Effects of p75NTR deficiency on cholinergic innervation of the amygdala and anxiety-like behavior.

The p75 neurotrophin receptor (p75NTR) is a low-affinity receptor that is capable of binding neurotrophins. Two different p75NTR knockout mouse lines are available either with a deletion in Exon III (p75NTRExIII-/- ) or in Exon IV (p75NTRExIV-/- ). In p75NTRExIII knockout mice, only the full-length p75NTR is deleted, whereas in p75NTRExIV knockout mice, the full-length as well as the truncated isoform of the receptor is deleted. Deletion of p75NTR has been shown to affect, among others, the septohippocampal cholinergic innervation pattern and neuronal plasticity within the hippocampus. We hypothesize that deletion of p75NTR also alters the morphology and physiology of a further key structure of the limbic system, the amygdala. Our results indicate that deletion of p75NTR also increases cholinergic innervation in the basolateral amygdala in adult as well as aged p75NTRExIII-/- and p75NTRExIV-/- mice. The p75NTRExIV-/- mice did not display altered long-term potentiation (LTP) in the basolateral amygdala as compared to age-matched control littermates. However, p75NTRExIII-/- mice display stronger LTP in the basolateral amygdala compared to age-matched controls. Bath-application of K252a (a trk antagonist) did not inhibit the induction of LTP in the basolateral amygdala, but reduced the level of LTP in p75NTRExIII-/- mice to levels seen in respective controls. Moreover, p75NTRExIII-/- mice display altered behavior in the dark/light box. Thus, deletion of p75NTR in mice leads to physiological and morphological changes in the amygdala and altered behavior that is linked to the limbic system.

Analysis of nerve supply pattern in human lymphatic vessels of young and old men.

BACKGROUND: The present work deals with innervation patterns along collector lymphatic vessels from cervical, mesenteric, and femoral regions, and lymph capillaries in young and elderly subjects. METHODS AND RESULTS: Morphological and morphometric analysis of nerve fibers along lymph vessels was performed by immunohistochemistry for PGP 9.5, NPY, TH, ChAT, VIP, SP, and dopamine. Nerves containing NPY and TH were frequent, whereas immunoreactivity for ChAT and VIP were few. SP-positive fibers were widely distributed in the medial and endothelial layers. Dopamine neurotransmitters were observed in a few short nerve fibers. A more diffuse presence of nerve fibers in mesenteric and femoral lymph vessels, compared to cervical ones, was detected. In lymph capillary vessels, a few nerve fibers positive for neuropeptides and neurotransmitters were detected, whereas no dopamine and VIP immunoreactive fibers were detected. A wide reduction of all specific nerve fibers analyzed was detected in lymph vessels from elderly subjects. CONCLUSIONS: The presence on lymph vessels of sympathetic and parasympathetic nerve systems can be declared. The differences observed in lymphatic vessel innervation patterns may note the involvement in lymph flow regulation, calling attention in aging, when nerve fibers reduction may cause functional default of lymph vessels.

Cholinergic modulation of hippocampal cells and circuits.

Septo-hippocampal cholinergic fibres ramify extensively throughout the hippocampal formation to release acetylcholine upon a diverse range of muscarinic and nicotinic acetylcholine receptors that are differentially expressed by distinct populations of neurones. The resultant modulation of cellular excitability and synaptic transmission within hippocampal circuits underlies the ability of acetylcholine to influence the dynamic properties of the hippocampal network and results in the emergence of a range of stable oscillatory network states. Recent findings suggest a multitude of actions contribute to the oscillogenic properties of acetylcholine which are principally induced by activation of muscarinic receptors but also regulated through activation of nicotinic receptor subtypes.

Quantitative immunohistochemical investigation of the intrinsic vasodilator innervation of the guinea pig lingual artery.

