Cholinergic Properties Research Articles

Potential Clinical Use of an Adrenergic/Cholinergic Agent (HP 128) in the Treatment of Alzheimer's Disease

A novel compound designated HP 128, which manifests adrenergic and cholinergic properties, was administered for 10 days to patients with Alzheimer's disease in a double-blind, placebo-controlled trial. All patients who entered the trial had previously failed to respond to a structurally related cholinesterase inhibitor without adrenergic properties (HP 029). The primary purpose of the study was to assess the safety and tolerance of HP 128. Efficacy measures were obtained to generate hypotheses for possible future studies. In the dosage range examined, HP 128 was safe and well tolerated. Effects on clinical measures of dementia severity were equivocal.

Developmental expression of muscarinic cholinergic receptors and coupling to phospholipase C in rat sweat glands are independent of innervation

During development, the innervation of rat sweat glands undergoes a striking change from noradrenergic to cholinergic function. The acquisition of secretory responsiveness by the glands is temporally correlated with the appearance of cholinergic properties. In addition, responsiveness fails to appear in the absence of innervation. To investigate the basis of the onset of functional transmission and secretory responsiveness and its possible relationship to innervation, we analyzed the development of muscarinic cholinergic receptors in sweat glands, examined their expression in the glands of adult rats sympathectomized at birth, and assayed the ability of muscarinic agonists to increase phosphoinositide (PI) turnover. Autoradiographic and in situ hybridization analysis revealed that muscarinic ligand binding sites were first detectable as glands begin to form on postnatal day 4 (P4). Between P4 and P14, receptor concentration increased in parallel with mRNA for the m3 receptor subtype. On P14, the concentration of ligand binding sites approached adult levels, although only a small proportion of glands at this age secrete in response to nerve stimulation or cholinergic agonists. When the pharmacological properties of muscarinic receptors in sweat glands of adult rats sympathectomized at birth were compared to those of normal glands, the concentration and affinity determined with [N-methyl-3H]-scopolamine and the Ki values determined with the subtype-selective muscarinic antagonists 4-DAMP, pirenzepine, and AF DX-116 were similar. In addition, the molecular subtype was unchanged as was the level of m3 message. Studies of PI turnover in response to muscarinic stimulation indicated that the receptors expressed in sweat glands isolated from sympathectomized and acutely denervated, as well as control, rats were functionally coupled to phospholipase C. The absence of sympathetic innervation therefore does not appear to influence either the development of muscarinic receptors or their coupling to PI turnover. Our results suggest that functional sympathetic cholinergic innervation plays a central role in the development and maintenance of secretory function at a step distal to signal transduction across the cell membrane.

The neuropeptide VIP modulates the neurotransmitter phenotype of cultured chick sympathetic neurons

The present report provides evidence for a novel function for the neuropeptide vasoactive intestinal peptide (VIP). We demonstrate that VIP increases the cholinergic and the noradrenergic properties of cultured chick sympathetic neurons without changing neuronal survival and metabolism. VIP induces a 10- to 15-fold increase in the activity of choline acetyltransferase and an approximately twofold increase in the activity of tyrosine hydroxylase. Forskolin, an activator of adenylate cyclase, mimics all the effects of VIP on these cells. In addition, the effects of forskolin and VIP at optimal concentrations are not additive. Furthermore, VIP induces a rapid increase in the intracellular cAMP levels. Thus VIP acts via a cAMP-dependent pathway to enhance the cholinergic and noradrenergic properties of cultured chick sympathetic neurons.

