Ontogenetic development of the guinea pig uterine innervation. An immunohistochemical study of different neuronal markers, neuropeptides and S-100 protein

The ontogenetic development of the guinea pig uterine autonomic innervation was studied immunohistochemically using neurofibrillary protein (NF) and neuron specific enolase (NSE) as general neuronal markers, tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) as specific markers for adrenergic innervation and S-100 protein as marker for Schwann cell structure and/or function. In addition, comparisons were made of the development of the different populations of peptide-containing nerves. The structure and time of appearance were similar for nerves with NF-, NSE-, TH- and DBH-immunoreactivities, which were first present in the organ periphery as coarse nerve trunks, then extending centrally and branching into non-varicose nerves. From these, varicose nerves developed first in relation to vessels and then in association with the myometrial smooth musculature. Development was completed earlier in the cervix than in the uterine horns suggesting differences in local environment. In comparison, S-100 nerve-immunoreactivity appeared later but attained complete development more rapidly than axonal structures. Neuropeptide Y-immunoreactive nerves showed a similar developmental pattern to presumed adrenergic nerves, further verifying the assumption of intraneuronal localization of NPY in uterine adrenergic nerves. Other peptide-containing nerves were developed later probably reflecting differences in neuronal growth properties.     

Ontogenetic development of the guinea pig uterine innervation

Summary The ontogenetic development of the guinea pig uterine autonomic innervation was studied immunohistochemically using neurofibrillary protein (NF) and neuron specific enolase (NSE) as general neuronal markers, tyrosine hydroxylase (TH) and dopamine -hydroxylase (DBH) as specific markers for adrenergic innervation and S-100 protein as marker for Schwann cell structure and/or function. In addition, comparisons were made of the development of the different populations of peptide-containing nerves.The structure and time of appearance were similar for nerves with NF-, NSE-, TH- and DBH-immunoreactivities, which were first present in the organ periphery as coarse nerve trunks, then extending centrally and branching into non-varicose nerves. From these, varicose nerves developed first in relation to vessels and then in association with the myometrial smooth musculature. Development was completed carlier in the cervix than in the uterine horns suggesting differences in local environment. In comparison, S-100 nerve-immunoreactivity appeared later but attained complete development more rapidly than axonal structures. Neuropeptide Y-immunoreactive nerves showed a similar developmental pattern to presumed adrenergic nerves, further verifying the assumption of intraneuronal localization of NPY in uterine adrenergic nerves. Other peptide-containing nerves were developed later probably reflecting differences in neuronal growth properties.     

Effects of pregnancy on the extrinsic innervation of the guinea pig uterus. A histochemical, immunohistochemical and ultrastructural study

Summary The extrinsic innervation of the guinea pig uterus was studied by immunohistochemical, ultrastructural and enzyme histochemical methods.The extrinsic innervation was organized in two major ways. One consisted of nerve trunks and non-varicose nerve fibres running in the suspensory ligament, and the other of a plexus of varicose nerve fibres surrounding vessels, and non-vessel-related non-varicose nerve fibres in the mesouterus. The use of different neuronal and Schwann cell markers showed that the extrinsic innervation was predominantly adrenergic and contained only few peptidergic nerves. Acetylcholinesterase-positive (cholinergic) nerves were only found around the uterine artery.In late pregnancy, the extrinsic nerves of the mesouterus adjacent to foetus-containing uterine horns underwent pronounced degenerative changes comprising both Schwann cell and axonal structures. In comparison, no changes were found in extrinsic nerves of mesouteri adjacent to non-foetus-bearing uterine horns or in extrinsic nerves in the suspensory ligaments. Further, chemical sympathectomy produced axonal degeneration but no changes in the Schwann cells.In conclusion, the pregnancy-induced nerve degeneration is of a very special type different from that following chemical sympathectomy and represents a local phenomenon related to the conceptus. Hypothetically, this could be of importance for counteracting disturbances in placental blood flow.     

