Effects of the Neuropeptides Pituitary Adenylate Cyclase Activating Polypeptide and Vasoactive Intestinal Peptide in Male Fertility.

Background and Objectives: The neuroendocrine system plays a crucial role in regulating various bodily functions, including reproduction, with evidence suggesting its significant involvement in male fertility and sperm development. Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) are expressed in both male and female reproductive tissues, influencing penile erection and regulating steroidogenesis in males. Therefore, our study aimed to compare the protein levels of VIP and PACAP in seminal plasma between healthy controls and sub-fertile patients. Additionally, we sought to correlate the levels of these biomarkers with clinical, functional, and laboratory findings in the participants. Materials and Methods: The study included a total of 163 male participants for analysis. The participants were further stratified into subgroups of fertile and sub-fertile men of four subgroups according to the 2021 WHO guidelines. Seminal plasma concentrations of the neuropeptides VIP and PACAP were measured using human enzyme-linked immunosorbent assay technique. Results: The findings showed statistically significant differences in total sperm count, sperm concentration, total motility, and vitality (p < 0.001) between the fertile group and the sub-fertile group. Specifically, significant differences found between healthy males and oligoasthenospermic patients (p = 0.002), and between asthenospermic and oligoasthenospermic patients (p = 0.039). An ROC analysis showed associated sensitivity and specificity values of 62.2% and 55.6%, respectively, to PACAP seminal levels differentiated between sub-fertile patients from fertile males (p = 0.028). No significant difference in seminal levels of VIP was found between the sub-fertile and fertile groups. Conclusions: Previous research leads to the point of PACAP active involvement in spermatogenesis. In accordance to our study, in human semen samples, we have seen a significance change in PACAP levels amongst patients with low sperm count or with both low sperm count and low motility, hinting at its contribution and acting as a possible factor in this complex process. Thus, alterations in the levels or actions of these neuropeptides have been associated with certain reproductive disorders in males.

Plasma levels of neurogenic inflammation related neuropeptides in pediatric patients with community-acquired pneumonia and their potential diagnostic value in distinguishing viral and bacterial pneumonia.

Neurogenic inflammation is involved in the development and progression of respiratory inflammatory diseases. However, its role in community-acquired pneumonia (CAP) remains unclear. We therefore aimed to investigate plasma levels of neurogenic inflammation-related neuropeptides, calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY), and procalcitonin (PCT) in pediatric patients with CAP and to assess their diagnostic value in viral and bacterial/mixed pneumonia. A total of 124 pediatric patients with CAP (1 month-18 years old) and 56 healthy children of similar ages were prospectively enrolled. The patients were classified as viral (n = 99) and bacterial/mixed (n = 25) pneumonia. Plasma levels of the peptides were quantified by ELISA. ROC analysis was performed to evaluate possible diagnostic value of the peptides. While plasma levels of CGRP, VIP and PCT were significantly higher in patients with CAP than in the control group, respectively, NPY levels were significantly lower. Moreover, plasma levels of all neuropeptides and PCT were significantly higher in bacterial pneumonia patients compared to viral pneumonia patients. ROC analysis revealed that CGRP, SP and NPY had a diagnostic value in distinguishing viral and bacterial/mixed pneumonia. CONCLUSIONS: Our findings suggest that these neuropeptides may be implicated in pediatric CAP. CGRP, SP and NPY together may be a promising candidate in distinguishing viral and bacterial/mixed pneumonia, however, for this, further studies are needed. WHAT IS KNOWN: • Neurogenic inflammation contributes to the development and progression of respiratory inflammatory diseases such as chronic obstructive pulmonary disease and bronchial asthma. WHAT IS NEW: • Plasma levels of neurogenic inflammation related neuropeptides calcitonin gene-related peptide, substance P, vasoactive intestinal peptide and neuropeptide Y are changed in pediatric community-acquired pneumonia. Calcitonin gene-related peptide, substance P and neuropeptide Y are promising candidates in distinguishing viral and bacterial/mixed pneumonia.

