Balanced Caloric Restriction Minimizes Changes Caused by Aging on the Colonic Myenteric Plexus.

Aging can promote significant morphofunctional changes in the gastrointestinal tract (GIT). Regulation of GIT motility is mainly controlled by the myenteric neurons of the enteric nervous system. Actions that aim at decreasing the aging effects in the GIT include those related to diet, with caloric restriction (CR). The CR is achieved by controlling the amount of food or by manipulating the components of the diet. Therefore, the objective of this study was to evaluate different levels of CR on the plasticity of nicotinamide adenine dinucleotide phosphate- (NADPH-) reactive myenteric neurons in the colon of Wistar rats during the aging process using ultrastructural (transmission electron microscopy) and morphoquantitative analysis. Wistar male rats (Rattus norvegicus) were distributed into 4 groups (n = 10/group): C, 6-month-old animals; SR, 18-month-old animals fed a normal diet; CRI, 18-month-old animals fed a 12% CR diet; CRII, 18-month-old animals fed a 31% CR diet. At 6 months of age, animals were transferred to the laboratory animal facility, where they remained until 18 months of age. Animals of the CRI and CRII groups were submitted to CR for 6 months. In the ultrastructural analysis, a disorganization of the periganglionar matrix with the aging was observed, and this characteristic was not observed in the animals that received hypocaloric diet. It was observed that the restriction of 12.5% and 31% of calories in the diet minimized the increase in density and cell profile of the reactive NADPH neurons, increased with age. This type of diet may be adapted against gastrointestinal disturbances that commonly affect aging individuals.

Developmental determinants of the independence and complexity of the enteric nervous system.

Enteric nervous system (ENS) development is relevant to Hirschsprung's disease (HSCR; congenital aganglionosis of the terminal bowel), which is still imperfectly treated. Mutations in genes encoding the RET receptor tyrosine kinase and endothelin receptor type B (EDNRB) are involved in HSCR pathogenesis; however, also important in ENS development are molecules that mediate events that are more restricted than those of RET and EDNRB, act later in development and which might not be HSCR-associated. Examples are molecules that function in the guidance of enteric neural crest-derived cells (ENCDCs) and vagal axons, and in regulating the terminal differentiation of enteric neurons from ENCDCs. It is probable that highly prevalent disorders of gastrointestinal sensation and motility result from subtle defects in ENS development.

Changes in the number and volume of NPY and VIP neurons from periprostatic accessory vegetative ganglia in pre- and peripubertal rats. A stereological study.

The amount of neurons of periprostatic accessory ganglia in pre- and peripubertal rats was studied to ascertain whether the development of these autonomic ganglia is androgen-dependent. Stereological estimates of the volumes and number of neurons immunoreactive to protein gene product 9.5 (PGP 9.5), neuropeptide Y (NPY), and vasoactive intestinal polypeptide (VIP) were carried out. Immunostaining of androgen receptors (AR) in the ganglia was also performed. The ganglionic neurons from the two groups studied were immunoreactive to PGP 9.5, NPY, and VIP. Almost all the neurons were immunostained for AR. The ganglionic volume showed a significant increase in peripubertal prostate in comparison with the prepubertal gland. No significant changes were observed with respect to the absolute number of neurons immunoreactive to all the antigens. The neuronal volume was significantly increased in peripubertal rats in comparison with prepubertal animals. These findings led us to the following conclusions: There is no evidence of neurogenesis during pubertal development in the periprostatic accessory ganglia of the rat. The increase of ganglionic volume in puberty is due to the growth in neuronal volume. There were no differences between the sizes of NPY and VIP neurons in pubertal periprostatic accessory ganglia. The development of periprostatic vegetative neurons is androgen-dependent.

Prenatal development of the human epicardiac Ganglia.