Differential modulation of the cholinergic activity of rat CNS neurons in culture

Treatment of septal cultures prepared from 17-day-old embryos with two different antimitotic agents, cytosine arabinoside (ara C) and 5′-fluoro-2′-deoxyuridine (FUdR), caused a 2-fold increase in the level of choline acetyltransferase (CAT) activity and no change in the glutamic acid decarboxylase (GAD) activity. In these cultures, there was also a large decrease in the number of astrocytes as determined by immunofluorescence for glial fibrillary acidic protein (GFAP). Furthermore, when epidermal growth factor (EGF) was added to the septal cultures to increase the astrocyte population, the CAT activity decreased. Therefore, it would appear that the astrocytes are responsible for producing this down-regulation on cholinergic neurons. In order to determine whether all CNS cholinergic neurons can be inhibited in this manner, cultures were prepared from two other CNS regions that contain a high percentage of cholinergic neurons, i.e. the striatum and the ventral spinal cord. When these cultures were treated with the antimitotic agents, there was little modification of the CAT or GAD activities. These results suggest that the astrocytic microenvironment of the septal neurons exerts an inhibitory effect on the CAT activity either via a soluble factor or via cell-cell contact. Such studies are an important demonstration that non-neuronal cells may alter cholinergic properties during CNS development.

An improved invertebrate synaptosomal preparation with cholinergic properties

Our laboratory has previously characterized a high affinity choline uptake system (HAChUS) in Limulus tissues and synaptosomes. We report here on the characterization of the HAChUS in synaptosomes prepared selectively from central nervous system tissues shown to be enriched for presumed cholinergic functions; namely the protocerebrum, corpora pedunculata and abdominal ganglia. Synaptosomes were prepared from these tissues by means of a modification of the subfractionation procedure developed by Dowdall and Whittaker. In our modification, we harvested a PP 2L fraction exclusively from the S 2 fraction. Compared to the P 2L fraction, the PP 2L was greater than three times more efficient in [ 3H]choline uptake and was significantly more sensitive to inhibition with micromolar concentrations of hemicholinium-3. The PP 2L fraction HAChUS was shown to have characteristics common to the HAChUS of identified cholinergic tissues.

Modulation of neuropeptide expression in avian embryonic sympathetic cultures

Two distinct neuropeptide-related phenotypes are found in avian paravertebral sympathetic ganglia, corresponding to somatostatin- (SS) and vasoactive intestinal polypeptide- (VIP) expressing cells. We have detected the same cell phenotypes in cultures of embryonic quail sympathetic ganglia and have used this system to study the modulation of their expression by the environment. The cell phenotypes were identified using immunocytochemistry and induced catecholamine fluorescence and quantitative data were obtained by radioimmunoassay. Dissociation of the ganglia caused a profound increase in the expression of VIP but had no effect on SS levels. Addition of corticosterone (10 −6 M) increased the expression of SS without modifying VIP levels. In contrast, depolarization of the cells induced changes in levels of both neuropeptides. The modulation of VIP correlates with the modulation of cholinergic properties. The regulation of neuropeptide expression in the avian system shows both similarities and differences to what has been found in the mammalian system.

Structure-activity relationships for various N-alkylcarbamyl esters of choline with selective nicotinic cholinergic properties

La methylcarbamylcholine (MCC) radiomarquee est utilisee pour explorer les sites nicotiniques au niveau du cerveau. L'addition de substituants aromatiques et cycloalkyls au niveau du N-carbamyl, attribue a ces substances des proprietes antagonistes de celles de la carbamylcholine. Dans cette etude, les auteurs explorent les effets d'une N-substitution, par des chaines alkyl de longueur variees, des esthers de carbamate de choline. Les produits ont ete evalues pour leurs capacites a entrer en competition avec la MCC, la nicotine et le quinuclidinylbenzylate au niveau des sites de fixation, ainsi que pour leur capacite a produire une prostration chez les rongeurs

Differential increase in the cholinergic properties of cultured chick sympathetic neurons by forskolin

Sympathetic neurons from the chick superior cervical ganglion (SCG) in culture synthesize and accumulate catecholamine (CA), and to a lesser extent, acetylcholine (AcCho). The noradrenergic and cholinergic properties of these cells can be differentially modulated by various external factors such as neuronal density, chick eye extract, liver-conditioned-medium, and inosine. We report here that forskolin, an activator of adenylate cyclase, selectively promotes AcCho, but not CA production in the chick SCG neurons. This effect is rapid since an increase in the AcCho content is already observed 60 min after the addition of forskolin to 2-week-old cultures. Rapid gene induction or protein phosphorylation via a cAMP-dependent pathway may thus be involved in the differential regulation of acetylcholine metabolism in cultured chick sympathetic neurons.

