Distribution and changes with age of calcitonin gene-related peptide- and substance P-immunoreactive nerves of the rat urinary bladder and lumbosacral sensory neurons

In the distal parts of the urinary tract, nerves containing calcitonin gene-related peptide (CGRP) or substance P (SP) are sensory with their cell bodies located in lumbosacral dorsal root ganglia. These two neuropeptides are recognised as being present in pelvic sensory nerves, and may be involved in the mediation of pain, stretch and/or vasodilatation. We have used indirect immunohistochemical techniques to examine the distribution and regional variation of nerves immunoreactive (-ir) for CGRP and SP in the urinary bladder and in neurons in lumbosacral dorsal root ganglia (L1-L2 & L6-S1) of young adult (3 months) and aged (24 months) male rats. Semi-quantitative estimations of nerve densities were made for CGRP-ir and SP-ir fibres innervating the dome, body and base of the urinary bladder. Quantitative studies were also used to examine the effects of age on the percentage of dorsal root ganglion neurons immunoreactive for CGRP and SP. There were very few immunoreactive axons in the dome and the overall density of innervation increased progressively towards the base of the bladder. The density of innervation in the aged rats revealed a slight reduction in CGRP and SP innervation of the detrusor muscle but was otherwise comparable to the young group. However, immunostaining of the lumbosacral dorsal root ganglia revealed that the percentage of CGRP- and SP-ir neuronal profiles showed a significant (P < 0.05) reduction from (mean +/- S.D) 44.5 +/- 2; 23.3 +/- 2 in young adult to 25.0 +/- 2.9; 14.8 +/- 1.6 in aged rats, respectively. These findings suggest that the involvement of CGRP and SP in urinary bladder innervation is relatively unchanged in old age, but their expression in dorsal root ganglion neurons is affected by age. The afferent micturition pathway from the pelvic region via these lumbosacral ganglia may be perturbed as a result.     

‘Neuropeptide tyrosine’ (NPY) is co-stored with noradrenaline in vascular but not in parenchymal sympathetic nerves of brown adipose tissue

By using immunohistochemistry it is shown that both the parenchymal and vascular sympathetic innervation in the interscapular depot of brown adipose tissue in the rat contain the catecholamine-synthesizing enzyme tyrosine-hydroxylase (TH). In contrast, 'neuropeptide tyrosine' (NPY) is selectively present in the vascular sympathetic nerves of the tissue--but not in nerves around brown fat cells. This is consistent with the presence of two populations of neurons (containing either TH alone or TH plus NPY) in the stellate ganglion, which is the probable origin of the sympathetic nerves in the interscapular brown adipose tissue. Furthermore, the perivascular NPY-positive nerves in the brown adipose tissue disappeared after 6-hydroxydopamine treatment, demonstrating their noradrenergic nature. Taken together, these findings suggest that sympathetic nerves to blood vessels and brown fat cells represent two separate subpopulations of autonomic neurons.     

Postnatal development of sympathetic and sensory innervation of the rhesus monkey ovary.

We have used immunofluorescence to study the postnatal development of the sympathetic and sensory innervation to the rhesus monkey (Macaca mulatta) ovary. Sympathetic nerves were identified as adrenergic by their content of tyrosine hydroxylase (TH)-like immunoreactivity and as peptidergic by the presence of neuropeptide Y (NPY). Fibers containing substance P (SP) or calcitonin gene-related peptide (CGRP)-like immunoreactivity were considered as sensory, whereas vasoactive intestinal peptide (VIP)-positive fibers were only defined as peptidergic because VIP may be present in both sympathetic and sensory nerves. Ovaries from neonatal (2-mo-old), juvenile (9-18-mo-old), peripubertal (3-3.5-yr-old), adult (9-14-yr-old), and senescent (20-27-yr-old) monkeys were studied. At all ages, with the exception of senescence, TH-, NPY-, and VIP-containing fibers were associated with follicles in different developmental stages. In peripubertal and adult animals, some primordial follicles were found to be selectively innervated by VIPergic fibers that almost completely encircled each follicle. Both sympathetic and VIP fibers were also detected in the interstitial tissue and associated with the ovarian vasculature at all ages. The number of sympathetic and VIP fibers increased significantly (p < 0.01) between 2 mo and 9-18 mo of age, and again increased (p < 0.01) around the age of puberty (approximately 3 yr of age). After this time, the number of NPY and TH fibers remained constant. Conversely, the number of VIP fibers decreased (p < 0.05) by 9-14 yr of age, but remained constant thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)     

