Developmental changes in the transmitter properties of sympathetic neurons that innervate the periosteum.

During the development of sweat gland innervation, interactions with the target tissue induce a change from noradrenergic to cholinergic and peptidergic properties. To determine whether the change in neurotransmitter properties that occurs in the sweat gland innervation occurs more generally in sympathetic neurons, we identified a new target of cholinergic sympathetic neurons in rat, the periosteum, which is the connective tissue covering of bone, and characterized the development of periosteal innervation of the sternum. During development, sympathetic axons grow from thoracic sympathetic ganglia along rib periosteum to reach the sternum. All sympathetic axons displayed catecholaminergic properties when they reached the sternum, but these properties subsequently disappeared. Many axons lacked detectable immunoreactivities for vesicular acetylcholine transporter and vasoactive intestinal peptide when they reached the sternum and acquired them after arrival. To determine whether periosteum could direct changes in the neurotransmitter properties of sympathetic neurons that innervate it, we transplanted periosteum to the hairy skin, a noradrenergic sympathetic target. We found that the sympathetic innervation of the transplant underwent a noradrenergic to cholinergic and peptidergic change. These results suggest that periosteum, in addition to sweat glands, regulates the neurotransmitter properties of the sympathetic neurons that innervate it.     

Developmental expression of the high affinity choline transporter in cholinergic sympathetic neurons

Choline uptake by the high affinity choline transporter (CHT) is the rate-limiting step in acetylcholine synthesis. Induction of CHT is therefore a critical step in cholinergic differentiation, and we examined the developmental expression of CHT in cholinergic sympathetic neurons that innervate rodent sweat glands. During postnatal development the earliest sympathetic axons in the rear footpads are noradrenergic, containing intense tyrosine hydroxylase immunoreactivity and lacking CHT-immunoreactivity (CHT-IR). By postnatal day 7 (P7) in mouse, and P10 in rat, weak CHT-IR appeared in axons associated with the sweat gland anlagen. CHT staining intensity increased during the following weeks in conjunction with plexus arborization and gland maturation. The pattern of CHT-immunoreactivity (CHT-IR) in the sweat gland innervation was similar to staining for the vesicular acetylcholine transporter and vasoactive intestinal peptide. Immunoblots of tissue from sympathectomized rats confirmed that most of the CHT in footpad was contained in sympathetic neurons. Although CHT expression has been reported in noradrenergic sympathetic neurons of the superior cervical ganglion, these data indicate that in the sympathetic neurons projecting to sweat glands CHT is present at detectable levels only after association with the glands.     

Sympathetic cholinergic target innervation requires GDNF family receptor GFR alpha 2

Many cholinergic parasympathetic and enteric neurons require neurturin signaling through GDNF family receptor GFRalpha2 for target innervation. Since a distinct minority of sympathetic neurons are cholinergic, we examined whether GFRalpha2 is important for their development. We detected GFRalpha2 in neonatal sympathetic cholinergic neurons and neurturin mRNA in their target tissues, sweat glands in footpads, and periosteum. Lack of GFRalpha2 in mice did not affect the number of sympathetic cholinergic neurons, but their soma size was decreased in comparison to wild types. In adult and in 3-week-old GFRalpha2 knockout mice, the density of sympathetic cholinergic innervation was reduced by 50-70% in the sweat glands, and was completely absent in the periosteum. Sympathetic noradrenergic innervation of blood vessels in the footpads was unchanged. The density of sympathetic axons in sweat glands was unaffected at postnatal day P4 reflecting successful growth into the target area. Our results indicate that the cholinergic subpopulation of sympathetic neurons requires GFRalpha2 signaling for soma size and for growth or maintenance of target innervation. Thus, neurturin may be a general target-derived innervation factor for postganglionic cholinergic neurons in all parts of the autonomic nervous system.     