Selective Expression of Factors Preventing Cholinergic Dedifferentiation

Chicken retina neurons from 8-9-day-old embryos developed prominent cholinergic properties after several days in stationary dispersed cell (monolayer) culture. These cells accumulated [3H]choline by a high-affinity, hemicholinium-sensitive transport system, converted [3H]choline to [3H]-acetylcholine [( 3H]ACh), released [3H]ACh in response to depolarization stimuli, and developed choline acetyltransferase (ChAT) activity to levels comparable to those of the intact retina. The cholinergic state, however, was not permanent. After 7 days in culture, the capacity for [3H]ACh release decreased drastically and continued to diminish with longer culture periods. Loss of this capacity seemed not to be due to loss of cholinergic neurons, because high-affinity choline uptake was unchanged. However, a substantial decrease of ChAT activity was observed as a function of culture age, and probably accounted for the low level of ACh synthesis in long-lasting cultures. The loss of ChAT activity could be prevented in at least two different ways: (a) Maintaining the neurons in rotary (aggregate) rather than stationary culture completely blocked the loss of enzyme activity and gave a developmental profile identical to the known "in situ" pattern of differentiation; and (b) Conditioned medium from aggregate cultures significantly reduced the drop in ChAT activity of neurons maintained in stationary, dispersed cell cultures. Activity that stabilized cholinergic differentiation was nondialyzable, heat-sensitive, and not mimicked by functional nerve growth factor. Production of activity by aggregates was developmentally regulated; medium obtained from aggregates after 3 days in culture had no effect on cholinergic differentiation, whereas medium obtained from aggregates between 6 and 10 days in culture produced a fivefold increase of ChAT in monolayers.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential development of cholinergic neurons from cranial and trunk neural crest cells in vitro

Several studies have suggested that the development of cholinergic properties in cranial parasympathetic neurons is determined by these cells' axial level of origin in the neural crest. All cranial parasympathetic neurons normally derive from cranial neural crest. Trunk neural crest cells give rise to sympathetic neurons, most of which are noradrenergic. To determine if there is an intrinsic difference in the ability of cranial and trunk neural crest cells to form cholinergic neurons, we have compared the development of choline acetyltransferase (ChAT)-immunoreactive cells in explants of quail cranial and trunk neural crest in vitro. Both cranial and trunk neural crest explants gave rise to ChAT-immunoreactive cells in vitro. In both types of cultures, some of the ChAT-positive cells also expressed immunoreactivity for the catecholamine synthetic enzyme tyrosine hydroxylase. However, several differences were seen between cranial and trunk cultures. First, ChAT-immunoreactive cells appeared two days earlier in cranial than in trunk cultures. Second, cranial cultures contained a higher proportion of ChAT-immunoreactive cells. Finally, a subpopulation of the ChAT-immunoreactive cells in cranial cultures exhibited neuronal traits, including neurofilament immunoreactivity. In contrast, neurofilament-immunoreactive cells were not seen in trunk cultures. These results suggest that premigratory cranial and trunk neural crest cells differ in their ability to form cholinergic neurons.