[Neuroimmunology of allergic rhinitis : Part 1: Cellular and humoral basic principles]. / Neuroimmunologie der allergischen Rhinitis : Teil 1: Zelluläre und humorale Grundlagen.

Allergic rhinitis (AR) is a very common disease with a high prevalence worldwide. It is an IgE-mediated type 2 inflammatory disease following exposure to inhalant allergens. A multitude of different neuropeptides including substance P, vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP), nerve growth factor (NGF), and neuromedin U (NMU) can be released via peripheral axon or central reflexes, interact with immune cells, and thus contribute to neurogenic inflammation which causes the nasal hyperreactivity (NHR) characteristic of AR. Independent production of neuroendocrine hormones and neuropeptides by immune cells has also been demonstrated. Neuro-immune cell units arise when immune and neuronal cells colocalize, for which typical anatomic regions are, e.g., the mast cell-nerve functional unit. The focus of this review is the elucidation of neuroimmune communication mechanisms in AR.

Histological study on regional specificity of the mucosal nerve network in the rat large intestine.

Our previous studies and others have revealed detailed characteristics of the mucosal nerve network in the small intestine, but much remains unknown about the corresponding network in the large intestine. We herein investigated regional differences in the expression of neurochemical markers, the nerve network structure, and the cells in contact with nerve fibers by histological analysis using both immunohistochemistry and serial block-face scanning electron microscopy (SBF-SEM). Immunohistochemistry revealed that immunopositive structures for protein gene product 9.5, vasoactive intestinal peptide (VIP), calretinin and vesicular acetylcholine transporter were more prevalent in the lamina propria of the ascending colon than the cecum and descending colon (DC). There was no significant difference in the frequency of most neurochemical markers between the cecum and DC, but the frequencies of VIP+ structures were higher in the cecum than in the DC. SBF-SEM analysis showed that the nerve network structure was more developed on the luminal side of the DC than the cecum. The cells that nerve fibers abundantly contacted were subepithelial and lamina propria fibroblast-like cells and macrophages. In addition, nerve fibers in the cecum were in more frequent contact with immune cells such as macrophages and plasma cells than nerve fibers in the DC. Thus, the present histological analysis suggested that the mucosal nerve network in the large intestine possessed both regional universality and various specificities, and revealed the intimate relationship between the nerve network and immune cells, especially in the cecum.

A subpopulation of cortical VIP-expressing interneurons with highly dynamic spines.

Structural synaptic plasticity may underlie experience and learning-dependent changes in cortical circuits. In contrast to excitatory pyramidal neurons, insight into the structural plasticity of inhibitory neurons remains limited. Interneurons are divided into various subclasses, each with specialized functions in cortical circuits. Further knowledge of subclass-specific structural plasticity of interneurons is crucial to gaining a complete mechanistic understanding of their contribution to cortical plasticity overall. Here, we describe a subpopulation of superficial cortical multipolar interneurons expressing vasoactive intestinal peptide (VIP) with high spine densities on their dendrites located in layer (L) 1, and with the electrophysiological characteristics of bursting cells. Using longitudinal imaging in vivo, we found that the majority of the spines are highly dynamic, displaying lifetimes considerably shorter than that of spines on pyramidal neurons. Using correlative light and electron microscopy, we confirmed that these VIP spines are sites of excitatory synaptic contacts, and are morphologically distinct from other spines in L1.

Cell-type-specific nicotinic input disinhibits mouse barrel cortex during active sensing.

Fast synaptic transmission relies upon the activation of ionotropic receptors by neurotransmitter release to evoke postsynaptic potentials. Glutamate and GABA play dominant roles in driving highly dynamic activity in synaptically connected neuronal circuits, but ionotropic receptors for other neurotransmitters are also expressed in the neocortex, including nicotinic receptors, which are non-selective cation channels gated by acetylcholine. To study the function of non-glutamatergic excitation in neocortex, we used two-photon microscopy to target whole-cell membrane potential recordings to different types of genetically defined neurons in layer 2/3 of primary somatosensory barrel cortex in awake head-restrained mice combined with pharmacological and optogenetic manipulations. Here, we report a prominent nicotinic input, which selectively depolarizes a subtype of GABAergic neuron expressing vasoactive intestinal peptide leading to disinhibition during active sensorimotor processing. Nicotinic disinhibition of somatosensory cortex during active sensing might contribute importantly to integration of top-down and motor-related signals necessary for tactile perception and learning.