Neonatal 6-hydroxydopamine treatment eliminates cholinergic sympathetic innervation and induces sensory sprouting in rat sweat glands

Previous studies of the development of cholinergic sympathetic innervation of sweat glands in rat footpads suggested that these terminals initially exhibit noradrenergic properties which are lost as the glands and their innervation mature. We have treated neonatal and adult rats with 6-hydroxydopamine (6-OHDA), a toxic congener of norepinephrine, and compared its effects on the cholinergic sympathetic innervation of sweat glands and the noradrenergic sympathetic innervation of the iris, salivary gland, and blood vessels. As reported by others, 6-OHDA treatment of neonates caused the destruction of noradrenergic fibers in the iris and salivary gland but did not affect other fibers projecting to these targets that stain for acetylcholinesterase (AChE). We found that 6-OHDA treatment of neonatal animals also caused the destruction of the sympathetic axons in immature sweat glands that possess catecholamine histofluorescence and tyrosine-hydroxylase-like immunoreactivity. Furthermore, when such animals were examined as adults, we found no AChE staining, vasoactive intestinal peptide (VIP)-like immunoreactivity, or characteristic sympathetic axonal varicosities. However, the denervated glands were invested by a plexus of sensory axons, some of which exhibited substance P-like immunoreactivity (SP-IR). An increase in the number of SP-IR fibers also occurred in the sympathetically denervated irides of these animals. Chronic treatment of neonates with guanethidine, another adrenergic sympathetic neurotoxin, resulted in similar loss of cholinergic sweat gland innervation. Treatment of adults rats with doses of 6-OHDA identical to those used to treat neonates caused the loss of noradrenergic fibers from the iris, salivary gland, and many blood vessels but did not noticeably affect AChE and VIP staining or axonal ultrastructure in the sweat glands. However, treatment with higher doses of 6-OHDA did cause significant axonal degeneration. The response of the sympathetic innervation of developing but not mature sweat glands to 6-OHDA provides evidence for a transition from noradrenergic to cholinergic phenotype during the development of sympathetic neurons in vivo similar to the transition observed in cell culture. The sprouting of sensory axons may be caused by NGF-like trophic influences present in some sympathetically denervated tissues.     

Absence of cholinergic sympathetic innervation from limb muscle vasculature in rats and mice

Although the existence of cholinergic sympathetic vasodilatory innervation in limb muscle vasculature is well established for some species, previous pharmacological studies have failed to reveal the presence of such innervation in rats. Recently, Schafer and colleagues [Schafer, M.K., Eiden, L.E., Weihe, E., 1998. Cholinergic neurons and terminal fields revealed by immunohistochemistry for the vesicular acetylcholine transporter. II. The peripheral nervous system. Neuroscience 84(2), 361-376] reported that vesicular acetylcholine transporter immunoreactivity (VAChT-IR), a marker for cholinergic terminals, is present in the innervation of the microvasculature of rat hindlimb skeletal muscle and concluded that rats possess cholinergic sympathetic innervation of limb muscle vasculature. Because of our interest in identifying targets of cholinergic sympathetic neurons, we have analyzed the transmitter properties of the innervation of muscle vessels in rat and mouse limbs. We found that the innervation of vasculature in muscle is noradrenergic, exhibiting robust catecholamine histofluorescence and immunoreactivity for tyrosine hydroxylase (TH) and the peptide transmitters, neuropeptide Y (NPY) and occasionally vasoactive intestinal peptide (VIP). In contrast, cholinergic phenotypic markers,VAChT-IR and acetylcholinesterase (AChE) activity, are absent. Neuron cell bodies in sympathetic ganglia, retrogradely labeled with injections of tracer into limb muscles, also lacked VAChT but contained TH-IR. The innervation of large extramuscular feed arteries in hindlimbs was also devoid of cholinergic markers, as were the cell bodies of sympathetic neurons innervating extramuscular femoral arteries. These results, like those of previous physiological studies, provide no evidence for the presence of cholinergic sympathetic innervation of muscle vasculature in rats or mice.     

c-ret regulates cholinergic properties in mouse sympathetic neurons: evidence from mutant mice

The search for signalling systems regulating development of noradrenergic and cholinergic sympathetic neurons is a classical problem of developmental neuroscience. While an essential role of bone morphogenetic proteins for induction of noradrenergic properties is firmly established, factors involved in the development of cholinergic traits in vivo are still enigmatic. Previous studies have shown that the c-ret receptor and cholinergic properties are coexpressed in chick sympathetic neurons. Using in situ hybridization we show now that a loss-of-function mutation of the c-ret receptor in mice dramatically reduces numbers of cells positive for choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT) in stellate ganglia of homozygous newborn animals. The number of neurons positive for tyrosine hydroxylase (TH) mRNA, the rate-limiting enzyme of noradrenaline synthesis, is reduced to a smaller degree and expression levels are not detectably altered. Already at embryonic day 16 (E16), ChAT and VAChT-positive cells are affected by the c-ret mutation. At E14, however, ChAT and VAChT mRNAs are detectable at low levels and no difference is observed between wildtype and mutant mice. Our data suggest that c-ret signalling is necessary for the maturation of cholinergic sympathetic neurons but dispensable for de novo induction of ChAT and VAChT expression.     