Target influences on transmitter choice by sympathetic neurons developing in the anterior chamber of the eye

In contrast to the majority of sympathetic neurons which are noradrenergic, the sympathetic neurons which innervate sweat glands are cholinergic. Previous studies have demonstrated that during development the sweat gland innervation initially contains catecholamines which are lost as cholinergic function appears. The neurotransmitter phenotype of sweat gland neurons further differs from the majority in that they contain vasoactive intestinal peptide (VIP) rather than neuropeptide Y (NPY). In the experiments described here, we addressed the question of whether sympathetic targets influence the neurotransmitter-related properties of the neurons which innervate them; in particular, do sweat glands play a role in reducing the expression of noradrenergic properties and inducing the expression of cholinergic properties and VIP in sympathetic neurons? This was accomplished by cotransplanting to the anterior chamber of the eye of host rats the superior cervical ganglia (SCG) which contains neurons that normally innervate targets other than the sweat glands and differentiate noradrenergically and footpad tissue from neonatal rats. Sweat glands developed in the transplanted footpad tissue and became innervated by the cotransplanted SCG neurons. The transplanted neurons and sweat gland innervation initially exhibited catecholamine histofluorescence which declined with further development in the anterior chamber. After 4 weeks, choline acetyltransferase (ChAT) and VIP immunoreactivities were evident. These observations suggest that as in the neurons which innervate the glands in situ, noradrenergic properties were suppressed and cholinergic function was induced in the neurons which innervated the glands in oculo. To distinguish a specific influence of the sweat glands on transmitter choice, SCG were also cotransplanted with the pineal gland, a normal target of the ganglion. Neurons cotransplanted with the pineal gland continued to exhibit catecholamine histofluorescence and contained NPY immunoreactivity. At least some neurons in SCG/pineal cotransplants, however, developed ChAT immunoreactivity. The target-appropriate expression of catecholamines and peptides in these experiments is consistent with the hypothesis that some transmitter properties are influenced by target tissues. The indiscriminant expression of ChAT, however, suggests that at least in oculo, additional factors can influence transmitter choice.

Modification of the indolamine content in neuroblastoma � glioma hybrid NG108-15 cells upon induced differentiation

1. The neuroblastoma x glioma hybrid NG108-15 cell line has been widely studied as a neuronal model for its serotonergic, cholinergic, and peptidergic properties. 2. The catecholamine and serotonin content and that of their major metabolites have been determined by high-performance liquid chromatography with electrochemical detection (HPLC-EC) in NG108-15 cells under differentiated and undifferentiated conditions. 3. Cellular contents of L-DOPA, norepinephrine, (NE), L-epinephrine (EPI), and dopamine (DA) in differentiated cells, induced by 1 mM dibutyryl cyclic AMP (dBcAMP), are 149, 40, 129, and 124%, respectively, higher than those in undifferentiated cells. 4. 3,4-Dihydroxyphenethylacetic acid (DOPAC), the major metabolite of DA, is detectable only in differentiated cells. Similarly, DOPAC is present only in culture medium from differentiated cells, and not that of undifferentiated cells. 5. Serotonin (5-HT) is detectable only in undifferentiated cells; and the level of 5-hydroxyindoleacetic acid (5-HIAA), the major metabolite of 5-HT, is also 12.7% higher is undifferentiated cells. 6. Comparative analyses of differentiated and undifferentiated cells in monolayer cultures and undifferentiated cells cultured in the presence of 1 mM dBcAMP under suspension conditions suggest that change in the indolamine content is due to cellular changes upon morphological differentiation. 7. The clonal NG108-15 cell line is also catecholaminergic, in addition to cholinergic and serotonergic; and a shift of neurotransmitter pattern from serotonin to dopamine production occurs during morphological differentiation.

Interferon induces astrocyte maturation causing an increase in cholinergic properties of cultured human spinal cord cells

It has been well established that interferon-γ (IFN-γ) can modify the immune status of cells in the central nervous system (CNS) by inducing major histocompatibility antigens. Furthermore, it has been shown that endogenous IFN can be produced in the brain following viral infection and a form of IFN- α β can be produced by astrocytes in culture. Here we show that IFN can induce astrocyte maturation and alter neurotransmitter properties in cultured CNS neurons at a given developmental stage. IFN causes a dose-dependent increase in choline acetyltransferase activity and glial fibrillary acidic protein (GFAP) immunoreactivity in cultures of human embryonic spinal cord neurons. The GABAergic activity and the Thy1 immunoreactivity remain unchanged. IFN-γ does not act directly on the neurons but via the nonneuronal cells, probably the astrocytes, which in turn stimulate the cholinergic traits. These studies could be important for demonstrating an action of the immune system on glial cell maturation and on the neurotransmitter phenotype expression in CNS neurons.