The vasculature of the guinea pig tongue is supplied by parasympathetic vasodilator nerve fibres of intrinsic origin. Here, we investigated first to what extent neuropeptides and the synthesizing enzymes of NO, CO and acetylcholine are contained and colocalized within periarterial lingual vasodilator axons of intrinsic origin. Then it was determined whether perivascular innervation by these fibre types changes with vascular diameter, in particular in comparison with the sensory substance P (SP)-positive and sympathetic noradrenergic vascular innervation. To this end, single, double and triple labelling histochemical techniques were performed on control tongues and tongues kept in short-term organotypic culture to induce degeneration of extrinsically originating nerve fibres. Cell bodies of intrinsic microganglia and their periarterial axons contained, simultaneously, NO synthase, vasoactive intestinal peptide and the acetylcholine-synthesizing enzyme choline acetyltransferase. Additionally, neuropeptide Y (NPY) was observed in a small percentage (12%) of neurons that increased to 39% after 36 h of organotypic culture. The CO synthesizing enzyme heme oxygenase-2 was detected only in perikarya but not in periarterial axons. Intrinsic vasodilator fibres were invariably present at arteries down to a luminal diameter of 150 microm, and reached 65% of section profiles of smallest arterioles, while noradrenergic and substance P-positive axons reached 80% of arteriolar profiles. These findings show that the intrinsic lingual vasodilator innervation of the guinea pig is far extending although slightly less developed than that by sensory and sympathetic axons, and differs both in this aspect and in patterns of colocalization from that reported for other organs, e.g. lung and pelvic organs.

Spatial segregation within the sacral parasympathetic nucleus of neurons innervating the bladder or the penis of the rat as revealed by three-dimensional reconstruction.

The purpose of the present investigations was (1) to examine the spatial organization of preganglionic neurons of the sacral parasympathetic nucleus in the lumbosacral spinal cord of male adult rats and (2) to search, in this nucleus, for a possible segregation of sub-populations of neurons innervating the penis or the bladder, respectively. To estimate their spatial organization, neurons of the sacral parasympathetic nucleus were retrogradely labeled by wheat germ agglutinin coupled to horseradish peroxidase applied to the central end of the sectioned pelvic nerve. The sub-populations of lumbosacral neurons innervating the corpus cavernosum of the penis or the dome of the bladder were identified using transsynaptic retrograde labeling by pseudorabies virus injected into these organs in different rats. In both wheat germ agglutinin-labeled and pseudorabies virus-labeled rats, serial coronal sections were cut through the spinal L5-S1 segments. Labeled neurons were revealed by histochemistry (peroxidase experiments) or immunohistochemistry (pseudorabies virus experiments). By means of a three-dimensional reconstruction software developed in our laboratory, three-dimensional models were calculated from each spinal section image series. They revealed the spatial organization of (i) preganglionic neurons and (ii) neurons innervating the bladder or the penis. The different three-dimensional models were subsequently merged into a single one which revealed the segregation, within the sacral parasympathetic nucleus, of the sub-populations of neurons. Neurons labeled by virus injected into the penis extended predominantly from the rostral part of the L6 segment to the rostral part of the S1 segment while those labeled by bladder injections were distributed predominantly from the caudal part of the L6 segment to the caudal part of the S1 segment. These results support the hypothesis of a viscerotopic organization of sacral neurons providing the spinal control of pelvic organs.

Distribution, chemical coding and origin of nitric oxide synthase-containing nerve fibres in the guinea pig nasal mucosa.

The distribution, chemical coding and origin of nitric oxide synthase (NOS)-containing nerve fibres in the respiratory mucosa of the nasal septum of the guinea pig were examined using nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry and immunohistochemistry. A rich supply of NADPH-d-positive nerve fibres was observed around blood vessels and in nasal glands where nerve fibres frequently penetrated into the epithelia of acini and intralobular ducts. NADPH-d reactivity was also found in the nerve fibres located under or within the respiratory epithelium. Combined immunofluorescence and histochemical staining of the same preparation demonstrated virtually complete overlapping of NOS immunoreactivity and NADPH-d reactivity in nerve fibres, indicating that NADPH-d can be used as a marker for NOS-containing neurons. Double-labelling using antibodies to vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), and calcitonin gene-related peptide (CGRP) revealed that NADPH-d-positive nerve fibres frequently contained VIP or NPY, but not CGRP. Pterygopalatine ganglionectomy significantly reduced the number of NADPH-d-positive nerve fibres innervating the respiratory epithelium as well as blood vessels and nasal glands. Neither superior cervical ganglionectomy nor sensory denervation by capsaicin treatment affected the distribution of NADPH-d-positive fibres. These results indicate that NOS-containing nerve fibres innervating the respiratory epithelium as well as blood vessels and nasal glands in the guinea pig originate mainly from the pterygopalatine ganglion, and suggest that NO may play a significant role as a neurotransmitter and/or neuromodulator in the control of the respiratory epithelium as well as vasculature and nasal glands.

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.

Relative amounts of mRNA encoding four subtypes of muscarinic receptors (m2-m5) in human peripheral blood mononuclear cells.