Chemical coding of the human gastrointestinal nervous system: cholinergic,VIPergic, and catecholaminergic phenotypes

The aim of this investigation was to identify the proportional neurochemical codes of enteric neurons and to determine the specific terminal fields of chemically defined nerve fibers in all parts of the human gastrointestinal (GI) tract. For this purpose, antibodies against the vesicular monoamine transporters (VMAT1/2), the vesicular acetylcholine transporter (VAChT), tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), serotonin (5-HT), vasoactive intestinal peptide (VIP), and protein gene product 9.5 (PGP 9.5) were used. For in situ hybridization (35)S-labeled VMAT1, VMAT2, and VAChT riboprobes were used. In all regions of the human GI tract, 50-70% of the neurons were cholinergic, as judged by staining for VAChT. The human gut unlike the rodent gut exhibits a cholinergic innervation, which is characterized by an extensive overlap with VIPergic innervation. Neurons containing VMAT2 constituted 14-20% of all intrinsic neurons in the upper GI tract, and there was an equal number of TH-positive neurons. In contrast, DBH was absent from intrinsic neurons. Cholinergic and monoaminergic phenotypes proved to be completely distinct phenotypes. In conclusion, the chemical coding of human enteric neurons reveals some similarities with that of other mammalian species, but also significant differences. VIP is a cholinergic cotransmitter in the intrinsic innervation of the human gut. The substantial overlap between VMAT2 and TH in enteric neurons indicates that the intrinsic catecholaminergic innervation is a stable component of the human GI tract throughout life. The absence of DBH from intrinsic catecholaminergic neurons indicates that these neurons have a dopaminergic phenotype.     

Human and monkey cholinergic neurons visualized in paraffin-embedded tissues by immunoreactivity for VAChT,the vesicular acetylcholine transporter

The predicted C-terminal dodecapeptide of the human vesicular acetylcholine transporter (VAChT), deduced from the unique open reading frame of the recently cloned human VAChT cDNA, was conjugated through an N-terminal cysteine to keyhole limpet hemocyanin and used as an immunogen to generate polyclonal antihuman VAChT antibodies in rabbits. The distribution of the VAChT antigen in representative regions of the cholinergic nervous system was examined and compared to that of the acetylcholine biosynthetic enzyme choline acetyltransferase (ChAT), a specific marker for cholinergic neurons. VAChT immunoreactivity was localized in cell bodies of neurons in the basal forebrain and ventral horn of the spinal cord, regions in which major cholinergic projection systems to the cerebral cortex and to skeletal muscle, respectively, originate. The primate caudate nucleus contained numerous VAChT-positive interneurons. VAChT immunoreactivity was visualized in both cell bodies and extensive terminals in striatal interneurons, in contrast to formalin-fixed, deparaffinized sections stained for ChAT, in which cell bodies and fibers were stained but nerve terminals were less well visualized than with the VAChT antiserum. VAChT-positive nerve fibers were visualized in routinely immersion-fixed, paraffin-embedded human cerebral cortex, comparable to the density of fibers observed in perfusion-fixed Bouin’s-postfixed monkey cerebral cortex. Extensive investment of virtually all principal ganglion cells of thoracic sympathetic ganglia of monkey and human with VAChT-positive nerve terminals was observed. VAChT-positive cell bodies, presumably corresponding to cholinergic sympathetic sudomotor neurons, were a significant fraction of the total principal cell population in monkey and human thoracic sympathetic ganglia. VAChT is a specific marker for cholinergic neurons in human and rhesus monkey, visualizing especially nerve terminals more extensively than antibodies against the cholinergic biosynthetic enzyme ChAT, in routinely fixed tissue. VAChT immunoreactivity in cholinergic nerve terminals of the central and peripheral nervous systems ought to prove useful for visualizing cholinergic synapses and neuroeffector junctions, and their functional status during development and in neurodegenerative and autonomic disease.     