Cholinergic properties of rat atrial subcellular preparations

Several presynaptic cholinergic markers were measured in subcellular fractions of the rat cardiac atrium. A P 2 fraction consisting of isolated terminals, free mitochondria, glycogen and other subcellular organelles was prepared first by sucrose density centrifugation. No intact cells were recovered in this fraction, as determined by electron microscopy. Hemicholinium-3 sensitive choline transport coupled to acetylcholine synthesis was concentrated over 10-fold in the P 2 fraction compared to minced atria when expressed per mg protein. Six subfractions were recovered after centrifugation of the P 2 fraction over a sucrose-metrizamide density gradient. One of these (fraction 4) consistently contained higher levels of choline acetyltransferase activity and hemicholinium-3 sensitive [ 3H]acetylcholine synthesis than were present in the P 2 fraction of the other five subfractions. Electron microscopy of fraction 4 revealed isolated nerve terminals among other subcellular organelles. Kinetic analysis of total [ 3H]choline uptake in the P 2 fraction revealed two apparent uptake processes, with K t s of approximately 11 μ m and 219 μ m. [ 3H]Acetylcholine synthesis was partially sodium-dependent in the P 2 fraction, and reached a maximal level between choline concentrations of 100 to 200 μ m. Some of the newly synthesized [ 3H]-acetylcholine in the P 2 fraction was released by 50 m m K + depolarization in a calcium-dependent manner, arguing for a neuronal localization. This depolarization-induced release was attenuated by 10 to 100 μ m oxotremorine in an atropine-sensitive manner, but was not affected by 1 μ m tetrodotoxin.

Hippocampal membranes contain a neurotrophic activity that stimulates cholinergic properties of fetal rat septal neurons cultured under serum-free conditions.

Primary cultures of fetal rat septal neurons were used to identify a membrane-associated cholinergic neurotrophic activity. Under serum-free culture conditions, approximately 98% of the septal cells are neurons, and approximately 6% of the neurons are cholinergic as determined immunocytochemically. Crude membranes prepared from rat hippocampal homogenates stimulate choline acetyltransferase (ChAT) activity in treated septal neurons. The membrane-associated trophic activity is apparent at lower protein concentrations than activity present in the soluble fraction and is unevenly distributed in various brain regions; it is highest in hippocampus and striatum and negligible in cerebellum. Membrane trophic activity is developmentally regulated, is heat and trypsin sensitive, and increases the rate of expression of ChAT in septal neurons. Upon gel filtration chromatography of a high-salt membrane extract, trophic activity elutes as a broad peak in the 500 kilodalton (kD) molecular mass range. Stimulation of septal neuronal ChAT activity by either crude membranes or partially purified preparations is not inhibited by antibodies against nerve growth factor (NGF), and its maximal activity is additive to maximally active doses of NGF. The results indicate that hippocampal membranes contain cholinergic neurotrophic activity which may be important for the development of septal cholinergic neurons.

Chapter 24 Muscle-derived trophic factors influencing cholinergic neurons in vitro and in vivo

This chapter review the effects of two different factors derived from muscle which enhance cholinergic properties of neurons in vitro. In the absence of muscle extract the development of choline acetyltransferase (CAT) in tissue culture follows a biphasic time course in which there is an initial decrease in CAT activity followed by a period of steadily increasing activity. The addition of muscle extract to the cultures prevents the initial decline and stimulates the subsequent development of CAT activity. A factor (cholinergic development factor, CDF) in the muscle extract which produces such effects has been purified and partially characterized. While CDF was being purified from rat skeletal muscle, attempts were made to purify neurotrophic activity from autopsied human skeletal muscle. Chick ciliary neurons were used to assay the enhancement of acetylcholine synthesis, CAT. Although neurotrophic factors may exhibit a range of specific behaviors in vitro such as neuron survival or enhancement of cholinergic activity, for complete validation such factors must be shown to be capable of influencing neuronal survival in vivo. An in vivo effect could eliminate a trivial explanation of the in vitro phenomena, i.e. that purified extracts may provide essential components missing or perturbed in the tissue culture environment, or that they act in a manner normally inoperative in vivo.