TH-, NPY-, SP-, and CGRP-immunoreactive nerves in interscapular brown adipose tissue of adult rats acclimated at different temperatures: an immunohistochemical study

Interscapular brown adipose tissue (IBAT), a site of nonshivering thermogenesis in mammals, is neurally controlled. The co-existence of sympathetic and peptidergic innervation has been demonstrated in different brown adipose depots. We studied the morphological profile of IBAT innervation and tested by immunohistochemical methods whether cold and warm stimulation are accompanied by modifications in the density of parenchymal noradrenergic nerve fibers. We also studied the immunoreactivity of afferent fibers—which contain calcitonin gene-related peptide (CGRP) and substance P (SP)<197>in different functional conditions. IBAT was obtained from adult rats (6 weeks old) acclimated at different temperatures (4°, 20°, and 28°C). Tissue activity was evaluated by studying the immunolocalization of uncoupling protein (UCP-1), a specific marker of brown adipose tissue. Noradrenergic and peptidergic innervation were seen to arise from morphologically different nerves. Fibers staining for tyrosine hydroxylase (TH) were thin, unmyelinated hilar nerves, and CGRP- and SP-positive fibers were in thick nerves containing both myelinated and unmyelinated fibers. Under cold stimulation, noradrenergic neurons produce greater amounts of TH, and their axons branch, resulting in increased parenchymal nerve fibers density. Neuropeptide Y (NPY) probably co-localizes with TH in noradrenergic neurons, but only in the perivascular nerve fiber network. The parenchymal distribution of NPY to interlobular arterioles and capillaries suggests that this peptide must have other functions besides that of innervating arteriovenous anastomoses, as hypothesized by other researchers. The different distribution of CGRP and SP suggests the existence of different sensory neuronal populations. The detection of CGRP at the parenchymal level is in line with the hypothesis of a trophic action of this peptide.     

Neuropeptide Y: presence in sympathetic and parasympathetic innervation of the nasal mucosa

Summary The occurrence of neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI) in the sympathetic and parasympathetic innervation of the nasal mucosa was studied in various species including man. A dense network of NPY-immunoreactive (IR) fibres was present around arteries and arterioles in the nasal mucosa of all species studied. NPY was also located in nerves around seromucous glands in pig and guinea-pig, but not in rat, cat and man. The NPY-IR glandular innervation corresponded to about 20% of the NPY content of the nasal mucosa as revealed by remaining NPY content determined by radioimmunoassay after sympathectomy. These periglandular NPY-positive fibres had a distribution similar to the VIP-IR and PHI-IR nerves but not to the noradrenergic markers tyrosine hydroxylase (TH) or dopamine--hydroxylase (DBH). The NPY nerves around glands and some perivascular fibres were not influenced by sympathectomy and probably originated in the sphenopalatine ganglion where NPY-IR and VIP-IR ganglion cells were present. The venous sinusoids were innervated by NPY-positive fibres in all species except the cat. Dense NPY and DBH-positive innervation was seen around thick-walled vessels in the pig nasal mucosa; the latter may represent arterio-venous shunts. Double-labelling experiments using TH and DBH, and surgical sympathectomy revealed that the majority of NPY-IR fibres around blood vessels were probably noradrenergic. The NPY-positive perivascular nerves that remained after sympathectomy in the pig nasal mucosa also contained VIP/PHI-IR. The major nasal blood vessels, i.e. sphenopalatine artery and vein, were also densely innervated by NPY-IR fibres of sympathetic origin. Perivascular VIP-IR fibres were present around small arteries, arterioles, venous sinusoids and arterio-venous shunt vessels of the nasal mucosa whereas major nasal vessels received only single VIP-positive nerves. The trigeminal ganglion of the species studied contained only single TH-IR or VIP-IR but no NPY-positive ganglion cells. It is concluded that NPY in the nasal mucosa is mainly present in perivascular nerves of sympathetic origin. In some species, such as pig, glandular and perivascular parasympathetic nerves, probably of VIP/PHI nature, also contain NPY.     