Cortical VIP+ /ChAT+ interneurons: From genetics to function.

One of the urgent tasks of neuroscience is to understand how neuronal circuits operate, what makes them fail, and how to repair them when needed. Achieving this goal requires identifying the principal circuitry elements and their interactions with one another. However, what constitutes 'an atom' of a neuronal circuit, a neuronal type, is a complex question. In this review we focus on a class of cortical neurons that are exclusively identified by the expression of vasoactive intestinal polypeptide (VIP) and choline acetyltransferase (ChAT). The genetic profile of these VIP+ /ChAT+ interneurons suggests that they can release both γ-aminobutyric acid (GABA) and acetylcholine (ACh). This hints to a specific potential role in the cortical circuitry. Yet the VIP+ /ChAT+ interneurons are sparse (a mere 0.5% of the cortical neurons), which raises questions about their potential to significantly affect the circuit function. In view of recent developments in genetic techniques that allow for direct manipulation of these neurons, we provide a thorough and updated picture of the properties of the VIP+ /ChAT+ interneurons. We discuss their genetic profile, their physiological and structural properties, and their input-output mapping in sensory cortices and the medial prefrontal cortex (mPFC). Then, we examine possible amplification mechanisms for mediating their function in the cortical microcircuit. Finally, we discuss directions for further exploration of the VIP+ /ChAT+ population, focusing on its function during behavioral tasks as compared to the VIP+ /ChAT- population.

Neuromodulation Leads to a Burst-Tonic Switch in a Subset of VIP Neurons in Mouse Primary Somatosensory (Barrel) Cortex.

Neocortical GABAergic interneurons expressing vasoactive intestinal polypeptide (VIP) contribute to sensory processing, sensorimotor integration, and behavioral control. In contrast to other major subpopulations of GABAergic interneurons, VIP neurons show a remarkable diversity. Studying morphological and electrophysiological properties of VIP cells, we found a peculiar group of neurons in layer II/III of mouse primary somatosensory (barrel) cortex, which showed a highly dynamic burst firing behavior at resting membrane potential that switched to tonic mode at depolarized membrane potentials. Furthermore, we demonstrate that burst firing depends on T-type calcium channels. The burst-tonic switch could be induced by acetylcholine (ACh) and serotonin. ACh mediated a depolarization via nicotinic receptors whereas serotonin evoked a biphasic depolarization via ionotropic and metabotropic receptors in 48% of the population and a purely monophasic depolarization via metabotropic receptors in the remaining cells. These data disclose an electrophysiologically defined subpopulation of VIP neurons that via neuromodulator-induced changes in firing behavior is likely to regulate the state of cortical circuits in a profound manner.

Somatostatin receptors (SSTR1-5) on inhibitory interneurons in the barrel cortex.