The aim of this study was to determine the developmental anatomy of intrinsic cardiac ganglia with respect to epicardiac ganglionated nerve plexus in the human fetuses at different gestation stages. Twenty fetal hearts were investigated applying a technique of histochemistry for acetylcholinesterase to visualize the epicardiac neural ganglionated plexus with its subsequent examinations on total (non-sectioned) hearts. Most epicardiac ganglia embodied multilayered neurons and were oval in shape, but some ganglia involved neurons lying in one layer or had the irregular appearance because of their extensions along inter-ganglionic nerves. The mean ganglion area of fetuses at gestation stages of 15-40 weeks was 0.03 +/- 0.008 mm(2). The largest epicardiac ganglia, reaching in area 0.4 mm(2), were concentrated on the dorsal surface of both atria. The particular fused or "dual" ganglia were identified at the gestation stages of 23-40 weeks, but they composed only 2.3 +/- 0.7% of all found epicardiac ganglia. A direct positive correlation was determined between the fetal age and the ganglion area (mm(2)) as well as between the fetal age and the number of inter-ganglionic nerves. The revealed appearance of epicardiac ganglia in the human fetuses at 15-40 weeks of gestation confirms their prenatal development and presumable intrinsic remodelling.

Gangliogenesis in the enteric nervous system: roles of the polysialylation of the neural cell adhesion molecule and its regulation by bone morphogenetic protein-4.

The neural crest-derived cells that colonize the fetal bowel become patterned into two ganglionated plexuses. The hypothesis that bone morphogenetic proteins (BMPs) promote ganglionation by regulating neural cell adhesion molecule (NCAM) polysialylation was tested. Transcripts encoding the sialyltransferases, ST8Sia IV (PST) and ST8Sia II (STX), which polysialylate NCAM, were detectable in fetal rat gut by E12 but were downregulated postnatally. PSA-NCAM-immunoreactive neuron numbers, but not those of NCAM, were developmentally regulated similarly. Circular smooth muscle was transiently (E16-20) PSA-NCAM-immunoreactive when it is traversed by migrating precursors of submucosal neurons. Neurons developing in vitro from crest-derived cells immunoselected at E12 with antibodies to p75(NTR) expressed NCAM and PSA-NCAM. BMP-4 promoted neuronal NCAM polysialylation and clustering. N-butanoylmannosamine, which blocks NCAM polysialylation, but not N-propanoylmannosamine, which does not, interfered with BMP-4-induced neuronal clustering. Observations suggest that BMP signaling enhances NCAM polysialylation, which allows precursors to migrate and form ganglionic aggregates during the remodeling of the developing ENS.

Constitutively expressed catalytic proteasomal subunits are up-regulated during neuronal differentiation and required for axon initiation, elongation and maintenance.

Inhibition of the proteasome by lactacystin, a specific blocker of the catalytic beta-subunits, results in transient neurite outgrowth by neuronal cell lines. Vice versa, as demonstrated in this study, treatment of pheochromocytoma (PC12) cells with nerve growth factor (NGF) or other differentiating agents reduces proteasomal activity. This is accompanied by an increase in mRNA and protein levels of the catalytically active subunits beta1, beta2 and beta5, but not of their inducible counterparts, indicating changes in subunit composition of the proteasome during neuronal differentiation. In contrast to neuronal cell lines, however, pre-treatment of primary neurons with proteasome inhibitors completely prevents axon formation, and lower concentrations of lactacystin (0.5-5 microm) significantly reduce axonal elongation and branching in vitro. Furthermore, established axonal networks degenerate rapidly and long-term survival of peripheral neurons is impaired in the presence of proteasome inhibitors. Axonal pathology is reminiscent of the morphological changes observed in neurodegenerative disorders and supports a crucial role of the constitutive catalytic subunits in axon initiation, maintenance and regeneration.

Structural changes in the nucleus ambiguus of the medulla oblongata and intracardiac ganglia in growing rats in immobilization stress.

The aim of the present work was to study qualitative and quantitative measures of histological changes in the nucleus ambiguus (NA) of the medulla oblongata and the cardiac autonomic ganglia (CAG) in growing animals in conditions of immobilization stress of different durations. Experiments were performed on 56 mongrel white rats aged 30 days. Immobilization stress was produced by placing rats in a special apparatus with a controllable internal volume for 3, 7, 15, and 30 days. The NA and CAG were studied on paraffin sections using neurohistological methods and quantitative analysis. Morphological and morphometric changes, consisting of a delay in the growth of neurocyte cell and nucleus volumes, as compared with controls, were seen both in the NA of the medulla oblongata and the CAG and were evaluated as impairments in the histogenesis of nerve tissue during postnatal ontogenesis. The extent of structural changes in these components of the autonomous nervous system was directly dependent on the duration of the experimental treatment.

Architecture and age-related analysis of the neuronal number of the guinea pig intrinsic cardiac nerve plexus.

