Gut microbiota and neuroinflammation in pathogenesis of hypertension: A potential role for hydrogen sulfide.

Inflammation and gut dysbiosis are hallmarks of hypertension (HTN). Hydrogen sulfide (H2S) is an important freely diffusing molecule that modulates the function of neural, cardiovascular and immune systems, and circulating levels of H2S are reduced in animals and humans with HTN. While most research to date has focused on H2S produced endogenously by the host, H2S is also produced by the gut bacteria and may affect the host homeostasis. Here, we review an association between neuroinflammation and gut dysbiosis in HTN, with special emphasis on a potential role of H2S in this interplay.

Cholinergic Pathway Suppresses Pulmonary Innate Immunity Facilitating Pneumonia After Stroke.

BACKGROUND AND PURPOSE: Temporary immunosuppression has been identified as a major risk factor for the development of pneumonia after acute central nervous system injury. Although overactivation of the sympathetic nervous system was previously shown to mediate suppression of systemic cellular immune responses after stroke, the role of the parasympathetic cholinergic anti-inflammatory pathway in the antibacterial defense in lung remains largely elusive. METHODS: The middle cerebral artery occlusion model in mice was used to examine the influence of the parasympathetic nervous system on poststroke immunosuppression. We used heart rate variability measurement by telemetry, vagotomy, α7 nicotinic acetylcholine receptor-deficient mice, and parasympathomimetics (nicotine, PNU282987) to measure and modulate parasympathetic activity. RESULTS: Here, we demonstrate a rapidly increased parasympathetic activity in mice after experimental stroke. Inhibition of cholinergic signaling by either vagotomy or by using α7 nicotinic acetylcholine receptor-deficient mice reversed pulmonary immune hyporesponsiveness and prevented pneumonia after stroke. In vivo and ex vivo studies on the role of α7 nicotinic acetylcholine receptor on different lung cells using bone marrow chimeric mice and isolated primary cells indicated that not only macrophages but also alveolar epithelial cells are a major cellular target of cholinergic anti-inflammatory signaling in the lung. CONCLUSIONS: Thus, cholinergic pathways play a pivotal role in the development of pulmonary infections after acute central nervous system injury.

Interleukin-1ß mediates virus-induced m2 muscarinic receptor dysfunction and airway hyperreactivity.

Respiratory viral infections are associated with the majority of asthma attacks. Inhibitory M2 receptors on parasympathetic nerves, which normally limit acetylcholine (ACh) release, are dysfunctional after respiratory viral infection. Because IL-1ß is up-regulated during respiratory viral infections, we investigated whether IL-1ß mediates M2 receptor dysfunction during parainfluenza virus infection. Virus-infected guinea pigs were pretreated with the IL-1ß antagonist anakinra. In the absence of anakinra, viral infection increased bronchoconstriction in response to vagal stimulation but not to intravenous ACh, and neuronal M2 muscarinic receptors were dysfunctional. Pretreatment with anakinra prevented virus-induced increased bronchoconstriction and M2 receptor dysfunction. Anakinra did not change smooth muscle M3 muscarinic receptor response to ACh, lung viral loads, or blood and bronchoalveolar lavage leukocyte populations. Respiratory virus infection decreased M2 receptor mRNA expression in parasympathetic ganglia extracted from infected animals, and this was prevented by blocking IL-1ß or TNF-α. Treatment of SK-N-SH neuroblastoma cells or primary cultures of guinea pig parasympathetic neurons with IL-1ß directly decreased M2 receptor mRNA, and this was not synergistic with TNF-α treatment. Treating guinea pig trachea segment with TNF-α or IL-1ß in vitro increased tracheal contractions in response to activation of airway nerves by electrical field stimulation. Blocking IL-1ß during TNF-α treatment prevented this hyperresponsiveness. These data show that virus-induced hyperreactivity and M2 dysfunction involves IL-1ß and TNF-α, likely in sequence with TNF-α causing production of IL-1ß.

α7-cholinergic receptor mediates vagal induction of splenic norepinephrine.