It is known that lymphocytes express functional muscarinic cholinergic receptors. In this study, RT-PCR method was applied to study the presence and relative levels of mRNA encoding muscarinic receptor subtypes in human peripheral blood mononuclear cells (PBMCs). Our results, confirmed by DNA sequencing, demonstrate the presence of m2, m3, m4, and m5 receptor subtypes in human PBMCs. The relative levels of muscarinic receptor subtypes fit the following pattern: m3 > m5 > m4 > m2. Our data provide strong evidence confirming previous pharmacological studies that suggested the existence of several subtypes of muscarinic receptors on human PBMCs. We cannot exclude the possibility that expression of receptor subtype depends on the lineage and/or activation status of the cell.

Direct measurement of acetylcholine release in detrusor smooth muscles isolated from rabbits.

In the present study, we measured acetylcholine (ACh) released from rabbit detrusor smooth muscle strips induced by electrical field stimulation (EFS) using high-performance liquid chromatography coupled with microdialysis procedure. There were frequency- and duration-dependent increases in contractile response and ACh release. There was a significant, but not simple correlation between EFS-induced contraction and ACh release. Atropine caused a decrease and increase in the contractile response and ACh release, respectively. Pretreatment with propranolol increased ACh release, but pretreatment with phentolamine had no significant effect. These results demonstrate that this method is applicable to direct measurement of ACh release by EFS, and that neurotransmitters other than ACh may relate to EFS-induced contraction. In addition, it is suggested that there are prejunctional inhibitory muscarinic receptors and beta-adrenoceptors, which contribute to ACh release induced by EFS in the rabbit detrusor smooth muscles.

Inhibition of vagal vasodilatation by a selective neuropeptide Y Y2 receptor agonist in the bronchial circulation of anaesthetised dogs.

Neuropeptide Y (NPY) is both co-stored and co-released with noradrenaline from sympathetic nerve terminals. In the cardiovascular system, NPY acts on two main receptor subtypes. At postjunctional, or Y1 receptors, NPY can cause both direct vasoconstriction and the potentiation of various constrictor agents. NPY acting at the presynaptic, or Y2 receptor, inhibits the release of neurotransmitter from autonomic nerves. In the present paper, we have used both sympathetic stimulation and the selective NPY Y2 receptor agonist, N-acetyl [Leu28,Leu31] NPY24-36, to examine the role of NPY in the inhibition of vagally mediated vasodilatation in the bronchial circulation of the anaesthetised dog. Stimulation of the cardiac end of the cervical vagus nerve at 1 Hz for 15 s (1 ms, 70 V) increased bronchial vascular conductance by 45%. This increase in flow was abolished by atropine. Sympathetic stimulation for 2.5 min at 16 Hz (1 ms, 20 V) produced a significant (P < 0.05) and prolonged (9 min) inhibition of the subsequent parasympathetically evoked vasodilatation. Similarly, the NPY Y2 receptor agonist, N-acetyl [Leu28,Leu31] NPY24-36, produced a significant (P < 0.05) and prolonged (15 min) inhibition of parasympathetically evoked vasodilatation. When vagus was stimulated at 2.5 Hz for 30 s (1 ms, 70 V), an atropine-resistant, but capsaicin-sensitive vasodilatation was observed. Neither sympathetic stimulation nor the NPY Y2 receptor agonist could be demonstrated to inhibit this vasodilatation. These results suggest that NPY can inhibit cholinergic parasympathetic vasodilatation in the bronchial circulation by an action on NPY Y2 receptors.

Parasympathetic non-adrenergic, non-cholinergic mechanisms in salivary glands and their role in reflex secretion.

Parasympathetic atropine-resistant vasodilatation is a well-known phenomenon in salivary glands. Less well known is the fact that there also exists an atropine-resistant secretory response to parasympathetic stimulation: some glands secrete saliva, albeit at a reduced rate, whereas others just release protein and acinar secretory granules. A number of peptides, including vasoactive intestinal peptide (VIP) and substance P, are likely to be involved in the so called non-adrenergic, non-cholinergic (NANC) transmission of the parasympathetic secretory impulses. We here report a series of experiments where we have found these NANC-mechanisms to be mobilized during reflex secretion. Loss of acinar granules, morphometrically assessed, and reduction in glandular amylase activity indicated secretory activity in the parotid gland of the conscious rat, in the presence of atropine and adrenoceptor antagonists, in response to food intake. As judged by these indices, the NANC mechanisms were potentially responsible for the whole parasympathetic response and, thus, for the major part of the marked response in the absence of the antagonists. Furthermore, the reflex mobilization of the NANC mechanisms depended on mastication rather than on taste, and involved the release of VIP and substance P. The results presented give weight to a physiological role for these mechanisms, and support the idea that, under normal conditions, the NANC mechanisms act in concert with cholinergic and adrenergic mechanisms to generate the most purposeful secretory reflex response to the afferent stimulation.