Effects of anticonvulsants on cholinergic and GABAergic properties in the neuronal cell clone NG108-15

The effects of anticonvulsant drugs on growth, cholinergic, and GABAergic properties were examined in the neuronal cell clone NG108-15. Cells were exposed for 4 days to valproic acid, phenobarbital, phenytoin, or carbamazepine in concentrations equivalent to therapeutic free levels in human serum. Experiments were also performed with varying concentrations of a recently proposed antiepileptic, gamma-vinyl GABA. Of these five anticonvulsants, cell growth (total protein and cell counts) was decreased with valproic acid and phenytoin but only valproic acid and gamma-vinyl GABA altered neurotransmitter markers. Therapeutic concentrations of valproic acid increased choline acetyltransferase activity to 142% of control but had no effect on either the activity of glutamate decarboxylase or the level of GABA. The effects of a higher (toxic) concentration of valproic acid (200 micrograms/ml) were similar to those induced by the differentiating agent dibutyryl cyclic AMP: both decreased cell growth, enhanced the activity of choline acetyltransferase and reduced the activity of glutamate decarboxylase. Gamma-vinyl GABA had no effect on cholinergic markers but, at 1300 micrograms/ml, increased GABA levels to 135% of control despite the reduction of glutamate decarboxylase to 68% of control. In the NG108-15 cell clone, anticonvulsants have varying effects on cell growth, differentiation, and neurotransmitter systems. Our findings do not support the proposal that the mechanism of action for valproic acid, phenobarbital, phenytoin, and carbamazepine is via alteration of GABA levels.

Cholinergic properties of embryonic chick sensory neurons

Experiments were carried out to determine the cholinergic properties of sensory neurons of the chick embryo by measuring the choline acetyltransferase activity (ChAT) and [ 3H]choline uptake. The choline acetyltransferase activity in the dorsal root ganglia of an 8-day-old chick embryo was 24.2 ± 2.52, which increased to 45.4 ± 9.69 pmol ACh/mg protein/min in the ganglia of 12-day-old embryos. Sensory neurons derived from dorsal root ganglia of 10-day-old embryos and maintained in a serum-free culture medium supplemented with insulin, transferrin and nerve growth factor (NGF) also contained significant amounts of ChAT (21.9 pmol ACh/mg protein/min). Omission of NGF resulted in neuronal death, and the enzyme activity could not be measured in these cultures. A specific inhibitor of ChAT, hydroxyethyl naphthylvinyl pyridine (NVP), when added to the assay mix produced a dose-dependent inhibition of ChAT from cultured neurons. Cultured sensory neurons incubated with [ 3H]choline followed by repeated washouts took up and retained [ 3H]choline. The uptake of [ 3H]choline was reduced by about 45% when NaCl, in the incubation medium, was replaced by LiCl. A specific inhibitor of choline uptake, hemicholinium-3, caused about 75% inhibition of [ 3H]choline uptake. It is implied that sensory neurons of the chick dorsal root ganglia express cholinergic properties during development.