Ganglion cells immunoreactive for catecholamine-synthesizing enzymes,neuropeptide Y and vasoactive intestinal polypeptide in the rat adrenal gland

Immunohistochemistry has been used to demonstrate tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) immunoreactivities, and acetylcholinesterase (AChE) activity was demonstrated in rat adrenal glands. The TH, DBH, NPY and VIP immunoreactivities and AChE activity were observed in both the large ganglion cells and the small chromaffin cells whereas PNMT immunoreactivity was found only in chromaffin cells, and not in ganglion cells. Most intraadrenal ganglion cells showed NPY immunoreactivity and a few were VIP immunoreactive. Numerous NPY-immunoreactive ganglion cells were also immunoreactive for TH and DBH; these cells were localized as single cells or groups of several cells in the adrenal cortex and medulla. Use of serial sections, or double and triple staining techniques, showed that all TH- and DBH-immunoreactive ganglion cells also showed NPY immunoreactivity, whereas some NPY-immunoreactive ganglion cells were TH and DBH immunonegative. NPY-immunoreactive ganglion cells showed no VIP immunoreactivity. AChE activity was seen in VIP-immunopositive and VIP-immunonegative ganglion cells. These results suggest that ganglion cells containing noradrenaline and NPY, or NPY only, or VIP and acetylcholine occur in the rat adrenal gland; they may project within the adrenal gland or to other target organs. TH, DBH, NPY, and VIP were colocalized in numerous immunoreactive nerve fibres, which were distributed in the superficial adrenal cortex, while TH-, DBH- and NPY-immunoreactive ganglion cells and nerve fibres were different from VIP-immunoreactive ganglion cells and nerve fibres in the medulla. This suggests that the immunoreactive nerve fibres in the superficial cortex may be mainly extrinsic in origin and may be different from those in the medulla.     

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.     

Involvement of perivascular neuropeptide Y nerve fibres in uterine arterial vasoconstriction in conjunction with pregnancy

The perivascular neuropeptide Y (NPY) innervation and its relation to adrenergic nerves of uterine arteries from non-pregnant and pregnant guinea pigs was analyzed immunocytochemically. The NPY content of the uterine artery was, in addition, measured radioimmunologically (RIA). Vasomotor effects of NPY per se and in combination with other vasoconstrictors were examined using a sensitive in vitro method. Pregnancy did not visibly affect density and distribution of NPY-immunoreactive fibres. The NPY fibres contained in addition immunoreactivity to dopamine-beta-hydroxylase (marker for noradrenergic neurons). RIA revealed a slight decrease of NPY content during pregnancy, probably due to the increased smooth muscle volume of uterine arteries. The contractile effect of NPY on uterine arteries was weak, while vasoconstriction induced by various agonists was potentiated by NPY, particularly during pregnancy. It is concluded that perivascular NPY-containing nerve fibres may be involved in the dramatic blood flow alterations that occur in the uterine circulation in connection with pregnancy and partus.     

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.     

Tyrosine hydroxylase and neuropeptide-Y immunoreactivity in pineal glands developing in situ and in pineal grafts

Summary Postnatal development of the innervation of the pineal gland in situ as well as the reinnervation of pineal grafts by tyrosine hydroxylase (TH)- and neuropeptide Y (NPY)-immunoreactive nerve fibers were examined using the avidin-biotin-peroxidase immunohistochemical technique. TH-immunoreactive nerve fibers appeared in the pineal gland on the second postnatal day (P2) in both hamsters and gerbils. NPY-immunoreactive nerve fibers first appeared in the pineal gland of gerbils on P2 and in the hamsters on P3. By the seventh postnatal day (P7), the pineal glands of both hamsters and gerbils were richly innervated by TH- and NPY-fibers that appeared as smooth fibers or fibers with sporadic varicosities. By the age of 4 weeks, the innervation of the pineal glands of hamsters and gerbils by TH-and NPY-fibers was fully developed. Abundant TH- and NPY-fibers formed a dense meshwork in the parenchyma of the superficial and deep pineals. The great majority of the fibers bore a large number of varicosities. More NPY-fibers were found in the pineal glands of gerbils than hamsters. NPY fibers were distributed evenly throughout the pineal glands of the gerbil, but they were more often located in the central region of the superficial pineal of the hamster. For the pineal grafts, superficial pineals from neonatal and 4-week-old hamsters were transplanted to different sites in the third cerebral ventricle (infundibular recess, posterior third ventricle) or beneath the renal capsule. The pineal grafts from 4-week-old donors appeared to undergo severe degeneration and eventually disappeared. The pineal grafts from neonatal hamsters, however, successfully survived and became well integrated into their new locations. Abundant TH-and NPY-fibers in the host brain were found surrounding the pineal grafts placed in the third cerebral ventricle, but were only rarely seen entering the parenchyma of the grafts. A few TH-fibers were demonstrated in the renal grafts 4 weeks after transplantation. These studies describe the postnatal development of the innervation of the pineal glands in situ by TH-and NPY-nerve fibers, and demonstrate a lack of reinnervation of cerebroventricular pineal grafts by TH and NPY fibers from adjacent host brain.Portions of the results of this paper were previously reported in abstract form at the 1990 Meeting of The American Association of Anatomists (Anat Rec 226:57A)     

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.     