Inhibitory interneurons in the cerebral cortex contain specific proteins or peptides characteristic for a certain interneuron subtype. In mice, three biochemical markers constitute non-overlapping interneuron populations, which account for 80-90% of all inhibitory cells. These interneurons express parvalbumin (PV), somatostatin (SST), or vasoactive intestinal peptide (VIP). SST is not only a marker of a specific interneuron subtype, but also an important neuropeptide that participates in numerous biochemical and signalling pathways in the brain via somatostatin receptors (SSTR1-5). In the nervous system, SST acts as a neuromodulator and neurotransmitter affecting, among others, memory, learning, and mood. In the sensory cortex, the co-localisation of GABA and SST is found in approximately 30% of interneurons. Considering the importance of interactions between inhibitory interneurons in cortical plasticity and the possible GABA and SST co-release, it seems important to investigate the localisation of different SSTRs on cortical interneurons. Here, we examined the distribution of SSTR1-5 on barrel cortex interneurons containing PV, SST, or VIP. Immunofluorescent staining using specific antibodies was performed on brain sections from transgenic mice that expressed red fluorescence in one specific interneuron subtype (PV-Ai14, SST-Ai14, and VIP-Ai14 mice). SSTRs expression on PV, SST, and VIP interneurons varied among the cortical layers and we found two patterns of SSTRs distribution in L4 of barrel cortex. We also demonstrated that, in contrast to other interneurons, PV cells did not express SSTR2, but expressed other SSTRs. SST interneurons, which were not found to make chemical synapses among themselves, expressed all five SSTR subtypes.

Novel long-range inhibitory nNOS-expressing hippocampal cells.

The hippocampus, a brain region that is important for spatial navigation and episodic memory, benefits from a rich diversity of neuronal cell-types. Through the use of an intersectional genetic viral vector approach in mice, we report novel hippocampal neurons which we refer to as LINCs, as they are long-range inhibitory neuronal nitric oxide synthase (nNOS)-expressing cells. LINCs project to several extrahippocampal regions including the tenia tecta, diagonal band, and retromammillary nucleus, but also broadly target local CA1 cells. LINCs are thus both interneurons and projection neurons. LINCs display regular spiking non-pyramidal firing patterns, are primarily located in the stratum oriens or pyramidale, have sparsely spiny dendrites, and do not typically express somatostatin, VIP, or the muscarinic acetylcholine receptor M2. We further demonstrate that LINCs can strongly influence hippocampal function and oscillations, including interregional coherence. The identification and characterization of these novel cells advances our basic understanding of both hippocampal circuitry and neuronal diversity.

Movement and VIP Interneuron Activation Differentially Modulate Encoding in Mouse Auditory Cortex.

Information processing in sensory cortex is highly sensitive to nonsensory variables such as anesthetic state, arousal, and task engagement. Recent work in mouse visual cortex suggests that evoked firing rates, stimulus-response mutual information, and encoding efficiency increase when animals are engaged in movement. A disinhibitory circuit appears central to this change: inhibitory neurons expressing vasoactive intestinal peptide (VIP) are activated during movement and disinhibit pyramidal cells by suppressing other inhibitory interneurons. Paradoxically, although movement activates a similar disinhibitory circuit in auditory cortex (ACtx), most ACtx studies report reduced spiking during movement. It is unclear whether the resulting changes in spike rates result in corresponding changes in stimulus-response mutual information. We examined ACtx responses evoked by tone cloud stimuli, in awake mice of both sexes, during spontaneous movement and still conditions. VIP+ cells were optogenetically activated on half of trials, permitting independent analysis of the consequences of movement and VIP activation, as well as their intersection. Movement decreased stimulus-related spike rates as well as mutual information and encoding efficiency. VIP interneuron activation tended to increase stimulus-evoked spike rates but not stimulus-response mutual information, thus reducing encoding efficiency. The intersection of movement and VIP activation was largely consistent with a linear combination of these main effects: VIP activation recovered movement-induced reduction in spike rates, but not information transfer.

Vasoactive intestinal peptide infusion reverses existing myocardial fibrosis in the rat.