Developmental discord among markers for cholinergic differentiation: In vitro time courses for early expression and responses to skeletal muscle extract

The effects of skeletal muscle extract on the development of CAT, ACh synthesis, high affinity choline uptake, and AChE activities were studied in dissociated ventral spinal cord cultures prepared from 14-day gestational rat embryos. In the absence of muscle extract, the development of CAT and AChE follow biphasic time courses in which they show initial declines followed by periods of steadily increasing activity. In contrast, ACh synthesis and high affinity choline uptake both gradually increase throughout the entire culture period. The presence of muscle extract both prevents the initial decline of CAT and AChE as well as stimulates the rates of development of all four cholinergic markers; however, the degrees and time courses of stimulation differ markedly. The effects of muscle extract on the kinetic and pharmacological properties of ACh synthesis and choline uptake in rat ventral cord cultures were also investigated. Cells treated with muscle extract for 2 days express both high affinity ( K m = 1.6 μM) and low affinity ( K m = 22 μM) choline uptake mechanisms. Control cells, on the other hand, express only low affinity uptake at this stage but develop a high affinity uptake mechanism by Day 7. During this time both ACh synthesis and high affinity choline uptake become increasingly sensitive to inhibition by hemicholinium-3. These results demonstrate that skeletal muscle factors enhance the development of cholinergic properties in embryonic spinal cord cultures. However, differences in sensitivity to muscle extract concentration, time courses of development, and degrees of stimulation suggest that these changes may involve distinct cellular mechanisms which are differentially affected by skeletal muscle factors.

Development and properties of the secretory response in rat sweat glands: Relationship to the induction of cholinergic function in sweat gland innervation

Previous studies suggest that the sympathetic innervation of the sweat glands in the rat is initially noradrenergic and during development undergoes a transition in neurotransmitter phenotype to become cholinergic. To characterize this system and its development further, we have examined the adrenergic and cholinergic components of the secretory response in adult and immature rats and have studied the onset of sweating in the plantar sweat glands of developing rats. Stimulation of the sciatic nerve in adult rats elicited a secretory response which was completely blocked by the cholinergic antagonist, atropine, and was unaffected by adrenergic antagonists, indicating that nerve-evoked secretion was cholinergic. In adult rats, the sweat glands were quite sensitive to cholinergic agonists. In addition to acetylcholine, the mature sweat gland innervation contains vasoactive intestinal peptide (VIP). In some rats, the injection of VIP alone elicited a secretory response which was blocked by atropine, suggesting that the response to VIP was mediated cholinergically. In contrast to cholinergic agonists, the glands responded relatively infrequently and with reduced volumes of sweat to the α- and β-adrenergic agonists 6-fluoronorepinephrine and isoproterenol. However, when VIP, which is a potent vasodilator, was simultaneously injected with adrenergic agonists, glands in many of the injected footpads exhibited a secretory response. The response to adrenergic agonists in combination with VIP was reduced by atropine and by phentolamine plus propranolo, but was blocked completely only by a combination of the three antagonists, indicating that both adrenergic and cholinergic mechanisms were involved. In immature rats, sweating evoked by nerve stimulation first appeared at 14 days of age in 25% of the rats tested. Both the percentage of rats sweating and the number of active glands increased rapidly. At 16 days, 50% of the rats tested exhibited some active glands, and by 21 days all rats tested exhibited a secretory response. In 16-day-old rats, nerve-evoked sweating was almost completely inhibited by local injection of 1 μ M atropine, but was unaffected by phentolamine and propranolol in concentrations up to 10 μ M. Similarly, the glands were sensitive to 10 μ M muscarine, but they exhibited no secretory response to the α-adrenergic agonists, clonidine and 6-fluoronorepinephrine, nor to the β-adrenergic agonist, isoproterenol, at concentrations up to 50 μ M. The simultaneous injection of VIP with adrenergic agonists did not reveal an adrenergically mediated secretory response in 16-day-old animals. Thus, in immature as well as adult rats, the neurally evoked secretory process was mediated through a muscarinic cholinergic mechanism. The onset of nerve- and agonist-induced sweating in developing rats lagged behind the development of other cholinergic properties in the sweat glands, raising the possibility that the cholinergic responsiveness of the gland cells is induced by the release of acetylcholine from the gland innervation. Consistent with this hypothesis is the observation reported here that in adult animals in which the glands were sympathetically denervated at birth, the glands did not respond to either sciatic nerve stimulation or cholinergic agonists.