Regional-dependent increase of sympathetic innervation in rat white adipose tissue during prolonged fasting.

White adipose tissue (WAT) is innervated by the sympathetic nervous system. A role for WAT sympathetic noradrenergic nerves in lipid mobilization has been suggested. To gain insight into the involvement of nerve activity in the delipidation process, WAT nerves were investigated in rat retroperitoneal and epididymal depots after prolonged fasting. A significant increase in tyrosine hydroxylase (TH) content was found in epididymal and, especially, retroperitoneal WAT by Western blotting. Accordingly, an increased immunoreactivity for TH was detected by immunohistochemistry in epididymal and, especially, retroperitoneal vascular and parenchymal noradrenergic nerves. Neuropeptide Y (NPY)-containing nerves were found around arteries and in the parenchyma. Double-staining experiments and confocal microscopy showed that most perivascular and some parenchymal noradrenergic nerves also contained NPY. Detection of protein gene product (PGP) 9.5, a general marker of peripheral nerves, by Western blotting and PGP 9.5-TH by double-staining experiments showed significantly increased noradrenergic nerve density in fasted retroperitoneal, but not epididymal depots, suggesting that formation of new nerves takes place in retroperitoneal WAT in fasting conditions. On the whole, these data confirm the important role of sympathetic noradrenergic nerves in WAT lipid mobilization during fasting but also raise questions about the physiological role of regional-dependent nerve adjustments and their functional significance in relation to white adipocyte secretory products.     

Neuropeptide Y,enkephalin and noradrenaline coexist in sympathetic neurons innervating the bovine spleen

Summary The subcellular distribution of noradrenaline (NA), neuropeptide Y (NPY), Met and Leu-enkephalin (ENK), substance P (SP), somatostatin (SOM), and vasoactive intestinal polypeptide (VIP) was investigated in homogenates of bovine splenic nerve. The distribution of noradrenergic peptide-containing nerves in the bovine celiac ganglion, splenic nerve and terminal areas in spleen was studied by indirect immunofluorescence histochemistry using antisera to tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH), NPY, enkephalin peptides, SP, SOM, VIP and peptide HI (PHI).After density gradient centrifugation, high levels of NPY and ENK-like immunoreactivity (LI) were found in high-density gradient fractions, coinciding with the main NA peak. SP, SOM and VIP were found in fractions with a lower density, VIP being also enriched in a heavy fraction; the latter three peptides were present in low concentrations.Immunohistochemistry revealed that staining for NPYLI and ENK-LI partly overlapped that for TH and DBH in celiac ganglia, splenic nerve axons and terminal areas of spleen. Almost all principal ganglion cells were TH- and DBH-immunoreactive. Many were also NPY-immunoreactive, whereas a smaller number were ENK-positive. In the celiac ganglion patches of dense SP-positive networks and some VIP/PHI- and ENK-immunoreactive fibers were seen around cell bodies.The results indicate that NPY and ENK are stored with NA in large dense-cored vesicles in unmyelinated axons of bovine splenic nerve. SP, SOM and VIP appear in different organelles in axon populations separate from sympathetic noradrenergic nerves.     

Patterns of co-existence of peptides and differences of nerve fibre types associated with noradrenergic and non-noradrenergic (putative cholinergic) neurons in the major pelvic ganglion of the male rat

Summary The pelvic ganglia supply cholinergic and noradrenergic nerve pathways to many organs. Other possible transmitters are also present in these nerves, including peptides. Multiple labelling immunofluorescence techniques were used in this study of the male rat major pelvic ganglion (MPG) to examine: (1) the peptides present in noradrenergic (tyrosine hydroxylase (TH)-positive) and non-noradrenergic (putative cholinergic) neurons, and (2) the types of peptide-containing nerve fibres closely associated with these two groups of neurons. The distribution of the peptide galanin (GAL) within the MPG was also investigated. All of the TH-neurons contained neuropeptide Y (NPY), but none of the other tested peptides. However, many NPY neurons did not contain TH and may have been cholinergic. TH-negative neurons also displayed vasoactive intestinal peptide (VIP), enkephalin (ENK) or GAL. VIP and NPY formed the most common types of putative cholinergic pelvic neurons, but few cells contained both peptides. Many ENK neurons exhibited VIP, NPY or GAL. Varicose nerve terminals surrounding ganglion cells contained ENK, GAL, somatostatin (SOM) and cholecystokinin (CCK). These peptide-immunoreactive fibres were more often associated with the non-noradrenergic (putative cholinergic) than the noradrenergic neurons; two types (SOM and CCK) were preferentially associated with the non-noradrenergic NPY neurons. GAL was distributed throughout the MPG, in small neurons, scattered small, intensely fluorescent (SIF) cells, and both varicose and non-varicose nerve fibres. The nerve fibres were concentrated near the pelvic and penile nerves; most of the varicose fibres formed baskets surrounding individual GAL-negative somata.     