Classically, sympathetic and parasympathetic systems act in opposition to maintain the physiological homeostasis. In this article, we report that both systems work together to restrain systemic inflammation in life-threatening conditions such as sepsis. This study indicates that vagus nerve and cholinergic agonists activate the sympathetic noradrenergic splenic nerve to control systemic inflammation. Unlike adrenalectomy, splenectomy and splenic neurectomy prevent the anti-inflammatory potential of both the vagus nerve and cholinergic agonists, and abrogate their potential to induce splenic and plasma norepinephrine. Splenic nerve stimulation mimics vagal and cholinergic induction of norepinephrine and re-establishes neuromodulation in α7 nicotinic acetylcholine receptor (α7nAChR)-deficient animals. Thus, vagus nerve and cholinergic agonists inhibit systemic inflammation by activating the noradrenergic splenic nerve via the α7nAChR nicotinic receptors. α7nAChR represents a unique molecular link between the parasympathetic and sympathetic system to control inflammation.

Autonomic nervous system modulation affects the inflammatory immune response in mice with acute Chagas disease.

The aim of the present study was to evaluate the effects of changes to the autonomic nervous system in mice during the acute phase of Chagas disease, which is an infection caused by the parasite Trypanosoma cruzi. The following types of mice were inoculated with T. cruzi (CHG): wild-type (WT) and vesicular acetylcholine transporter knockdown (KDVAChT) C57BL/6j mice; wild-type non-treated (NT) FVB mice; FVB mice treated with pyridostigmine bromide (PYR) or salbutamol (SALB); and ß(2)-adrenergic receptor knockout (KOß2) FVB mice. During infection and at 18-21 days after infection (acute phase), the survival curves, parasitaemia, electrocardiograms, heart rate variability, autonomic tonus and histopathology of the animals were evaluated. Negative control groups were matched for age, genetic background and treatment. The KDVAChT-CHG mice exhibited a significant shift in the electrocardiographic, autonomic and histopathological profiles towards a greater inflammatory immune response that was associated with a reduction in blood and tissue parasitism. In contrast, the CHG-PYR mice manifested reduced myocardial inflammation and lower blood and tissue parasitism. Similar results were observed in CHG-SALB animals. Unexpectedly, the KOß2-CHG mice exhibited less myocardial inflammation and higher blood and tissue parasitism, which were associated with reduced mortality. These findings could have been due to the increase in vagal tone observed in the KOß2 mice, which rendered them more similar to the CHG-PYR animals. In conclusion, our results indicate a marked immunomodulatory role for the parasympathetic and sympathetic autonomic nervous systems, which inhibit both the inflammatory immune response and parasite clearance during the acute phase of experimental Chagas heart disease in mice.

Neurotransmitters in airway parasympathetic neurons altered by neurotrophin-3 and repeated allergen challenge.

Changes in airway nerves associated with chronic inflammation may underlie the pathogenesis and symptoms of lower airway diseases, such as asthma. The molecules most likely causing such alterations are neurotrophins (NTs) and/or related neurokines. In several species, including humans, lower airway parasympathetic postganglionic neurons that project axons to airway smooth muscle are either cholinergic or nonadrenergic noncholinergic (NANC), the latter synthesizing vasoactive intestinal peptide and nitric oxide, but not acetylcholine. In guinea pig trachealis smooth muscle, cholinergic nerve terminals arise from ganglionic neurons located near the tracheal smooth muscle, whereas the source of NANC nerve fibers is from neurons in ganglia located in the adjacent myenteric plexus of the esophagus, making this an ideal species to study regulation of parasympathetic neurotransmitter phenotypes. In the present study, we determined that, 48 hours after repeated allergen challenge, the NANC phenotype of airway parasympathetic ganglionic neurons changed to a cholinergic phenotype, and NT-3 mimicked this change. Nerve growth factor, brain-derived neurotrophic factor, leukemia inhibitory factor, or IL-1ß had no effect on either phenotype, and they did not induce these neurons to synthesize substance P or tyrosine hydroxylase. These results indicate a role for inflammation and NT-3 in regulating biochemical and anatomical characteristics of principal neurons in adult airway parasympathetic ganglia.

Historical, pathophysiological, and therapeutic aspects of vidian neurectomy.