Annual variations of the NPYergic innervation of the pineal gland in the European hamster (Cricetus cricetus): a quantitative immunohistochemical study

A prominent innervation of the pineal gland of the European hamster with nerve fibres containing neuropeptide Y (NPY) and tyrosine hydroxylase (TH) was demonstrated by means of immunohistochemistry. Nearly all the TH- and NPY-immunoreactive nerve fibres in the superficial pineal gland disappeared after bilateral superior cervical ganglionectomy, showing that the majority of NPY- and TH-immunoreactive nerve fibres belonged to the sympathetic nervous system. Since, in the European hamster, preliminary studies of the NPY-fibre density in the pineal gland had indicated seasonal changes, the density of NPY-immunoreactive nerve fibre profiles was ascertained in the superficial pineal gland in a series of animals between the first part of November and late April. The highest density of NPY-immunoreactive nerve fibre profiles was observed during midwinter. On the other hand, during the same period of the year, the number of sympathetic TH-immunoreactive sympathetic nerve fibre profiles did not exhibit seasonal variation, nor did substitution of testosterone, during the sexually inactive period, affect the density of NPY-containing nerve fibres in the gland. Our results show the presence of a testosterone-independent annual variation in the content of NPY in the sympathetic nerve fibres innervating the pineal gland of the European hamster. This variation can be correlated with the changes in the daily pattern of melatonin production observed by others in the same species at this period of the year.     

S-100-immunoreactive nerves in the guinea-pig uterus with reference to ultrastructural correlations: Effects of chemical sympathectomy and pregnancy

Summary In the guinea-pig uterus, by use of an indirect immunofluorescence method, S-100 immunoreactivity was found to be restricted to nerves that corresponded in number, distribution and type to adrenergic axons and preterminals. With advancing pregnancy S-100 immunoreactivity completely disappeared in uterine tissue adjacent to a fetus, in parallel with an ultrastructural degeneration of the adrenergic innervation. In the cervix and the uterine horn devoid of a fetus, however, the number and distribution of S-100-immunoreactive nerves was seemingly unchanged and no ultrastructural changes were found in adrenergic nerves. In contrast, chemical sympathectomy produced by 6-hydroxydopamine did not change S-100 immunoreactivity of uterine nerves. These findings suggest that there are differences in the denervation effected by chemical and by pregnancy-induced sympathectomy. The latter probably represents a special type of adrenergic denervation by inducing a degeneration of Schwann cells in addition to destruction of neuronal structures. This may explain the differences in the speed of regeneration of uterine adrenergic nerves following the two types of denervation.     

‘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.     

The intramural innervation of the rabbit upper urinary tract

The role of the autonomic innervation of the upper urinary tract for pyeloureteral motility is not completely understood. It is still debatable if the autonomic nervous system might play a modulating role on the ureteral peristalsis. The aim of this study was to investigate the distribution and regional variation of the intramural innervation using whole-mount preparations of the rabbit upper urinary tract. Whole-mount preparation was performed at upper urinary tracts of healthy rabbits. Immunohistochemistry was employed using Neurofilament (NF), Tyrosine Hydroxylase (TH), Choline Acetyltransferase (ChAT) and Substance P (SP) antibodies. NADPH-diaphorase and Acetylcholinesterase (AChE) histochemistry was carried out at the specimens. The stains were evaluated using brightfield, fluorescence and laser confocal microscopy. NF-, TH-, ChAT- and SP-immunoreactive (-IR) nerves formed distinct neuronal plexuses at the submucosal and muscle layers. Perivascular TH-, ChAT- and SP-IR fibres were demonstrated. AChE positive nerves were revealed in all layers, but only moderate NADPH-diaphorase positive innervation was found. Renal pelvis, upper and lower ureter showed enriched intrinsic innervation. Ganglia were found at the ureteropelvic border and the distal ureter. Whole-mount preparation technique revealed detailed informations about morphology and regional variation of the intramural innervation of the rabbit upper urinary tract.     