Specific distribution pattern of nerve fibers containing catecholamine-synthesizing enzymes, neuropeptide Y (NPY) and C-terminal flanking peptide of NPY (CPON) in the pineal gland of the chinchilla (Chinchilla laniger)--an immunohistochemical study

The sympathetic nerve fibers originating from the superior cervical ganglia and supplying the pineal gland play the most important role in the control of the pineal activity in mammals. NPY and CPON are also present in the majority of the pinealopetal sympathetic neurons. In this study, immunohistochemical techniques were used to demonstrate the existence and coexistence of tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DbetaH) as well as NPY and CPON in the nerve fibers supplying the chinchilla pineal gland. Ten two-year-old female chinchillas housed in natural light conditions were used in the study. The pineals were fixed by perfusion. ABC immunohistochemical technique and immunofluorescence labelling method were employed. TH-immunoreactive (TH-IR) varicose nerve fibers were observed in the pineal gland as well as in the posterior commissural area. Within the chinchilla pineal gland, TH-IR nerve fibers were located in the capsule and connective tissue septa. Numerous varicose TH-IR branches penetrated into the parenchyma and formed a network showing the highest density in the proximal region of the gland. In the central and distal parts of the pineal parenchyma, a subtle network, composed of thin varicose nerve branches, was observed. Double immunostaining revealed that the majority of TH-IR nerve fibers was positive for DbetaH or NPY. TH- and DbetaH-positive neuron-like cells were observed in the proximal region of the gland. The pattern of pineal innervation immunoreactive to CPON was similar to the innervation containing NPY, TH and DbetaH. The chinchilla intrapineal innervation containing TH, DbetaH, NPY and CPON is characterized by the higher density in the proximal part of the gland than in the middle and distal ones. The specific feature of the chinchilla pineal is also the presence of single TH/DbetaH-immunoreactive neuron-like cells in the proximal part of the gland.     

Immunohistochemical properties and spinal connections of pelvic autonomic neurons that innervate the rat prostate gland

Autonomic innervation of the prostate gland supplies the acini, and non-vascular and vascular smooth muscle. The activity of each of these tissues is enhanced by sympathetic outflow, whereas the role of the parasympathetic nervous system in this organ is unclear. In the present study, a range of methods was applied in rats to determine the location of autonomic neurons supplying this gland, the immunohistochemical properties of these neurons, the spinal connections made with the postganglionic pathways and the distribution of various axon types within the gland. Injection of the retrograde tracer, FluoroGold, into the ventral gland visualised neurons within the major pelvic ganglion and sympathetic chain. Fluorescence immunohistochemical studies on the labelled pelvic neurons showed that most were noradrenergic (also containing neuropeptide Y, NPY), the others being non-noradrenergic and containing either vasoactive intestinal peptide (VIP) or NPY. Sympathetic dyelabelled neurons were identified by the presence of varicose nerve terminals stained for synaptophysin on their somata following lesion of sacral inputs. Parasympathetic innervation of dye-labelled neurons was identified by continued innervation after hypogastric nerve lesion. Most noradrenergic prostate-projecting neurons were sympathetic, as were many of the non-noradrenergic VIP neurons. Parasympathetic prostate-projecting neurons were largely non-noradrenergic and contained either VIP or NPY. All substances found in retrogradely labelled somata were located in axons within the prostate gland but had slightly different patterns of distribution. The studies have shown that there are a significant number of non-noradrenergic sympathetic prostate-projecting neurons, which contain VIP.     