Cardiovascular dysautonomia in Parkinson disease: from pathophysiology to pathogenesis

Signs or symptoms of impaired autonomic regulation of circulation often attend Parkinson disease (PD). This review covers biomarkers and mechanisms of autonomic cardiovascular abnormalities in PD and related alpha-synucleinopathies. The clearest clinical laboratory correlate of dysautonomia in PD is loss of myocardial noradrenergic innervation, detected by cardiac sympathetic neuroimaging. About 30-40% of PD patients have orthostatic hypotension (OH), defined as a persistent, consistent fall in systolic blood pressure of at least 20 mmHg or diastolic blood pressure of at least 10 mmHg within 3 min of change in position from supine to standing. Neuroimaging evidence of cardiac sympathetic denervation is universal in PD with OH (PD+OH). In PD without OH about half the patients have diffuse left ventricular myocardial sympathetic denervation, a substantial minority have partial denervation confined to the inferolateral or apical walls, and a small number have normal innervation. Among patients with partial denervation the neuronal loss invariably progresses over time, and in those with normal innervation at least some loss eventually becomes evident. Thus, cardiac sympathetic denervation in PD occurs independently of the movement disorder. PD+OH also entails extra-cardiac noradrenergic denervation, but this is not as severe as in pure autonomic failure. PD+OH patients have failure of both the parasympathetic and sympathetic components of the arterial baroreflex. OH in PD therefore seems to reflect a "triple whammy" of cardiac and extra-cardiac noradrenergic denervation and baroreflex failure. In contrast, most patients with multiple system atrophy, which can resemble PD+OH clinically, do not have evidence for cardiac or extra-cardiac noradrenergic denervation. Catecholamines in the neuronal cytoplasm are potentially toxic, via spontaneous and enzyme-catalyzed oxidation. Normally cytoplasmic catecholamines are efficiently taken up into vesicles via the vesicular monoamine transporter. The recent finding of decreased vesicular uptake in Lewy body diseases therefore suggests a pathogenetic mechanism for loss of catecholaminergic neurons in the periphery and brain. Parkinson disease (PD) is one of the most common chronic neurodegenerative diseases of the elderly, and it is likely that as populations age PD will become even more prevalent and more of a public health burden. Severe depletion of dopaminergic neurons of the nigrostriatal system characterizes and likely produces the movement disorder (rest tremor, slowness of movement, rigid muscle tone, and postural instability) in PD. Over the past two decades, compelling evidence has accrued that PD also involves loss of noradrenergic neurons in the heart. This finding supports the view that loss of catecholaminergic neurons, both in the nigrostriatal system and the heart, is fundamental in PD. By the time PD manifests clinically, most of the nigrostriatal dopaminergic neurons are already lost. Identifying laboratory measures-biomarkers-of the disease process is therefore crucial for advances in treatment and prevention. Deposition of the protein, alpha-synuclein, in the form of Lewy bodies in catecholaminergic neurons is a pathologic hallmark of PD. Alpha-synucleinopathy in autonomic neurons may occur early in the pathogenetic process. The timing of cardiac noradrenergic denervation in PD is therefore a key issue. This review updates the field of autonomic cardiovascular abnormalities in PD and related disorders, with emphasis on relationships among striatal dopamine depletion, sympathetic noradrenergic denervation, and alpha-synucleinopathy.     

Development of choline acetyltransferase (CAT) in the sympathetic innervation of rat sweat glands

It has been postulated that the developing sympathetic innervation of rat eccrine sweat glands changes from adrenergic to cholinergic under the influence of its target. In agreement with previous evidence that the sympathetic innervation of adult rat sweat glands is cholinergic, we found that choline acetyltransferase (CAT)-immunoreactive nerve fibers are present in adult glands, and that gland-rich chunks of adult footpads contain CAT enzyme activity. We were therefore interested in determining when CAT activity is first expressed in the developing gland innervation. Low levels of acetylating activity were observed in rat footpads as early as postnatal day 4, when sympathetic fibers first contact the glands. A greater than fourfold increase in CAT specific activity occurred between postnatal days 11 and 21. Neonatal treatment of rats with the adrenergic neurotoxin 6-hydroxydopamine (6-OHDA) eliminated most of the CAT activity in 14 and 19 d footpads. In contrast, the acetylating activity observed prior to day 11 was unaffected by neonatal 6-OHDA treatment, and only slightly reduced by the selective CAT inhibitor, naphthylvinylpyridine. These results indicate that the sympathetic fibers that innervate rat sweat glands do not acquire detectable levels of CAT activity until a full week after they contact the glands.     