The aims of the present study have been to determine the architecture of the guinea pig intrinsic cardiac nerve plexus (ICNP) and to test whether or not the heart of this species undergoes decrease in neuronal number with aging. Nine young (3-4 weeks of age) and nine adult (18-24 months of age) animals were examined employing histochemistry for acetylcholinesterase to reveal the ICNP in total hearts. The number of intracardiac neurons in seven animals was assessed via counting of the nerve cells both on total hearts and in serial sections of the atrial walls. The intracardiac neurons from adult guinea pigs were amassed within 329 +/- 15 ganglia. The hearts of young animals contained significantly fewer ganglia, only 211 +/- 27. In adult guinea pigs approximately 60% of the intracardiac neurons were distributed within ganglia of not more than 20 neurons, but the ganglia of such size accumulated only 45% of the neurons in young animals. The total number of the intracardiac neurons estimated per guinea pig heart was 2321 +/- 215, and this number did not differ significantly between young and adult animals. The nerves entering the guinea pig heart were found both in the arterial and venous part of the heart hilum. The nerves from the arterial part of the heart hilum proceeded into the ventricles, but the nerves from the venous part of the hilum formed a nerve plexus of the cardiac hilum located on the heart base. Within the guinea pig epicardium, intrinsic nerves divided into six routes and proceeded to separate atrial, ventricular and septal regions. In conclusion, findings of this study contradict the age-related decrease of the neuronal number in the guinea pig heart and illustrate the remarkable similarity in the architecture of the intracardiac nerve plexuses between guinea pig and rat.

Postnatal-related changes in the size and total number of neurons in the caudal mesenteric ganglion of dogs: total number of neurons can be predicted from body weight and ganglion volume.

Aging is mostly characterized by a progressive decline of neuronal function that involves both the central and the peripheral nervous system. The aging process is accompanied by changes in either the number or the size of neurons. However, these data are controversial and not very well known in the sympathetic ganglia of large mammals. Hence, the present investigation aimed to study the dog's caudal mesenteric ganglion (CMG) in three different periods of postnatal development, searching for qualitative and quantitative alterations. The CMG is responsible for the large intestine, internal anal sphincter, and partially the urogenital system innervations. Nine dead male dogs from the Veterinary Hospital of the College of Veterinary Medicine at University of São Paulo were divided into three well-defined age groups (1-2 months old, 1-2 years old, and 5-10 years old). The stereological study was pursued using the physical disector method combined to the Cavalieri principle. The postnatal development was accompanied by an increase in the nonneuronal tissue amount and in ganglion volume. Additionally, the total number of neurons also increased during aging (from 70,140 to 1,204,516), although the neuronal density showed an opposite trend (from 29,911 to 11,500 mm(-3)). Due to the interrelation between either body weight or ganglion volume and aging in the dogs investigated in this study, it was possible to predict the total number of neurons in CMG using both body weight and ganglion volume in an attempt to verify whether or not size and total number of neurons are both allometrically and aging ruled, i.e., if either the animal's body weight and ganglion volume or aging influence these parameters. The prediction of the total number of neurons was very close to the initially estimated values.

[Structural changes in the medullary nucleus ambiguus and cardiac autonomic ganglia of growing rats under influence of the immobilization stress].

The objective of this study was to evaluate the quantitative and qualitative histological changes in the medullary nucleus ambiguus (NA) and cardiac autonomic ganglia (CAG) of the growing organism under the influence of immobilization stress of different duration. The experiments were performed on 56 outbred albino rats with the initial age of 30 days. Immobilization stress was induced by placing the rats into special chambers with a controllable volume of the inner space for 3, 7, 15 and 30 days. NA and CAG were studied on paraffin sections using the neurohistological methods and a quantitative analysis. Morphological and morphometric changes have demonstrated the retardation of the growth of neurocyte cell bodies and of their nuclei both in medullary NA and CAG as compared to those in control animals, which are regarded as an indication of a disturbance of the histogenesis of the nervous tissue in postnatal development. The degree of structural changes of the components of autonomic nervous system studied was directly related to the duration of experimental exposure to immobilization stress.

Distribution and fate of cocaine- and amphetamine-regulated transcript peptide (CARTp)-expressing cells in rat urinary bladder: a developmental study.