Innervation of the proximal urethra of ovariectomized and estrogen-treated female rats

The proximal urethra plays a central role in maintaining urinary continence, and sympathetic excitatory innervation to urethral smooth muscle is a major factor in promoting tonic contraction of this organ. Elevated estrogen levels are often associated with incontinence in humans. Because elevated estrogen levels result in degeneration of sympathetic nerves from the closely related uterine smooth muscle, we examined the effects of chronic estrogen administration on proximal urethral innervation. Ovariectomized virgin female rats received either vehicle or 17 beta-estradiol for 1 week, and smooth muscle size and parasympathetic, sensory and sympathetic nerve densities were assessed quantitatively throughout the first 3 mm of the proximal urethral smooth muscle. In vehicle-infused ovariectomized rats, parasympathetic nerves immunoreactive for vesicular acetylcholine transporter were most abundant, while calcitonin gene-related peptide-immunoreactive sensory nerves and tyrosine hydroxylase-immunoreactive sympathetic nerves were less numerous. The densities of parasympathetic and sensory nerves remained constant along the proximal urethra, while sympathetic nerves showed a significant increase along a proximal-distal gradient. Administration of 17beta-estradiol for 7 days via subcutaneous osmotic pump did not change smooth muscle area in sections, and neither densities nor total innervation of any nerve population was altered. These findings reveal a rich cholinergic innervation of the proximal urethra, and a pronounced gradient in sympathetic innervation. Unlike the embryologically similar uterine smooth muscle, estrogen does not influence muscle size or composition of innervation, indicating that estrogen's actions on innervation are highly target-specific. Thus, estrogen's effects on urinary continence apparently occur independently of any significant remodeling of smooth muscle or resident innervation.     

Studies on colocalization of neuropeptide Y,vasoactive intestinal polypeptide,catecholamine-synthesizing enzymes and acetylcholinesterase in the larynx of the rat

Summary In the present immunohistochemical study, the distribution of nerve fibers containing neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) in the larynx was examined and compared with that of fibers containing tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (BDH), and with that of acetylcholinesterase (AChE)-positive nerve fibers, in intact and vagotomized rats and in rats subjected to removal of the superior cervical ganglion (SCG). Fibers showing TH/DBH-like immunoreactivity (LI) were only found in the walls of arteries and arterioles, whereas AChE-positive nerve fibers were located close to the acini and ducts of the glands, in blood vessel walls, in the perichondrium and in the lamina propria. NPY-LI and VIP-LI coexisted in local AChE-positive ganglionic cells and in a subpopulation of the AChE-positive fibers, NPY-LI also being present in some periarterial fibers showing TH/DBH-LI. Unilateral removal of the SCG eliminated the TH/DBH-innervation in the upper but not the lower parts of the larynx ipsilaterally, whereas the NPY-innervation of the arteries in the upper parts only partly disappeared and the NPY-innervation of the other structures remained unchanged. The distribution of VIP-innervation was unchanged after vagotomy and removal of the SCG. The results suggest that VIP is present in the postganglionic parasympathetic innervation, whereas NPY is present in both the postganglionic parasympathetic and sympathetic innervation of the rat larynx.     

An immunocytochemical study of cutaneous innervation and the distribution of neuropeptides and protein gene product 9.5 in man and commonly employed laboratory animals.

The cutaneous nerves of rat, cat, guinea pig, pig, and man were studied by immunocytochemistry to compare the staining potency of general neural markers and to investigate the density of nerves containing peptides. Antiserum to protein gene product 9.5 (PGP 9.5) stained more nerves than antisera to neurofilaments, neuron-specific enolase (NSE), and synaptophysin or histochemistry for acetylcholinesterase (AChE). Peptidergic axons showed species variation in density of distribution and were most abundant in pig and fewest in man. However, the specific peptides in nerves innervating the various structures were consistent between species. Nerve fibers immunoreactive for calcitonin gene-related peptide (CGRP) and/or vasoactive intestinal polypeptide (VIP) predominated in all the species; those immunoreactive to tachykinins (substance P and neurokinin A [NKA]) and neuropeptide tyrosine (NPY) were less abundant. Neonatal capsaicin, at the doses employed in this study, destroyed approximately 70% of CGRP- and tachykinin-immunoreactive sensory axons; whereas 6-hydroxydopamine (6-OHDA) at the doses employed resulted in a complete loss of NPY and tyrosine hydroxylase (TH) immunoreactivity without affecting VIP, CGRP, and tachykinins. Thus, this study confirms that antiserum to PGP 9.5 is the most suitable and practical marker for the demonstration of cutaneous nerves. Species differences exist in the density of peptidergic innervation, but apparently not for specific peptides. Not all sensory axons immunoreactive for CGRP and substance P/NKA are capsaicin-sensitive. However, all sympathetic TH- and NPY-immunoreactive axons are totally responsive to 6-OHDA; but no change was seen in VIP-immunoreactive axons, suggesting some demarcation of cutaneous adrenergic and cholinergic sympathetic fibers.     