Coexistence of calcitonin gene-related peptide and vasoactive intestinal peptide in cholinergic sympathetic innervation of rat sweat glands

Immunoreactivity for calcitonin gene-related peptide (CGRP) has been localized with indirect immunofluorescence techniques in the cholinergic sympathetic fibers that innervate eccrine sweat glands in the rat. This innervation also contains vasoactive intestinal peptide-like immunoreactivity (VIP-IR). A small proportion of principal neurons in stellate and lumbar sympathetic ganglia which provide innervation to the sweat glands contain detectable CGRP-immunoreactivity. The CGRP-IR neurons are immunoreactive for VIP; however, many VIP-IR neurons in these ganglia do not contain detectable levels of CGRP-IR.     

Distribution of the vesicular acetylcholine transporter (VAChT) in the central and peripheral nervous systems of the rat

Expression of the acetylcholine biosynthetic enzyme choline acetyltransferase (ChAT), the vesicular acetylcholine transporter (VAChT), and the high-affinity plasma membrane choline transporter uniquely defines the cholinergic phenotype in the mammalian central (CNS) and peripheral (PNS) nervous systems. The distribution of cells expressing the messenger RNA encoding the recently cloned VAChT in the rat CNS and PNS is described here. The pattern of expression of VAChT mRNA is consistent with anatomical, pharmacological, and histochemical information on the distribution of functional cholinergic neurons in the brain and peripheral tissues of the rat. VAChT mRNA-containing cells are present in brain areas, including neocortex and hypothalamus, in which the existence of cholinergic neurons has been the subject of debate. The demonstration that VAChT is a completely adequate marker for cholinergic neurons should allow the systematic delineation of cholinergic synapses in the rat nervous system when antibodies directed to this protein are available.     

Parasympathetic nerve fibers invade the upper dermis following sensory denervation of the rat lower lip skin

The sympathetic division of the autonomic nervous system is known to play a role in the genesis of neuropathic pain. In the skin of the rat lower lip (hairy skin), sympathetic and parasympathetic fibers normally innervate the same blood vessels in the lower dermis but do not occur in the upper dermis. However, we have shown that sympathetic fiber migration into the upper dermis occurs following mental nerve lesions (Ruocco et al. [2000] J. Comp. Neurol. 422:287-296). As sensory denervation has a dramatic effect on sympathetic fiber innervation patterns in the rat lower lip skin, we decided to investigate the possible changes in the other autonomic fiber type in the skin-the parasympathetic fiber. Sensory denervation of the rat lower lip was achieved by bilateral transection of the mental nerve, and animals were allowed to recover for 1-8 weeks. Lower lip tissue was processed for double-labeling light microscopic immunocytochemistry (ICC), using antibodies against substance P (SP), which labels a subpopulation of peptidergic sensory fibers, and against the vesicular acetycholine transporter (VAChT), as a marker for parasympathetic fibers. In sham-operated rats, SP-immunoreactive (IR) sensory fibers were found in the epidermis and upper and lower dermal regions, whereas VAChT-IR fibers were confined to the lower dermis. Mental nerve lesions induced the gradual disappearance of SP-IR fibers from all skin layers accompanied by the progressive migration of VAChT-IR fibers into the upper dermis. Cholinergic fiber migration was evident by the second week post surgery, and the ectopic innervation of the upper dermis by these fibers persisted even at the last time point studied (8 weeks) when SP-IR fibers have completely regrown. VAChT-IR fibers were observed in the upper dermis, well above the opening of the sebaceous glands into the hair follicles. These results show that considerable changes occur in the innervation patterns of parasympathetic fibers following mental nerve lesions.     