We examined the distribution and fate of cocaine- and amphetamine-regulated transcript peptide (CARTp)(55-102)-immunoreactive (IR) structures in the neonatal and adult rat urinary bladder. Double-labeling studies examining CARTp with tyrosine hydroxylase (TH), neuronal nitric oxide synthase (nNOS), or choline acetyltransferase (ChAT) were performed in wholemounts of urothelium or detrusor or cryostat sections of the bladder. In younger animals (postnatal day [P]1, P3), CARTp-IR cell bodies in detrusor smooth muscle were observed in large clusters ( approximately 100 cells/cluster) at the ureteral insertion and along thick bundles of nerve fibers at the bladder base. The total number of CARTp-IR cells was significantly reduced (by five-fold) at P14, and this reduced number persisted into adulthood. The decrease in the number of CARTp-expressing cells was complemented with positive staining for cleaved caspase-3, suggesting that apoptosis contributed to this decrease. At birth (P1), all CARTp-IR cells expressed the neuronal marker Hu. After birth, CARTp was expressed by some neurons (CARTp-IR, Hu-IR) that represent intramural ganglion cells and by cells that lacked a neuronal phenotype (CARTp-IR, Hu-) but did express TH. Neither of these cell populations expressed ChAT immunoreactivity in adult bladder. These cells (CARTp-IR, Hu-, TH-IR) may represent paraganglion or small intensely fluorescent (SIF) cells. The percentage of colocalization of CARTp-IR and nNOS or TH was dependent on postnatal age and showed an inverse relationship. At P1, 67.1 % of CARTp-IR cells expressed nNOS immunoreactivity. Decreased colocalization was observed with increasing postnatal age. In contrast, 19.5% of CARTp-IR cells expressed TH at P1, but colocalization increased with postnatal age. The suburothelial plexus lacked CARTp-IR nerve fibers until P14, when nerve fibers with varicosities were observed in the urethra and bladder neck region. In summary, we demonstrate 1) a decrease in the number of CARTp-IR cells in rat detrusor in early postnatal development; 2) apoptotic events in the bladder during early postnatal development; 3) rostral migration of CARTp-IR cells from the ureteral insertion toward the bladder body during postnatal development; 4) the presence of different populations of CARTp-IR cells, some with and others without a neuronal phenotype; and (5) age-dependent changes in chemical coding of CARTp-IR cells with postnatal development. This study demonstrates that CARTp-IR intramural ganglia and CARTp-IR paraganglion or SIF cells exist in the postnatal and adult rat bladder, although the role of these cell types remains to be determined.

Developmental changes in expression of GABAA receptor-channels in rat intrinsic cardiac ganglion neurones.

The effects of gamma-aminobutyric acid (GABA) on the electrophysiological properties of intracardiac neurones were investigated in the intracardiac ganglion plexus in situ and in dissociated neurones from neonatal, juvenile and adult rat hearts. Focal application of GABA evoked a depolarizing, excitatory response in both intact and dissociated intracardiac ganglion neurones. Under voltage clamp, both GABA and muscimol elicited inward currents at -60 mV in a concentration-dependent manner. The fast, desensitizing currents were mimicked by the GABA(A) receptor agonists muscimol and taurine, and inhibited by the GABA(A) receptor antagonists, bicuculline and picrotoxin. The GABA(A0) antagonist (1,2,5,6-tetrahydropyridin-4-yl)methyl phosphonic acid (TPMPA), had no effect on GABA-induced currents, suggesting that GABA(A) receptor-channels mediate the response. The GABA-evoked current amplitude recorded from dissociated neurones was age dependent whereby the peak current density measured at -100 mV was approximately 20 times higher for intracardiac neurones obtained from neonatal rats (P2-5) compared with adult rats (P45-49). The decrease in GABA sensitivity occurred during the first two postnatal weeks and coincides with maturation of the sympathetic innervation of the rat heart. Immunohistochemical staining using antibodies against GABA demonstrate the presence of GABA in the intracardiac ganglion plexus of the neonatal rat heart. Taken together, these results suggest that GABA and taurine may act as modulators of neurotransmission and cardiac function in the developing mammalian intrinsic cardiac nervous system.

Development of nerves expressing P2X3 receptors in the myenteric plexus of rat stomach.