Neuropeptide Y (NPY)-like immunoreactivity in guinea pig uterus is reduced during pregnancy in parallel with noradrenergic nerves

Neuropeptide Y (NPY) is a recently discovered neuropeptide with vasoconstrictor effects when given in vivo. It occurs in many sympathetic neurons, where it appears to coexist with noradrenaline (NA). It is wellknown that profound changes in the levels of uterine NA occur in many species during pregnancy. Therefore we have investigated the distribution of catecholamine neurons and NPY by immunohistochemistry in the pregnant and nonpregnant guinea pig uterus. In the virgin uterus NPY-like immunoreactivity was present in nerve fibres and terminals in the smooth muscle layers of the uterine horns and around blood vessels. The distribution of NPY fibres was very similar to that of noradrenergic nerves visualized with antibodies against the catecholamine synthesizing enzyme tyrosine hydroxylase (TH). In the pregnant uterus, NPY- and TH-like immunoreactivity disappeared almost completely. In the cervix, a slight decrease of immunoreactivity was observed, whereas in the ovaries no changes were noted between the pregnant and nonpregnant condition. The results indicate that NPY and catecholamines coexists in the adrenergic neurons of the guinea pig uterus, cervix and ovary and that they vary together in the myometrium during pregnancy. We suggest that NPY may be of functional importance for the pregnant uterus.     

Neuropeptide Y (NPY)-like immunoreactivity in guinea pig uterus is reduced during pregnancy in parallel with noradrenergic nerves

Summary Neuropeptide Y (NPY) is a recently discovered neuropeptide with vasoconstrictor effects when given in vivo. It occurs in many sympathetic neurons, where it appears to coexist with noradrenaline (NA). It is wellknown that profound changes in the levels of uterine NA occur in many species during pregnancy. Therefore we have investigated the distribution of catecholamine neurons and NPY by immunohistochemistry in the pregnant and nonpregnant guinea pig uterus. In the virgin uterus NPY-like immunoreactivity was present in nerve fibres and terminals in the smooth muscle layers of the uterine horns and around blood vessels. The distribution of NPY fibres was very similar to that of noradrenergic nerves visualized with antibodies against the catecholamine synthesizing enzyme tyrosine hydroxylase (TH). In the pregnant uterus, NPY- and TH-like immunoreactivity disappeared almost completely. In the cervix, a slight decrease of immunoreactivity was observed, whereas in the ovaries no changes were noted between the pregnant and nonpregnant condition. The results indicate that NPY and catecholamines coexists in the adrenergic neurons of the guinea pig uterus, cervix and ovary and that they vary together in the myometrium during pregnancy. We suggest that NPY may be of functional importance for the pregnant uterus.     

Ontogeny of intrinsic innervation in the human thymus and spleen.

The ontogeny of the innervation of human lymphoid organs has not been studied in detail. Our aim was to assess the nature and distribution of parenchymal nerves in human fetal thymus and spleen. We used the peroxidase immunohistochemical technique with antibodies specific to neuron-specific enolase (NSE), neurofilaments (NF), PGP9.5, S100 protein, and tyrosine hydroxylase (TH) and evaluated our results with image analysis. In human fetal thymus, NSE-, NF-, S100-, PGP9.5-, and TH-positive nerves were identified associated with large blood vessels from 18 gestational weeks (gw) onwards, increasing in density during development. Their branches penetrated the septal areas at 20 gw, reaching the cortex and the corticomedullary junction between 20 and 23 gw. Few nerve fibers were seen in the medulla in close association with Hassall's corpuscles. In human fetal spleen, NSE-, NF-, S100-, PGP9.5-, and TH-positive nerve fibers were localized in the connective tissue surrounding the splenic artery at 18 gw. Perivascular NSE-, NF-, S100-, PGP9.5-, and TH-positive nerve fibers were seen extending into the white pulp, mainly in association with the central artery and its branches, increasing in density during gestation. Scattered NSE-, NF-, S100-, PGP9.5-, and TH-positive nerve fibers and endings were localized in the red pulp from 18 gw onward. The predominant perivascular distribution of most parenchymal nerves implies that thymic and splenic innervation may play an important functional role during intrauterine life.     