TH and NPY in sympathetic neurovascular cultures: role of LIF and NT-3

The sympathetic nervous system is an important determinant of vascular function. The effects of the sympathetic nervous system are mediated via release of neurotransmitters and neuropeptides from postganglionic sympathetic neurons. The present study tests the hypothesis that vascular smooth muscle cells (VSM) maintain adrenergic neurotransmitter/neuropeptide expression in the postganglionic sympathetic neurons that innervate them. The effects of rat aortic and tail artery VSM (AVSM and TAVSM, respectively) on neuropeptide Y (NPY) and tyrosine hydroxylase (TH) were assessed in cultures of dissociated sympathetic neurons. AVSM decreased TH (39 +/- 12% of control) but did not affect NPY. TAVSM decreased TH (76 +/- 10% of control) but increased NPY (153 +/- 20% of control). VSM expressed leukemia inhibitory factor (LIF) and neurotrophin-3 (NT-3), which are known to modulate NPY and TH expression. Sympathetic neurons innervating blood vessels expressed LIF and NT-3 receptors. Inhibition of LIF inhibited the effect of AVSM on TH. Inhibition of neurotrophin-3 (NT-3) decreased TH and NPY in neurons grown in the presence of TAVSM. These data suggest that vascular-derived LIF decreases TH and vascular-derived NT-3 increases or maintains NPY and TH expression in postganglionic sympathetic neurons. NPY and TH in vascular sympathetic nerves are likely to modulate NPY and/or norepinephrine release from these nerves and are thus likely to affect blood flow and blood pressure. The present studies suggest a novel mechanism whereby VSM would modulate sympathetic control of vascular function.     

Immunohistochemical characterisation of sympathetic and parasympathetic pelvic neurons projecting to the distal colon in the male rat

The pelvic ganglia are mixed ganglia containing both sympathetic and parasympathetic neurons that receive spinal input via the hypogastric (lumbar cord) and pelvic nerves (sacral cord), respectively. A recent study has utilised immunohistochemistry against synaptophysin (a protein associated with small vesicles) to visualise the preganglionic terminals in these ganglia. By selectively cutting the hypogastric or pelvic nerves and allowing subsequent terminal degeneration, the populations of parasympathetic and sympathetic preganglionic terminals, respectively, can be visualised. The present study has used this method in conjunction with retrograde labelling of pelvic neurons from the distal colon and double label immunofluorescence against tyrosine hydroxylase and vasoactive intestinal polypeptide (VIP) to identify and characterise the sympathetic and parasympathetic neurons projecting to the distal colon from the major pelvic ganglia of the male rat. Approximately equal numbers of distal colonic-projecting pelvic neurons are sympathetic and parasympathetic. Almost all noradrenergic neurons are sympathetic. Of the VIP neurons that project to the distal colon approximately one third are sympathetic, one third parasympathetic and the remaining third are possibly innervated by both the lumbar and sacral cord. Extrapolation from our results also suggests that the majority of non-noradrenergic neuropeptide Y neurons (which are known to comprise the remainder of the neurons) are parasympathetic. These studies have demonstrated that the pelvic ganglia are a major source of sympathetic innervation to the distal bowel and have further shown that the distal colon is another target for the non-noradrenergic sympathetic neurons of the pelvic ganglia.     

In vivo mechanisms of cutaneous vasodilation and vasoconstriction in humans during thermoregulatory challenges.