Congestive cardiac failure has become one of the major health challenges of the 21st century and new therapies are needed to address this problem. The concentration of vasoactive intestinal peptide (VIP) in the heart has been shown to decrease as fibrosis (the pathology leading to heart failure) increases and to become undetectable in end stage cardiomyopathy. We sought to determine whether replenishment of myocardial VIP might treat myocardial fibrosis and therefore represent a new therapeutic target. Wistar Kyoto rats on a high (4.4%) salt diet were randomised to zero time control, 4 week infusion of VIP (5 pmol/kg/min) or vehicle control infusion. Myocardial VIP concentration was measured by radioimmunoassay, fibrosis was quantitated by computerised histomorphometry and changes in pro-fibrotic mediators were measured by quantitative rt-PCR. Myocardial VIP increased significantly in VIP treated rats compared with vehicle treated controls (P < 0.01) while fibrosis in the VIP treated rats was significantly lower than in both the zero time control (P < 0.05) and the vehicle infused control (P < 0.0005). Although all six profibrotic mediators which were measured increased over the 4 week experimental period VIP infusion only affected angiotensinogen (Agt) and angiotensin receptor type 1a (AT1a) expression. In both instances VIP caused a significant decrease in messenger rna expression (Agt P < 0.01 and At1a P < 0.01) compared with vehicle infused controls. We conclude that VIP infusion increased myocardial VIP concentration and was able to reverse existing myocardial fibrosis suggesting a possible therapeutic role for a VIP based therapy in cardiac failure.

A novel class of inferior colliculus principal neurons labeled in vasoactive intestinal peptide-Cre mice.

Located in the midbrain, the inferior colliculus (IC) is the hub of the central auditory system. Although the IC plays important roles in speech processing, sound localization, and other auditory computations, the organization of the IC microcircuitry remains largely unknown. Using a multifaceted approach in mice, we have identified vasoactive intestinal peptide (VIP) neurons as a novel class of IC principal neurons. VIP neurons are glutamatergic stellate cells with sustained firing patterns. Their extensive axons project to long-range targets including the auditory thalamus, auditory brainstem, superior colliculus, and periaqueductal gray. Using optogenetic circuit mapping, we found that VIP neurons integrate input from the contralateral IC and the dorsal cochlear nucleus. The dorsal cochlear nucleus also drove feedforward inhibition to VIP neurons, indicating that inhibitory circuits within the IC shape the temporal integration of ascending inputs. Thus, VIP neurons are well-positioned to influence auditory computations in a number of brain regions.

Changes in VIP-, SP- and CGRP- like immunoreactivity in intramural neurons within the pig stomach following supplementation with low and high doses of acrylamide.

Acrylamide is one of the food toxins to which the human body is exposed. Although researchers' interest in acrylamide has been growing in recent years, the knowledge of its effect on the gastrointestinal tract, especially on intramural neurons which form the enteric nervous system is scarce. The aim of this experiment was to determine the influence of acrylamide, administered at doses equivalent to the human tolerable daily intake (TDI, 0.5 µg/kg b.w./day) and ten times higher than the TDI (5 µg/kg b.w./day), on the distribution of vasoactive intestinal peptide (VIP), substance P (SP), and calcitonin gene related peptide (CGRP) in intramural neurons of the domestic pig stomach. Using double immunofluorescent labelling we revealed that the ENS neurons underwent adaptive changes in response to the supplementation of acrylamide, which manifested themselves as increased expression of VIP, SP and CGRP, both in intramural neurons and by an increase in the nerve density in submucous and muscular layers in the porcine stomach. These substances take part in defensive reactions of neurons and transmission of sensory reactions may play an important role in protecting the stomach against the harmful effect of acrylamide. Moreover, it has been shown that acrylamide induces a significant response of ENS neurons even in TDI dose, which suggests that it is not neutral to the body. These findings may be the basis for further toxicological studies addressing the question if currently permitted minimal content of acrylamide in the food does jeopardize the health of human consumers?

Differential Innervation of Secretory Coils and Ducts in Human Eccrine Sweat Glands.