Vidian neurectomy yields dramatic relief of nasal hypersecretion in patients with allergic rhinitis. Clinical studies conducted on vidian neurectomized nasal mucosa have shown that nasal hypersecretion observed after challenging the nasal mucosa with antigen is caused by reflexively induced activation of the parasympathetic center secondary to stimulation of the sensory nerve terminals in the nasal mucosa by histamine. On the contrary, nasal mucosal swelling is caused mostly by the direct effects of chemical mediators on the nasal vasculature, although vascular reflex mediated by the noncholinergic parasympathetic nerve may be partially involved in the onset of nasal mucosal swelling after antigen challenge. Considering the long-term side effects of inhibition of lacrimation and possible partial recurrence of hyperreactive nasal symptoms observed after vidian neurectomy, less invasive endoscopic posterior nasal neurectomy is considered the treatment of choice for patients with allergic rhinitis who require surgical intervention.

Does an acute inflammatory response temporarily attenuate parasympathetic reactivation?

PURPOSE: Although observational studies suggest that inflammatory markers are associated with autonomic nervous system function, the causal relationship of this is not clear. We tested the hypothesis that acute inflammation will temporarily attenuate vagal reactivation as measured by heart rate recovery after exercise. METHODS: In this double-blind randomized study, 24 healthy subjects were assigned to receive either an influenza vaccine (n = 15) as a model to generate a systemic inflammatory response or a sham vaccine (n = 9). Heart rate recovery after exercise testing was used as an index of parasympathetic nervous function and was calculated as the difference between maximal heart rate during the test and heart rate 1 and 2 min after cessation of exercise. Both blood analysis and treadmill exercise stress tests were conducted before and 48 h after each vaccination. RESULTS: Inflammatory marker, log C-reactive protein (1.9 +/- 1.2 to 2.8 +/- 1.4, p < 0.05) was significantly increased after the influenza vaccine. Heart rate recovery 1 was significantly attenuated 48 h after the influenza vaccination (23.4 +/- 6.4 to 20.5 +/- 4.9, p < 0.05) but not sham vaccination. CONCLUSIONS: These findings show that acute inflammation is associated with a temporary deterioration in cardiac autonomic nervous system function in healthy subjects.

Atropine-enhanced, antigen challenge-induced airway hyperreactivity in guinea pigs is mediated by eosinophils and nerve growth factor.

Although anticholinergic therapy inhibits bronchoconstriction in asthmatic patients and antigen-challenged animals, administration of atropine 1 h before antigen challenge significantly potentiates airway hyperreactivity and eosinophil activation measured 24 h later. This potentiation in airway hyperreactivity is related to increased eosinophil activation and is mediated at the level of the airway nerves. Since eosinophils produce nerve growth factor (NGF), which is known to play a role in antigen-induced airway hyperreactivity, we tested whether NGF mediates atropine-enhanced, antigen challenge-induced hyperreactivity. Antibody to NGF (Ab NGF) was administered to sensitized guinea pigs with and without atropine pretreatment (1 mg/kg iv) 1 h before challenge. At 24 h after challenge, animals were anesthetized, vagotomized, paralyzed, and ventilated. Electrical stimulation of both vagus nerves caused bronchoconstriction that was increased in challenged animals. Atropine pretreatment potentiated antigen challenge-induced hyperreactivity. Ab NGF did not affect eosinophils or inflammatory cells in any group, nor did it prevent hyperreactivity in challenged animals that were not pretreated with atropine. However, Ab NGF did prevent atropine-enhanced, antigen challenge-induced hyperreactivity and eosinophil activation (assessed by immunohistochemistry). This effect was specific to NGF, since animals given control IgG remained hyperreactive. These data suggest that anticholinergic therapy amplifies eosinophil interactions with airway nerves via NGF. Therefore, therapeutic strategies that target both eosinophil activation and NGF-mediated inflammatory processes in allergic asthma are likely to be beneficial.

Neuronal eotaxin and the effects of CCR3 antagonist on airway hyperreactivity and M2 receptor dysfunction.