Development of neurones and fibres expressing P2X3 receptors in the myenteric plexus of rat stomach and coexistence of the P2X3 receptor with calbindin, calretinin and NOS during postnatal development, were investigated with immunostaining methods. Extrinsic nerves expressing P2X3 receptors appeared as early as E12 and were localised in the trunk and branches of the vagus nerve, which extended rapidly onto the whole rat stomach from E12 to E14. Intrinsic neurone cell bodies with P2X3-immunoreactivity in the myenteric ganglia were first demonstrated postnatally at P1, and at P14, when the number of neurones expressing the P2X3 receptor peaked at 45%. P2X3 receptor-immunoreactivity decreased subsequently, and at P60 only about 11% were P2X3-immunoreactive. Intraganglionic laminar nerve endings and intramuscular arrays were first demonstrated postnatally at P1 and P7, respectively. In the early postnatal days, there were many growth cone-like structures with strong P2X3 immunostaining associated with these endings and arrays. Double-immunostaining showed that 9-15% of P2X3-immunoreactive neurones in the gastric myenteric plexus expressed calbindin D-28 k only in the early postnatal days, while 14-21% of neurones from P1 to P60 increasingly expressed calretinin. About 20% of neurones with P2X3 immunoreactivity coexpressed NOS throughout perinatal development.

Synaptic dynamism measured over minutes to months: age-dependent decline in an autonomic ganglion.

Naturally occurring rearrangements of synaptic terminals are common in the nervous systems of young mammals, but little is known about their incidence in adults. Using transgenic mice that express yellow fluorescent protein (YFP) in axons, we repeatedly imaged nerve terminals in the parasympathetic submandibular ganglion. We found that the pattern of synaptic branches underwent significant rearrangements over several weeks in young adult mice. In older mice, rearrangements were less common, and synaptic patterns on individual neurons were recognizable for many months to years. Axonal branches frequently retracted or extended on a time scale of minutes in young adult mice, but seldom in mature animals. These results provide direct evidence for a decrease in plasticity of interneuronal connections as animals make the transition from young adulthood to middle age. The long-term stability of synaptic patterns could provide a structural basis for the persistence of memory in the adult nervous system.

Postnatal maturational changes in rat pelvic autonomic ganglion cells: a mixture of steroid-dependent and -independent effects.

Androgens have potent effects on the maturation and maintenance of a number of neural pathways involved in reproductive behaviors in males. Most studies in this area have focused on central pathways, but androgen receptors are expressed by many peripheral neurons innervating reproductive organs, and previous studies have demonstrated structural and chemical changes in these neurons at puberty and after castration. We have performed the first electrophysiological comparison of pelvic autonomic ganglion neurons in male rats before and after puberty and following pre- or postpubertal castration. Studies were performed in vitro on intact ganglia with hypogastric and pelvic nerves attached to allow synaptic activation of sympathetic or parasympathetic neurons, respectively. Pelvic ganglion neurons underwent many changes in their passive and active membrane properties over the pubertal period, and some of these changes were dependent on exposure to circulating androgens. The most pronounced steroid-dependent effects were on membrane capacitance (soma size) in sympathetic neurons and duration of the action potential afterhyperpolarization in tonic neurons. Our study also showed that rat pelvic ganglion cells and their synaptic inputs were more diverse than previously reported. In conclusion, this study demonstrated that rat pelvic ganglion neurons undergo considerable postnatal changes in their electrophysiological properties. The steroid dependence of some of these changes indicates that circulating androgens may influence reproductive behaviors at many locations within the nervous system not just in the brain and spinal cord.

N-terminal Slit2 promotes survival and neurite extension in cultured peripheral neurons.

To investigate the effect of the N-terminal Slit2 protein on neuronal survival and development, recombinant human N-terminal Slit2 (N-Slit2) was assayed against isolated embryonic chick dorsal root ganglion sensory, ciliary ganglion and paravertebral sympathetic neurons. N-Slit2 promoted significant levels of neuronal survival and neurite extension in all of these populations. The protein was also assayed against postnatal mouse dorsal root ganglion neurons and found to promote neuronal survival in a similar manner. These findings suggest the Slit proteins may play an important role during development of the nervous system, mediating cellular survival in addition to the well documented role these proteins play in axonal and neuronal chemorepulsion.

Maturation of postsynaptic nicotinic structures on autonomic neurons requires innervation but not cholinergic transmission.