BACKGROUND: Previous studies demonstrate that eccrine sweat glands are innervated by both cholinergic and adrenergic nerves. However, it is still unknown whether the secretory coils and ducts of eccrine sweat glands are equally innervated by the sympathetic nerve fibers. To well understand the mechanisms on sweat secretion and reabsorption, the differential innervation of secretory coils and ducts in human eccrine sweat glands was investigated in the study. METHODS: From June 2016 to June 2017, six human skins were fixed, paraffin-embedded, and cut into 5 µm-thick sections, followed by costaining for nerve fiber markers protein gene product 9.5 (PGP 9.5), tyrosine hydroxylase (TH) and vasoactive intestinal peptide (VIP), and eccrine sweat gland markers K7, S100P, and K14 by combining standard immunofluorescence with tyramide signal amplification (IF-TSA). Stained sections were observed under the microscope, photographed, and analyzed. RESULTS: The fluorescent signals of PGP 9.5, TH, and VIP were easily visualized, by IF-TSA, as circular patterns surrounding eccrine sweat glands, but only PGP 9.5 could be observed by standard IF. The IF-TSA method is more sensitivity than standard IF in detecting antigens expressed at low levels. PGP 9.5, TH, and VIP appeared primarily surrounding the secretory coils and sparsely surrounding the sweat ducts. CONCLUSION: Sweat secretion is mainly controlled by autonomic nerves whereas sweat reabsorption is less affected by nerve activity.

Neurochemical characterization of intramural nerve fibres in the porcine oesophagus.

The gastrointestinal (GI) tract is innervated by nerve processes derived from the intramural enteric neurons and neurons localized outside the digestive tract. This study analysed the neurochemical characterization of nerves in the wall of the porcine oesophagus using single immunofluorescence technique. Immunoreactivity to vesicular acetylcholine transporter (VAChT), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), somatostatin (SOM), galanin (GAL), neuronal isoform of nitric oxide synthase (nNOS), substance P (SP), leucine enkephalin (LENK), calcitonin gene-related peptide (CGRP) or dopamine beta-hydroxylase (DBH) was investigated in intramuscular and intramucosal nerves of the cervical, thoracic and abdominal oesophagus. The results indicate that all of the substances studied were present in the oesophageal nerves. The density of particular populations of fibres depended on the segment of the oesophagus. The most numerous were fibres immunoreactive to VIP in the longitudinal and circular muscle layers of the abdominal oesophagus: The number of these fibres amounted to 16.4 ± 0.8 and 18.1 ± 3.1, respectively. In turn, the least numerous were CGRP-positive fibres, which were present only in the circular muscle layer of the cervical oesophagus and mucosal layer of the abdominal oesophagus in the number of 0.3 ± 0. The obtained results show that nerves in the porcine oesophageal wall are very diverse in their neurochemical coding, and differences between particular parts of the oesophagus suggest that organization of the innervation clearly depends on the fragment of this organ.

Vasoactive Intestinal Polypeptide-Immunoreactive Interneurons within Circuits of the Mouse Basolateral Amygdala.

In cortical structures, principal cell activity is tightly regulated by different GABAergic interneurons (INs). Among these INs are vasoactive intestinal polypeptide-expressing (VIP+) INs, which innervate preferentially other INs, providing a structural basis for temporal disinhibition of principal cells. However, relatively little is known about VIP+ INs in the amygdaloid basolateral complex (BLA). In this study, we report that VIP+ INs have a variable density in the distinct subdivisions of the mouse BLA. Based on different anatomical, neurochemical, and electrophysiological criteria, VIP+ INs could be identified as IN-selective INs (IS-INs) and basket cells expressing CB1 cannabinoid receptors. Whole-cell recordings of VIP+ IS-INs revealed three different spiking patterns, none of which was associated with the expression of calretinin. Genetic targeting combined with optogenetics and in vitro recordings enabled us to identify several types of BLA INs innervated by VIP+ INs, including other IS-INs, basket and neurogliaform cells. Moreover, light stimulation of VIP+ basket cell axon terminals, characterized by CB1 sensitivity, evoked IPSPs in ∼20% of principal neurons. Finally, we show that VIP+ INs receive a dense innervation from both GABAergic inputs (although only 10% from other VIP+ INs) and distinct glutamatergic inputs, identified by their expression of different vesicular glutamate transporters.In conclusion, our study provides a wide-range analysis of single-cell properties of VIP+ INs in the mouse BLA and of their intrinsic and extrinsic connectivity. Our results reinforce the evidence that VIP+ INs are structurally and functionally heterogeneous and that this heterogeneity could mediate different roles in amygdala-dependent functions.SIGNIFICANCE STATEMENT We provide the first comprehensive analysis of the distribution of vasoactive intestinal polypeptide-expressing (VIP+) interneurons (INs) across the entire mouse amygdaloid basolateral complex (BLA), as well as of their morphological and physiological properties. VIP+ INs in the neocortex preferentially target other INs to form a disinhibitory network that facilitates principal cell firing. Our study is the first to demonstrate the presence of such a disinhibitory circuitry in the BLA. We observed structural and functional heterogeneity of these INs and characterized their input/output connectivity. We also identified several types of BLA INs that, when inhibited, may provide a temporal window for principal cell firing and facilitate associative plasticity, e.g., in fear learning.