Spatial distribution of nerve processes and beta-adrenoreceptors in the rat atrioventricular node.

Atrioventricular (AV) nodal conduction time is known to be modulated by the autonomic nervous system. The presence of numerous parasympathetic and sympathetic nerve fibres in association with conduction tissue in the heart is well authenticated. In this study, confocal microscopy was used to image the distribution of antibodies directed against the general neuronal marker PGP 9.5, tyrosine hydroxylase (TH), vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP) and beta1 and beta2-adrenoreceptors. Serial 12 microm sections of fresh frozen tissue taken from the frontal plane of the rat atrioventricular node, His bundle and bundle branches were processed for histology, acetylcholinesterase (AChE) activity and immunohistochemistry. It was found that the AV and ventricular conduction systems were more densely innervated than the atrial and ventricular myocardium as revealed by PGP 9.5 immunoreactivity. Furthermore, the transitional cell region was more densely innervated than the midnodal cell region, while spatial distribution of total innervation was uniform throughout all AV nodal regions. AChE-reactive nerve processes were found throughout the AV and ventricular conduction systems, the spatial distribution of which was nonuniform exhibiting a paucity of AChE-reactive nerve processes in the central midnodal cell region and a preponderance in the circumferential transitional cell region. TH-immunoreactivity was uniformly distributed throughout the AV and ventricular conduction systems including the central midnodal and circumferential transitional cell regions. Beta1-adrenoreceptors were found throughout the AV and ventricular conduction systems with a preponderance in the circumferential transitional cell region. Beta2-adrenoreceptors were localised predominantly in AV and ventricular conduction systems with a paucity of expression in the circumferential transitional cell region. These results demonstrate that the overall uniform distribution of total nerve processes is comprised of nonuniformly distributed subpopulations of parasympathetic and sympathetic nerve processes. The observation that the midnodal cell region exhibits a differential spatial pattern of parasympathetic and sympathetic innervation suggests multiple sites for modulation of impulse conduction within this region. Moreover, the localisation of beta2-ARs in the AV conduction system, with an absence of expression in the circumferential transitional cell layer, suggests that subtype-specific pharmacological agents may have distinct effects upon AV nodal conduction.

Reciprocal regulation of ciliary neurotrophic factor receptors and acetylcholine receptors during synaptogenesis in embryonic chick atria.

Ciliary neurotrophic factor (CNTF) has been implicated in the development, survival, and maintenance of a broad range of neurons and glia in the peripheral nervous system and the CNS. Evidence also suggests that CNTF may affect development of cells outside the nervous system. We have found that functional CNTF and its receptor are expressed in developing embryonic chick heart and may be involved in parasympathetic synapse formation. CNTF and CNTF receptor mRNA levels were highest at embryonic day 11 (E11)-E13, the period of parasympathetic innervation in chick atria. Levels of atrial CNTF receptor mRNA were fourfold greater at E13 than at E6 and at E13 were 2.5-fold higher in atria than in ventricle, corresponding to the higher degree of parasympathetic innervation occurring in atria. Treatment of isolated atria or cultured atrial myocytes with recombinant human or avian CNTF resulted in the tyrosine phosphorylation and nuclear translocation of the signal transducer and activator of transcription STAT3. The developmental increase in atrial CNTF receptor mRNA was enhanced by stimulating muscarinic receptors with carbachol in ovo and was inhibited by blocking muscarinic cholinergic receptors with atropine. Treatment of cultured atrial myocytes with CNTF resulted in a twofold increase in the levels of muscarinic receptors. Thus, CNTF was able to regulate a key component of parasympathetic synapses on atrial myocytes. These results suggest a postsynaptic role for CNTF in the onset of parasympathetic function in the developing heart and provide new clues to molecular mechanisms directing synapse formation at targets of the autonomic nervous system.

Stimulation of NTS activates NMDA and non-NMDA receptors in rat cardiac vagal neurons in the nucleus ambiguus.