Postsynaptic development at the neuromuscular junction depends on nicotinic transmission and secreted components from the presynaptic motor nerve terminal. Similarly, secreted components and synaptic activity are both thought to guide development of glutamatergic synapses in the CNS. Nicotinic synapses on chick ciliary neurons are structurally complex: a large presynaptic calyx engulfs the postsynaptic neuron and overlays a series of discrete mats of receptor-rich somatic spines tightly interwoven and folded against the soma. We used fluorescence imaging of alpha 7-containing nicotinic receptors and the spine constituent drebrin to monitor postsynaptic development. The results show that surgical disruption of the preganglionic input or removal of the ganglionic synaptic target tissue after synapses form in the ganglion does not disrupt the receptor-rich spine mats. Similarly, removal of the target tissue even prior to synapse formation in the ganglion does not prevent subsequent formation of the receptor clusters and associated spine constituents. Postsynaptic development is arrested, however, if normal innervation is prevented by ablating the preganglionic neurons prior to synapse formation. In this case the neurons express reduced levels of nicotinic receptors and cytoskeletal components and organize them only into early-stage clusters. Even low levels of residual innervation, however, can restore much of the normal postsynaptic receptor patterns. Chronic pharmacological blockade of cholinergic synaptic activity fails to replicate the effects of ablating the preganglionic nucleus. The results indicate that ciliary neurons are programmed to express postsynaptic components and can initiate clustering of alpha 7-containing receptors but need presynaptic guidance for maturation of the postsynaptic structure.

Testosterone and nerve growth factor have distinct but interacting effects on structure and neurotransmitter expression of adult pelvic ganglion cells in vitro.

Circulating testosterone has potent effects on the structure and function of many pelvic ganglion cells in adult rats in vivo. However not all androgen-sensitive pelvic neurones possess androgen receptors and testosterone effects may therefore be indirect, by an action on the target organs. Here we have examined if testosterone influences neuronal structure in vitro in pelvic ganglion cells cultured from adult male rats. We have also used multiple label immunofluorescence to monitor the expression of transmitter-synthesising enzymes and peptides under various culture conditions. Testosterone was a more potent stimulant of noradrenergic soma growth in culture than nerve growth factor. Whereas nerve growth factor increased the number, branching and length of neurites, testosterone stimulated growth of a small number of very short processes, each of which bore numerous short protrusions. Testosterone also impeded the longer neurite growth induced by nerve growth factor. Many pelvic ganglion cells altered their expression of transmitters/neuropeptides under different culture conditions. In particular, under control conditions or during nerve growth factor treatment, vasoactive intestinal peptide was up-regulated in noradrenergic and cholinergic neurones; testosterone impeded this up-regulation in noradrenergic neurones. Choline acetyltransferase immunoreactivity could only be visualised when nerve growth factor was present in the cultures, and cholinergic neurones showed less neurite outgrowth than noradrenergic neurones under all culture conditions. Nerve growth factor did not stimulate levels of this enzyme as strongly if testosterone was present. This study has shown that testosterone has potent effects on the structure of many pelvic ganglion cells in vitro. It is possible that these effects are mediated indirectly, e.g. by stimulating glial-derived substances, however our results suggest that the effects are not mediated by nerve growth factor. The results also show that testosterone influences some of the actions of nerve growth factor, suggesting that there may be complex interactions between steroid signalling and neurotrophic factors in maintaining neuronal structure and function in vivo.

Enkephalinergic sympathetic and parasympathetic innervation of the rat submandibular and sublingual glands.

Enkephalinergic innervation of the rat salivary glands was investigated by immunocytochemical techniques. Based upon immunostaining for enkephalin (ENK) and tyrosine hydroxylase (TH), 4 types of neurons could be distinguished in the submandibular ganglion: cells containing both ENK and TH (9% of all ganglion cells), cells containing only ENK (17%), cells containing only TH (4%) and cells lacking both ENK and TH (70%). Almost all of the ganglion neurons were also positive for AChE and so were most of the TH-positive cells. The ENK-positive fibers outnumbered the TH-positive fibers. Although TH-positive fibers displayed concurrent ENK immunoreactivity, fibers in the blood vessel walls were only immunoreactive for TH. Excision of the superior cervical ganglion resulted in a decrease of ENK fibers and the disappearance of most of the TH fibers from the submandibular gland. Most of the remaining ENK-positive fibers were immunonegative for TH, while the remaining TH-positive fibers were also positive for ENK. The salivary gland of the postnatal 8-week-old rats had a considerable number of ENK-positive neurons and fibers in the submandibular ganglion and acini.