Site-dependent differences in the composite fibers of male pelvic plexus branches: an immunohistochemical analysis of donated elderly cadavers.

BACKGROUND: Although the pelvic autonomic plexus branches are considered to be a mixture of sympathetic and parasympathetic nerves, little is known regarding the composite fibers of the pelvic plexus branches. This study aimed to investigate the immunohistochemical features of sympathetic and parasympathetic nerves in the pelvic autonomic plexus branches. METHODS: Using 10 donated elderly male cadavers, the detailed topohistology of nerve fibers at and around the bladder, seminal vesicle, prostate, and rectum was examined. Neuronal nitric oxide synthase (nNOS) and vasoactive intestinal polypeptide (VIP) were used as parasympathetic nerve markers; tyrosine hydroxylase (TH) was used as a sympathetic nerve marker. The myenteric plexus of the colon was utilized as a positive control. RESULTS: Most nerve fibers in the bladder, seminal vesicle, prostate, and rectum were both nNOS- and TH-positive. Thus, pelvic plexus branches were classified into two types: 1) triple-positive mixed nerves (nNOS+, VIP+, TH+, thick myelinated fibers + or -) and 2) double-positive mixed nerves (nNOS+, VIP-, TH+, thick myelinated fibers + or -). Notably, triple-positive nerves were localized within the posterosuperior part of the plexus (near the rectum) and travelled anteroinferiorly toward the posterolateral corner of the prostate. The posteriorly and inferiorly located nerves were predominantly composed of parasympathetic, rather than sympathetic, fibers. In contrast, nerve fibers within and along the bladder and seminal vesicle contained either no or few VIP-positive nerves. These superiorly located nerves were characterized by clear sympathetic nerve dominance. CONCLUSIONS: The nerves of the pelvic plexus branches were clearly classified into nerves around the bladder and seminal vesicle (VIP-negative) and nerves around the prostate (VIP-positive). Although nNOS- and VIP-positive nerve fibers are candidate cavernous nerves, cavernous nerve identity cannot be definitively concluded for these nerves in the periprostatic region.

Long-term in vivo recording of circadian rhythms in brains of freely moving mice.

Endogenous circadian clocks control 24-h physiological and behavioral rhythms in mammals. Here, we report a real-time in vivo fluorescence recording system that enables long-term monitoring of circadian rhythms in the brains of freely moving mice. With a designed reporter of circadian clock gene expression, we tracked robust Cry1 transcription reporter rhythms in the suprachiasmatic nucleus (SCN) of WT, Cry1-/- , and Cry2-/- mice in LD (12 h light, 12 h dark) and DD (constant darkness) conditions and verified that signals remained stable for over 6 mo. Further, we recorded Cry1 transcriptional rhythms in the subparaventricular zone (SPZ) and hippocampal CA1/2 regions of WT mice housed under LD and DD conditions. By using a Cre-loxP system, we recorded Per2 and Cry1 transcription rhythms specifically in vasoactive intestinal peptide (VIP) neurons of the SCN. Finally, we demonstrated the dynamics of Per2 and Cry1 transcriptional rhythms in SCN VIP neurons following an 8-h phase advance in the light/dark cycle.

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.