Neuronal interactions between neuropeptide Y (NPY) and catecholaminergic systems in the rat arcuate nucleus as shown by dual immunocytochemistry

Several recent studies have suggested interactions between catecholamine (CA) and neuropeptide Y (NPY) neuronal systems in the rat brain. In order to obtain morphological evidence for such CA/NPY interactions in the arcuate nucleus, we have used a double immunostaining procedure using an anti-tyrosine hydroxylase (TH) antiserum as a marker for catecholamine neurons and an anti-NPY antiserum. This double staining, where the first staining is silver-gold intensified, was detectable at both light and electron microscopic levels. In semi-thin sections, a substantial overlap and close proximity of TH-immunopositive neurons and NPY neuronal elements could be seen within the arcuate nucleus. At the electron microscopic level, direct appositions between TH- and NPY-immunoreactive structures could be detected. These appositions were of axosomatic, axodendritic or axoaxonic types without any synaptic membrane differentiation. Moreover, direct appositions between NPY-immunoreactive structures have also been observed. This morphological study showing appositions between TH and NPY neuronal systems suggest direct interactions between these two systems in the arcuate nucleus.     

Neuropeptide Y: presence in perivascular noradrenergic neurons and vasoconstrictor effects on skeletal muscle blood vessels in experimental animals and man

The presence of neuropeptide Y (NPY)-like immunoreactivity (-LI) in sympathetic perivascular nerves and the functional effects of NPY and noradrenaline (NA) on vascular tone were studied in skeletal muscle of various species. A dense network of NPY-LI was found around arteries and arterioles but not venules in the gluteus maximus muscle of man, gracilis muscle of dog, tenuissimus muscle of rabbit and quadriceps muscle of cat, rat, guinea pig and pig. The distribution of NPY-immunoreactive (-IR) nerves was closely correlated to the presence of tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH)-positive fibers, two markers for noradrenergic neurons. Double-staining experiments revealed that NPY- and TH-IR as well as NPY- and DBH-IR nerve fibers around arteries and arterioles were identical. The veins and venules, however, lacked or had a very sparse innervation of NPY-, TH- and DBH-positive fibers. The NPY- and TH-IR nerves in quadriceps muscle of the guinea pig were absent after treatment with 6-hydroxydopamine. Lumbosacral sympathetic ganglia from the same species contained many NPY-positive cells which were also TH- and DBH-IR. NPY-LI was also detected by radioimmunoassay in extracts of skeletal muscle from guinea pig, rabbit, dog, pig and man as well as of lumbosacral sympathetic ganglia. The content of NPY-LI in skeletal muscle was relatively low (0.1-0.4 pmol/g), whereas lumbosacral sympathetic ganglia had a much higher content (48-88 pmol/g). NPY (10(-7) M) contracted arterioles in the tenuissimus muscle of the rabbit to a similar extent (by 65%) as NA (10(-6) M), as studied by intravital microscopy in vivo. NPY had no effect on the corresponding venules while NA caused a slight contraction of these vessels. In vitro studies of small human skeletal muscle arteries and veins revealed that NPY was more potent than NA in contracting the arteries, and the highest concentration of NPY (5 x 10(-7) M) caused a contraction of a similar magnitude as NA 10(-5) M. NA contracted veins from human skeletal muscle, while NPY had only small effects. It is suggested that NPY, together with NA, could be of importance for sympathetic control of skeletal muscle blood flow.     

Co-existence and origin of peptidergic and adrenergic nerves in the guinea pig uterus

Summary The occurrence and distribution of peptidergic nerves in the guinea pig uterus was studied by means of immunocytochemistry using numerous neuropeptide antisera. Neuropeptide Y (NPY)-immunoreactive (IR) nerves were the most abundant, whereas substance P (SP)-, calcitonine gene-related peptide (CGRP)-, and neurokinin A (NKA)-IR nerves were less frequent, and peptide histidine isoleucine (PHI)-IR nerves were the most sparse. Chemical sympathectomy by means of 6-hydroxydopamine, and capsaicin treatment revealed the division of the peptidergic nerves into three separate populations: (1) NPY-IR nerves, which co-existed with adrenergic nerves, (2) SP-, CGRP-and NKA-IR nerves, which mutually co-existed, and (3) PHI-IR nerves. Parallel-running adrenergic/NPY-IR and SP-IR nerves could be found with very similar although not completely identical morphological appearance. Paracervical ganglia contained neurotensin-and dynorphin A-IR cell bodies in addition to cell bodies with immunoreactivities similar to those in prevertebral ganglia. Combined retrograde tracing with True blue and immunocytochemistry showed that the adrenergic and NPY-IR uterine nerves originate in paracervical and prevertebral ganglia. In the prevertebral ganglia the cellular origin was the same for adrenergic and NPY-IR nerves. In contrast, SP-, CGRP-,and NKA-IR nerves originated in dorsal root ganglia. At full-term pregnancy all the neuropeptide immunoreactivities had vanished, probably reflecting a fetus-induced general nerve degeneration.     

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.