Eosinophils cluster around airway nerves in patients with fatal asthma and in antigen-challenged animals. Activated eosinophils release major basic protein, which blocks inhibitory M2 muscarinic receptors (M2Rs) on nerves, increasing acetylcholine release and potentiating vagally mediated bronchoconstriction. We tested whether GW701897B, an antagonist of CCR3 (the receptor for eotaxin as well as a group of eosinophil active chemokines), affected vagal reactivity and M2R function in ovalbumin-challenged guinea pigs. Sensitized animals were treated with the CCR3 antagonist before inhaling ovalbumin. Antigen-challenged animals were hyperresponsive to vagal stimulation, but those that received the CCR3 antagonist were not. M2R function was lost in antigen-challenged animals, but not in those that received the CCR3 antagonist. Although the CCR3 antagonist did not decrease the number of eosinophils in lung tissues as assessed histologically, CCR3 antagonist prevented antigen-induced clustering of eosinophils along the nerves. Immunostaining revealed eotaxin in airway nerves and in cultured airway parasympathetic neurons from both guinea pigs and humans. Both IL-4 and IL-13 increased expression of eotaxin in cultured airway parasympathetic neurons as well as in human neuroblastoma cells. Thus, signaling via CCR3 mediates eosinophil recruitment to airway nerves and may be a prerequisite to blockade of inhibitory M2Rs by eosinophil major basic protein.

Botulinum toxin type-B improves sialorrhea and quality of life in bulbaronset amyotrophic lateral sclerosis.

BACKGROUND: Sialorrhea is a disabling problem in bulbaronset amyotrophic lateral sclerosis (ALS). Botulinum toxin (BTX) type A and B have been proposed as alternatives to traditional treatments. OBJECTIVES: To evaluate the efficacy and safety of BTX type B in the treatment of sialorrhea in patients with bulbar-onset ALS. METHODS: Open-label prospective study of BTX type B injections in parotids (1000 U) and submandibular (250 U) glands using anatomic landmarks. Primary outcome was rate of responders (improvement > 50% on visual analogue scales (VAS) of severity and disability of sialorrhea) 1 month post-treatment. Other outcomes included subjective (drooling and quality of daily living questionnaires) and objective (cotton roll weights and number of paper handkerchiefs used) evaluations. Safety evaluations included questionnaires regarding brain stem symptoms. RESULTS: Sixteen ALS patients were included. At 1 month the rate of responders was 75% with a mean reduction of 70% in severity and disabling VASs. Fifteen patients (94 %) reported some benefit with drooling reduction. In objective measurements there was a reduction over 60 % in saliva production and in the number of handkerchiefs used. Onset of effect occurred within 3 days. Most patients reported better quality of living. The most frequent side-effects were viscous saliva, local pain, chewing weakness and respiratory infection. There were no changes in blood pressure or cardiac rate. At 3 months, there was still a positive effect in all outcomes. All patients except one manifested their willingness to repeat treatment. CONCLUSIONS: Anatomic guided BTX type B injections seem effective and safe to treat sialorrhea in bulbar-onset ALS.

Eosinophil and airway nerve interactions in asthma.

Airway eosinophils are increased in asthma and are especially abundant around airway nerves. Nerves control bronchoconstiction and in asthma, airway hyperreactivity (where airways contract excessively to inhaled stimuli) develops when eosinophils alter both parasympathetic and sensory nerve function. Eosinophils release major basic protein, which is an antagonist of inhibitory M2 muscarinic receptors on parasympathetic nerves. Loss of M2 receptor inhibition potentiates parasympathetic nerve-mediated bronchoconstriction. Eosinophils also increase sensory nerve responsiveness by lowering neurons' activation threshold, stimulating nerve growth, and altering neuropeptide expression. Since sensory nerves activate parasympathetic nerves via a central neuronal reflex, eosinophils' effects on both sensory and parasympathetic nerves potentiate bronchoconstriction. This review explores recent insights into mechanisms and effects of eosinophil and airway nerve interactions in asthma.

The role of immune system parameters in the relationship between depression and coronary artery disease.

The relationship between depressive symptoms and coronary artery disease (CAD) is mediated in part by immune system parameters. This review describes research on the psychoneuroimmunological pathways accounting for the association between depression and CAD, and addresses conceptual and methodological issues. Relationships between central nervous system correlates of depression and immune system parameters are bidirectional and are mediated via neurohormonal and parasympathetic pathways. Evidence suggests that these associations can be affected by a) the clinical characteristics of depression (e.g., typical depression versus atypical depression and exhaustion), b) the duration and severity of depressive symptoms, and c) the stage of underlying CAD. Depressive symptoms are hypothesized to affect primarily the transition from stable CAD to acute coronary syndromes via plaque activation and prothrombotic processes, and may play an additional role in the response to injury at early stages of coronary atherosclerosis.