This review focuses on the neural and local mechanisms that have been demonstrated to effect cutaneous vasodilation and vasoconstriction in response to heat and cold stress in vivo in humans. First, our present understanding of the mechanisms by which sympathetic cholinergic nerves mediate cutaneous active vasodilation during reflex responses to whole body heating is discussed. These mechanisms include roles for cotransmission as well as nitric oxide (NO). Next, the mechanisms by which sympathetic noradrenergic nerves mediate cutaneous active vasoconstriction during whole body cooling are reviewed, including cotransmission by neuropeptide Y (NPY) acting through NPY Y1 receptors. Subsequently, current concepts for the mechanisms that effect local cutaneous vascular responses to direct skin warming are examined. These mechanisms include the roles of temperature-sensitive afferent neurons as well as NO in causing vasodilation during local heating of skin. This section is followed by a review of the mechanisms that cause local cutaneous vasoconstriction in response to direct cooling of the skin, including the dependence of these responses on intact sensory and sympathetic, noradrenergic innervation as well as roles for nonneural mechanisms. Finally, unresolved issues that warrant further research on mechanisms that control cutaneous vascular responses to heating and cooling are discussed.     

Estrogen regulates vaginal sensory and autonomic nerve density in the rat

Vaginal function is strongly influenced by reproductive hormone status. Vaginal dysfunction during menopause is generally assumed to occur because of diminished estrogen-mediated trophic support of vaginal target cells. However, peripheral neurons possess estrogen receptors and are potentially responsive to gonadal steroid hormones. In the present study, we investigated whether sensory and autonomic innervation of the vagina varies among rats during the estrus phase of the estrous cycle, following chronic ovariectomy, and after sustained estrogen replacement. Relative to rats in estrus, ovariectomized rats showed a 59% elevation in nerve density, as determined using the panneuronal marker PGP 9.5. This increase persisted even after correcting for differences in vaginal tissue size, indicating true axonal proliferation after ovariectomy rather than changes secondary to altered volume. Increased total innervation after ovariectomy was attributable to increased densities of sympathetic nerves immunostained for tyrosine hydroxylase (70%), cholinergic parasympathetic nerves immunoreactive for vesicular acetylcholine transporter (93%), and calcitonin gene-related peptide-immunoreactive sensory nociceptor nerves (84%). Myelinated primary sensory innervation revealed by RT-97 immunoreactivity did not appear to be affected. Sustained 17beta-estradiol administration reduced innervation density to an extent comparable to that of estrus, implying that estrogen is the hormone mediating vaginal neuroplasticity. These findings indicate that some aspects of vaginal dysfunction during menopause may be attributable to changes in innervation. Increased sympathetic innervation may augment vasoconstriction and promote vaginal dryness, while sensory nociceptor axon proliferation may contribute to symptoms of pain, burning, and itching associated with menopause and some forms of vulvodynia.     

Early postnatal exposure of mice to side-steam tobacco smoke increases neuropeptide Y in lung

Our recent study showed that prenatal and early postnatal exposure of mice to side-steam tobacco smoke (SS), a surrogate to environmental tobacco smoke (ETS), leads to increased airway responsiveness and sensory innervation later in life. However, the underlying mechanism initiated in early life that affects airway responses later in life remains undefined. The concomitant increase in nerve growth factor (NGF) after exposures suggests that NGF may be involved the regulation of airway innervation. Since NGF regulates sympathetic nerve responses, as well as sensory nerves, we extended previous studies by examining neuropeptide Y (NPY), a neuropeptide associated with sympathetic nerves. Different age groups of mice, postnatal day (PD) 2 and PD21, were exposed to either SS or filtered air (FA) for 10 consecutive days. The level of NPY protein in lung and the density of NPY nerve fibers in tracheal smooth muscle were significantly increased in the PD2-11SS exposure group compared with PD2-11FA exposure. At the same time, the level of NGF in lung tissue was significantly elevated in the PD2-11SS exposure groups. However, neither NPY (protein or nerves) nor NGF levels were significantly altered in PD21-30SS exposure group compared with the PD21-30FA exposure group. Furthermore, pretreatment with NGF antibody or K252a, which inhibits a key enzyme (tyrosine kinase) in the transduction pathway for NGF receptor binding, significantly diminished SS-enhanced NPY tracheal smooth muscle innervation and the increase in methacholine-induced airway resistance. These findings show that SS exposure in early life increases NPY tracheal innervation and alters pulmonary function and that these changes are mediated through the NGF.     

