surrounded by dense networks of vasoactive intestinal polypeptide/peptide histidine isoleucine (VIP/PHI)-positive fibers. VIP and PHI released from these fibers may contribute to the elevated levels of these two peptides measured in portal blood as compared to peripheral blood by radioimmunoassay. VIP and PHI may also be important in the control of blood flow through the median eminence.