Distribution of choline acetyltransferase immunoreactivity in the pigeon brain.

We have investigated the distribution of cholinergic perikarya and fibers in the brain of the pigeon (Columba livia). With this aim, pigeon brain sections were processed immunohistochemically by using an antiserum specific for chicken choline acetyltransferase. Our results show cholinergic neurons in the pigeon basal telencephalon, the hypothalamus, the habenula, the pretectum, the midbrain tectum, the dorsal isthmus,the isthmic tegmentum, and the cranial nerve motor nuclei. Cholinergic fibers were prominent in the dorsal telencephalon, the striatum, the thalamus, the tectum, and the interpeduncular nucleus. Comparison of our results with previous studies in birds suggests some major cholinergic pathways in the avian brain and clarifies the possible origin of the cholinergic innervation of some parts of the avian brain. In addition, comparison of our results in birds with those in other vertebrate species shows that the organization of the cholinergic systems in many regions of the avian brain (such as the basal forebrain, the epithalamus, the isthmus, and the hindbrain) is much like that in reptiles and mammals. In contrast, however, birds appear largely to lack intrinsic cholinergic neurons in the dorsal ("neocortex-like") parts of the telencephalon.

Localization and regulation of low affinity nerve growth factor receptor expression in the rat olfactory system during development and regeneration.

Nerve growth factor (NGF), a classic neurotrophic factor, promotes neuronal survival, maintenance, regeneration and differentiation in the peripheral nervous system and parts of the central nervous system. NGF activity is mediated by cell surface bound receptors including the low affinity NGF receptor (LNGFr) which is expressed by some peripheral and central neurons and is present on peripheral nerve Schwann cells during development and regeneration. The olfactory system is a useful model for the study of the role of LNGFr in neuronal development and regeneration. The growth of olfactory axons into the brain begins in the embryo and continues through the first few postnatal weeks. In mature animals there is persistent turnover and generation of olfactory receptor neurons (ORNs) and continuous growth of new axons into the olfactory bulb. These new axons grow along the preexisting olfactory pathway. In the mature olfactory system, LNGFr has been observed in the glomerular layer of the olfactory bulb, the target of ORNs. However, neither the cellular localization nor the developmental expression of LNGFr has been characterized. Here, we tested the hypothesis that LNGFr expression is developmentally regulated in the olfactory nerve and is reinduced following injury to the mature olfactory nerve. LNGFr-immunoreactivity (IR) was first observed in the olfactory mucosa at embryonic day (E)13 and in the olfactory nerve at E14. LNGFr-IR increased in the nerve during embryonic development, began to decrease at around postnatal day (P)5 and was scarcely detectable in normal adults. The staining pattern suggests that LNGFr is located on the olfactory nerve Schwann cells. Streaks of LNGFr-IR were present in the adult olfactory nerve. We reasoned that these streaks might represent transient reexpression of LNGFr associated with normal olfactory neuron turnover and replacement. Consistent with this hypothesis, LNGFr was robustly reexpressed in the adult olfactory nerve following lesion of the olfactory epithelium. Starting late in development (E21) and in the adult, LNGFr-IR was also observed on fibers in deep layers of the olfactory bulb. LNGFr-IR was also observed in neurons of the nucleus of the diagonal band (NDB) in the basal forebrain. NDB is the sole source of cholinergic afferents of the olfactory bulb. Thus, we tested the hypothesis that LNGFr in the deep layers of the olfactory bulb is located on NDB axons by making lesions of NDB. Following the lesion, LNGFr-IR disappeared in the deep layers of the olfactory bulb but remained in the glomerular layer.(ABSTRACT TRUNCATED AT 400 WORDS)

Sympathetic decentralization abolishes increased secretion of immunoglobulin A evoked by parasympathetic stimulation of rat submandibular glands.