While it is widely accepted that tonic and reflex changes in cardiac vagal activity play significant roles in cardiovascular function, little is known about the synaptic pathways in the brainstem responsible for the control of cardiac vagal neurons in the nucleus ambiguus (NA). In this study, we identified the principal post-synaptic receptors activated in cardiac vagal neurons upon stimulation of the nucleus tractus solitarius (NTS). Cardiac vagal neurons were identified by the presence of a retrograde fluorescent tracer and were visualized in rat brainstem slices. Perforated patch clamp techniques were used to record post-synaptic currents. NTS stimulation activated glutamatergic currents in cardiac vagal neurons with a typical delay of 8-18 ms. Post-synaptic responses were separated into NMDA and non-NMDA components using D-2-amino-5-phophonovalerate (AP5) and 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX), respectively. In conclusion, this study characterizes a monosynaptic glutamatergic pathway from NTS that activates NMDA and kainate/AMPA post-synaptic receptors in cardiac vagal neurons.

Functional evaluation of inhibition of autonomic transmitter release by autoantibody from Lambert-Eaton myasthenic syndrome.

The effects of the anti-voltage-gated Ca2+ channel (VGCC) antibody obtained from patients with Lambert-Eaton myasthenic syndrome (LEMS) on autonomic neurotransmission were studied in in-vitro experiments. The releases of acetylcholine (ACh) and norepinephrine from the autonomic nerves were evaluated by changes in the contractile responses of guinea pig taenia caeci and left atria to electric field stimulation, respectively. Incubations for 6 hours with LEMS serum and IgG, both of which contain anti-VGCC antibody, markedly suppressed the parasympathetic response but did not affect the sympathetic response. Pharmacological experiments with specific blockers to the VGCC subtypes showed that the Q-type VGCC is closely linked to the genesis of the parasympathetic response. We suggest that the anti-VGCC antibody from the LEMS patients specifically reduces the ACh release from the parasympathetic nerve by binding to the Q-type VGCC.

Nitrergic innervation of the cerebral arterial tree in the bent-winged bat (mammalia: Microchiroptera).

The distribution and origin of cerebrovascular nitrergic nerves were studied immunohistochemically and histochemically in the bent-winged bat. The supply of nitric oxide synthase (NOS)-immunoreactive (IR) and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd)-positive nerves to the bat major cerebral arteries differs from the general mammalian pattern in that it is preferential for the vertebrobasilar system (VBS) as opposed to the internal carotid system. Interestingly, a few nerve cells with bright NOS immunofluorescence and intense NADPHd activity were localized in the walls of the vertebral artery (VA) and basilar artery (BA) from many individual bats. Cerebral perivascular NOS-IR nerves were generally immunoreactive for vasoactive intestinal polypeptide (VIP). NOS-IR neurons intrinsic to the BA and VA expressed variable degrees of VIP immunoreactivity and showed no acetylcholinesterase (AChE) activity. Most cell bodies of the microganglia (MG) in the carotid canal and tympanic cavity, and those of the cranial and cervical facial ganglia, showed both NOS and VIP immunoreactivities and were stained intensely for NADPHd. From these and other findings, it is suggested that, in the bent-winged bat at least, the BA and VA of the cerebral arterial tree are frequently dually innervated by two neurochemically defined nitrergic neurons, the cranial parasympathetic VIP-IR and AChE-positive neurons, which are derived mainly from the MG via the internal carotid artery, and the intrinsic neurons, either IR or immunonegative for VIP but negative for AChE, which form an outflow tract from some caudally located ganglia projecting to the VBS via the VA.

Cultures of airway parasympathetic nerves express functional M2 muscarinic receptors.

To study the control of acetylcholine release from airway parasympathetic neurons, primary cultures of these cells were established. Guinea pig tracheas were disaggregated with collagenase and plated onto matrigel-coated plates in medium that contained cytosine arabinoside to inhibit growth of dividing cells. Over 7 to 10 days neurites grow from the cell bodies, reaching a length of 2 mm. The vast majority of the cells in these cultures were neurons, as identified by morphology and staining with Neurotag and with antibody to neuron-specific antigen protein gene product 9.5. Cultured neurons contained acetylcholine, which was released by electrical field stimulation. Thus these were parasympathetic neurons. Staining with antibodies to M1, M2, and M4 muscarinic receptors revealed the presence of only M2 receptors. Likewise, reverse transcription-polymerase chain reaction using primers for M1, M2, and M4 muscarinic receptors revealed mRNA only for M2 receptors. Blocking these M2 receptors using atropine potentiated the stimulated release of acetylcholine, demonstrating that the M2 receptors inhibit acetylcholine release, as they have been shown to do in vivo. Thus airway parasympathetic neurons can be grown in culture, they retain the ability to synthesize and release acetylcholine, and they express functional inhibitory M2 muscarinic receptors.