Comparison of the subregional distributions of the monoamine vesicular transporter and dopamine uptake complex in the rat striatum and changes during aging

Summary We have studied the heterogeneous distribution of the vesicular monoamine transporter, labelled with³H dihydrotetrabenazine (³H TBZOH) and the dopamine uptake complex, labelled with³H GBR12783 in the rat striatum. The ratio TBZOH/GBR12783 was higher in the anterior part of the striatum than in the caudal part. This discrepancy could not be explained by the contribution of serotoninergic innervation to³H TBZOH binding, since the ratio TBZOH/citalopram was also higher in the anterior striatum than in the caudal striatum. The monoamine vesicular transporter and the dopamine uptake complex were more abundant in the lateral regions than in the regions situated near the midline. In the caudal striatum, the ventral part was richer in vesicular transporter than the dorsal part. In aged rats (30 months), a significant decrease in the density of both transporters was noticed in the middle part of the striatum. In the anterior part of the striatum, the ratio TBZOH/GBR12783 was elevated in aged rats compared to adult ones. This could participate in a functional adaptation of the partially diminished population of dopaminergic neurons during aging.     

Overexpression of nerve growth factor in epidermis disrupts the distribution and properties of sympathetic innervation in footpads

Sympathetic and sensory neurons form distinct axonal arborizations in several peripheral targets. The developmental mechanisms responsible for partitioning sympathetic and sensory axons between potential target tissues are poorly understood. We have used rodent footpads to study this process because three populations of peripheral axons innervate topographically segregated targets in the footpad; cholinergic sympathetic axons innervate sweat glands, noradrenergic sympathetic axons innervate blood vessels, and sensory axons form a plexus at the epidermal/dermal junction. To examine how nerve growth factor (NGF), a trophic and survival factor for sympathetic and some sensory neurons, may contribute to the generation of the patterned distribution of axons among targets, we studied transgenic mice (K14-NGF mice) in which NGF expression was significantly increased in the epidermis. Whereas the temporal sequence in which sensory and sympathetic fibers arrived in the footpad was not affected, the normal partitioning of axons between target tissues was disrupted. The two sympathetic targets in footpads, sweat glands, and blood vessels lacked substantial innervation and instead a dense plexus of catecholaminergic sympathetic fibers was found commingled with sensory fibers in the dermis. Those sympathetic fibers present in sweat glands expressed an abnormal dual catecholaminergic/cholinergic phenotype. Our findings indicate that overexpression of NGF in skin interferes with the segregation of sensory and sympathetic axonal arbors and suggests a role for target-derived NGF in the establishment of distinct axonal territories. Our data also suggest that by determining where axon arbors form, NGF can indirectly influence the phenotypic properties of sympathetic neurons. J. Comp. Neurol. 393:231–243, 1998. © 1998 Wiley-Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.     

Generalised anhidrosis: different lesion sites demonstrated by microneurography and skin biopsy

Generalised anhidrosis (GA) shows a uniform clinical picture whether the pathogenesis involves intrinsic abnormalities of sweat glands or postganglionic sympathetic cholinergic nerve dysfunction. We describe two patients who presented intolerance to heat and anhidrosis. In the first patient, symptoms started at 33 years of age, and were associated with absent tendon reflexes and a mydriatic right pupil unreactive to light. The other patient had been unable to sweat since birth. GA was diagnosed on the basis of clinical findings and thermoregulatory tests. Microneurography and morphological analysis of the skin and its innervation disclosed a different lesion site underlying GA in the two patients, and distinguished between a postganglionic autonomic nerve fibre lesion and sweat gland dysfunction.     

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.     

Target regulation of neurotransmitter phenotype.

Studies of sympathetic neurons developing in cell culture revealed a surprising degree of transmitter plasticity and established the role of environmental factors in determining transmitter choice. The sympathetic neurons that innervate sweat glands undergo a change in neurotransmitter phenotype from noradrenergic to cholinergic during normal development similar to that observed in culture. Cross-innervation experiments indicate that the target sweat glands induce the switch and thereby specify the phenotype of the neurons that innervate them. Thus, both the transmitter plasticity and the role of environmental influences initially elucidated in culture are part of the developmental repertoire of sympathetic neurons in vivo. Further, these findings extend considerably our understanding of the role that targets may play during development; targets may not only determine how many neurons survive but also what their properties will be.