Sensory and Sympathetic Nerve Sprouting in the Rat Cornea Following Neonatal Administration of Capsaicin

Corneal sensory and sympathetic nerves exert opposing actions on corneal mitogenesis and wound healing. The mechanisms by which these nerves exert their actions are unknown; however, the release of axonally transported neuropeptides has been postulated. In the present study, we investigated changes in innervation densities of calcitonin gene-related peptide (CGRP-) and tyrosine hydroxylase (TH-)immunoreactive (IR) nerves of the rat cornea following neonatal capsaicin administration, and the relationships between these changes and the development of neuroparalytic keratitis. Newborn rats were injected with capsaicin on each of the first 3 days of life. Forty-eight hours after the last injection, corneal CGRP immunostaining had totally disappeared from the cornea, whereas TH immunostaining was relatively unaffected. Over the next several weeks, a dramatic reinnervation of the cornea took place. By 6–8 weeks both the CGRP-and TH-IR corneal innervation density in the capsaicin-treated animals exceeded that of age-matched control or normal animals; that is, the corneas had become “hyper-reinnervated”. The pattern of innervation that returned was grossly abnormal and was characterized by the presence of a bizarre subepithelial plexus of fine stromal sprouts; an abundance of myelinated axons; and complex, atypical, epithelial leash morphologies. Retrograde transport of wheatgerm agglutinin conjugated to horseradish peroxidase (WGA:HRP) from the central cornea in control and capsaicin-treated adult animals labeled an average of 143 and 47 trigeminal ganglion cells, respectively (with mean diameters of 25.7 × 0.49 μm and 34.3 × 0.72 μm), suggesting a 67% decrease in corneal afferent neurons in the capsaicin-treated animals. Transection of the ophthalmomaxillary nerve in adult capsaicin-treated animals completely eliminated corneal CGRP-IR staining, and extirpation of the superior cervical ganglion resulted in the loss of 70–80% of corneal TH-IR nerves, thus demonstrating the sensory and predominantly sympathetic origins, respectively, of these fiber populations. Chronic keratitis and neovascularization developed in the capsaicin-treated animals by approximately 3 weeks of age, achieved a maximum intensity between 4 and 6 weeks, and showed some gradual improvement thereafter. However, the keratitis never completely disappeared, even after 13 months. In conclusion, these data show that corneal sensory (CGRP-IR) and sympathetic (TH-IR) nerve fibers undergo extensive sprouting following partial corneal sensory denervation with the neurotoxin capsaicin. However, the resultant “hyper-reinnervation” is morphologically abnormal and, for reasons unknown, functionally incapable of preventing or totally reversing the keratitis.     

Evidence for neurotransmitter plasticity in vivo. II. Immunocytochemical studies of rat sweat gland innervation during development

Previous studies of the cholinergic sympathetic innervation of rat sweat glands provide evidence for a change in neurotransmitter phenotype from noradrenergic to cholinergic during development. To define further the developmental history of cholinergic sympathetic neurons, we have used immunocytochemical techniques to examine developing and mature sweat gland innervation for the presence of the catecholamine synthetic enzymes tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) and for two neuropeptides present in the mature cholinergic innervation, vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP). In 7-day old animals, intensely TH- and DBH-immunoreactive axons were closely associated with the forming glands. The intensity of both the TH and DBH immunofluorescence decreased as the glands and their innervation developed. Neither TH-IR nor DBH-IR disappeared entirely; faint immunoreactivity for both enzymes was reproducibly detected in mature animals. In contrast to noradrenergic properties, the expression of peptide immunoreactivities appeared relatively late. No VIP-IR or CGRP-IR was detectable in the sweat gland innervation at 4 or 7 days. In some glands VIP-IR first appeared in axons at 10 days, and was evident in all glands by 14 days. CGRP-IR was detectable only after 14 days. In addition to VIP-IR and CGRP-IR, we examined the sweat gland innervation for several neuropeptides which have been described in noradrenergic sympathetic neurons including neuropeptide Y, somatostatin, substance P, and leu- and met-enkephalin; these peptides were not evident in either developing or mature sweat gland axons. Our observations provide further evidence for the early expression and subsequent modulation of noradrenergic properties in a population of cholinergic sympathetic neurons in vivo. In addition, the asynchronous appearance during development of the two neuropeptide immunoreactivities raises the possibility that the expression of peptide phenotypes may be controlled independently.