Sympathetic and parasympathetic regulation of the uterine blood flow and contraction in the rat.

The effects of electrical stimulation of hypogastric sympathetic and pelvic parasympathetic nerves on uterine blood flow and contraction in anesthetized female non-pregnant normal cycling rats were examined. Electrical stimulation of the efferent pelvic nerve with supramaximal intensity induced marked increase of uterine blood flow accompanied by uterine contraction. On the other hand, the stimulation of efferent hypogastric nerve caused decrease of uterine blood flow accompanied by uterine contraction. These responses could only be elicited with stimulus intensity above the threshold for unmyelinated C fibers in both the hypogastric and pelvic nerves. Intravenous administration of atropine (0.5 mg/kg) totally blocked the response of uterine contraction elicited by pelvic and hypogastric nerve stimulation and also the increase of blood flow induced by pelvic nerve stimulation. Intravenous administration of phenoxybenzamine (0.5 mg/kg) blocked the decreased response of uterine blood flow induced by hypogastric nerve stimulation. It was concluded that uterine blood flow and contraction were regulated by both the parasympathetic and sympathetic nerves, but in different manners; blood flow is regulated reciprocally (1) by parasympathetic vasodilators mainly via activation of muscarinic cholinergic receptors, and (2) by sympathetic vasoconstrictors via activation of alpha-adrenergic receptors; contraction is produced by activation of both parasympathetic and sympathetic nerves via muscarinic cholinergic receptors.

Structure and chemical coding of human, canine and opossum gallbladder ganglia.

Immunohistochemistry and cholinesterase histochemistry were used to evaluate the structure and neurotransmitter content of the ganglionated plexuses of the human, canine, and opossum (Monodelphis domestica) gallbladders. In each species, the ganglionated plexus consisted of small (mean approximately 4 neurons/ganglion), irregularly dispersed ganglia that were interconnected by bundles of nerve fibers. The density of ganglia was about ten-fold higher in the opossum than in the human or the dog. Immunostaining for choline acetyltransferase (ChAT) was accomplished in the human, dog, opossum, and the guinea pig where all neurons were found to express ChAT-immunoreactivity. In the human, immunoreactivities for vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) were the most abundant followed by substance P (SP). In the dog, immunoreactivity for galanin (GAL) was the strongest, followed closely by VIP and then by SP. NPY-immunoreactive neurons were not observed in the dog, but immunoreactive nerve fibers were seen in the perivascular plexus. In the opossum, immunoreactivity for GAL was the most intense and abundant followed by SP, which was followed by VIP. NPY-immunoreactivity in the opossum was limited to scarce perivascular nerve fibers. Immunoreactivity for calcitonin-gene-related peptide (CGRP) was not observed in neuronal somata, but CGRP/SP-immunoreactive nerve fibers were a feature of each species studied. These findings, along with previously published work on the guinea pig, indicate that it is likely that all gallbladder neurons are cholinergic, and that VIP, SP, and NPY and/or GAL are commonly expressed in gallbladder neurons.

Galantide distinguishes putative subtypes of galanin receptors in mudpuppy parasympathetic neurons.

The effect of the chimeric ligand galantide on the galanin-induced activation of membrane K+ conductance and inhibition of voltage-dependent Ca2+ conductance has been studied using voltage-clamped dissociated mudpuppy parasympathetic neurons. Galantide did not activate the K+ conductance but produced a concentration-dependent antagonism (IC50 = 4 nM) of the galanin-induced increase in K+ conductance. Galantide acted like galanin and inhibited the voltage-dependent Ba2+ current (IBa). The inhibition of IBa also was concentration dependent (IC50 = 16 nM) and the maximum inhibition produced by galantide was approximately 40%. We also demonstrate that the galanin-(1-16) fragment increased the membrane K+ conductance and decreased IBa, suggesting that the NH2 portion of the galanin molecule is sufficient to mediate both actions. One interpretation of these observations is that different galanin receptors mediate the different effects of galanin on the mudpuppy parasympathetic neurons.