Salivary secretion of immunoglobulin A (IgA) in response to electrical stimulation of the parasympathetic nerve supply was assessed bilaterally in the submandibular glands of anaesthetized rats 1 week following unilateral pre-ganglionic sympathectomy (decentralization). Nerve-mediated stimulation on the non-denervated side increased IgA secretion several fold above an unstimulated rate of secretion whereas sympathetic decentralization reduced the parasympathetically stimulated secretion of IgA without affecting the basal rate. Glandular levels of IgA were increased following decentralization compared to the control glands. Salivary levels of free secretory component (FSC), the cleaved polymeric immunoglobulin receptor (plgR), were increased by parasympathetic stimulation and reduced by sympathectomy, though not as much as IgA. The decreased secretion of FSC suggests a reduced production of plgR and may account in part, for reduced IgA secretion following long-term removal of sympathetic nerve impulses.

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.

Choline acetyltransferase-immunoreactive neurons in the rat entopeduncular nucleus.

Using a monoclonal antibody against choline acetyltransferase, neurons of the rat entopenduncular nucleus were found to express choline acetyltransferase immunoreactivity. These cholinergic cells were located mostly in the rostral portion of the entopeduncular nucleus with a marked decrease towards its caudal portion. To identify their target sites, a retrograde fiber tracing technique was combined with immunohistochemistry for choline acetyltransferase. After injection of wheatgerm agglutinin conjugated with horseradish peroxidase into the habenula, some of the entopedunculo-habenular cells were found to be immunoreactive for choline acetyltransferase. The cells in the peripallidal region (the substantia innominata, nucleus basalis magnocellularis and ansa lenticularis) with choline acetyltransferase immunoreactivity did not contain horseradish peroxidase. Following injection of fluorescent tracer into the frontal cerebral cortex, retrogradely labeled cells were observed in the rostral part of the entopedunucular nucleus. A majority of these entopedunculo-cortical cells exhibited choline acetyltransferase immunoreactivity, similar to the cells of the peripallidal region projecting to the neocortex. Employing two different fluorescent tracers, entopedunculo-cortical cells were shown to constitute a distinct cell population from the numerous entopedunculo-habenular cells. The present study demonstrated, in the rat entopeduncular nucleus, the presence of cholinergic neurons that projected to the neocortex and habenula.

Eosinophils and airway nerves in asthma.

In the lungs, neuronal M2 muscarinic receptors limit the release of acetylcholine from postganglionic cholinergic nerves. However, these receptors are not functional under certain circumstances in animal models of hyperreactivity such as occurs after exposure of sensitised animals to an allergen or during a respiratory tract virus infection. This loss of M2 receptor function leads to an increase in acetylcholine release from cholinergic nerves and thus is a mechanism for the vagally mediated hyperreactivity seen in these animals. Studies in animal models of hyperreactivity have shown that eosinophils localise to the airway nerves of sensitised animals after antigen challenge. Inhibiting this localisation of eosinophils either with an antibody to the eosinophil survival cytokine IL-5 or the eosinophil adhesion molecule VLA-4 prevents loss of M2 muscarinic receptor function. It is likely that eosinophil MBP is responsible for the loss of M2 receptor function, since inhibiting eosinophil MBP with an antibody or neutralising MBP with heparin prevents this loss of function. These data are also supported by ligand binding studies where it has been shown that eosinophil MBP is an allosteric antagonist at neuronal M2 muscarinic receptors. Loss of function of lung neuronal M2 muscarinic receptors may also occur under certain circumstances in patients with asthma, although the mechanisms are not yet established.

A monoclonal antibody to a parasympathetic neurotrophic factor causes immunoparasympathectomy in mice.

A monoclonal antibody capable of blocking the biological activity of the ciliary neurotrophic factor purified from bovine cardiac muscle has been produced. This antibody, when administered perinatally to mice, causes a failure of the normal development of the parasympathetic innervation of the iris as determined by assay for the activity of the cholinergic marker enzyme choline acetyltransferase. The same treatment has no effect on the adrenergic neuronal marker, tyrosine hydroxylase. This immunoparasympathectomy suggests that the ciliary neurotrophic factor has an essential role in regulating the development of the mammalian parasympathetic nervous system.