Substance P in the human brainstem. Preliminary results of its immunohistochemical localization

The localization of substance P in the human post-mortem brainstem is studied by the indirect immunofluorescence technique. Positive neuronal cell bodies, nerve fibers and terminals are unevenly present over both sensory and effector areas and nuclei. The pattern of distribution of substance P in the human brainstem, similar to that seen in the laboratory animals, supports the hypothesis of a neurotransmitter role of this compound in man.     

Innervation of the rat gastrointestinal sphincters: Changes during development and aging

The effect of age on the adrenergic and peptidergic innervation of the lower oesophageal, pyloric and ileocaecal sphincters of the rat was investigated using immunohistochemical techniques. The distribution of nerve fibres containing the neuronal protein, growth associated protein-43, was also studied to determine the integrity of the enteric nervous system during development and aging. The four age groups examined were 2–3 days, 6 weeks, 3 months and 25 months old rats.Using protein gene product 9.5 antibody (a non-specific general neuronal marker), it was revealed that the myenteric ganglia in all sphincter regions were compactly arranged and were smaller in size at neonatal stage getting more spaced out and larger in size with age. There was no obvious change in the structure of the neuronal elements with age. In the lower oesophageal sphincter, calcitonin gene-related peptide- and substance P-like immunoreactive nerve fibres showed notable changes in density and fluorescence intensity with age, decreasing and increasing, respectively, with no obvious change in vasoactive intestinal polypeptide- and growth-associated protein-like immunoreactivity. A slight increase in dopamine-β-hydroxylase-like immunoreactivity was seen in old age. In the pyloric sphincter, there was an increase in calcitonin gene-related peptide- and substance P-like immunoreactivity with a less notable increase in dopamine-β-hydroxylase-like immunoreactivity. A decrease in vasoactive intestinal polypeptide- and growth-associated protein-43-like immunoreactivity in the circular muscle of the sphincter was seen in old age. In the ileocaecal sphincter there was a marked increase in growth associated protein-43-, vasoactive intestinal polypeptide-, dopamine-β-hydroxylase and substance P-like immunoreactivity. There was a decrease in the density of calcitonin gene-related peptide-like immuno-reactive nerve fibres in old age.In summary, two main conclusions can be drawn from the results of the present study. First, there was an age-related differential change in the density of immunoreactive nerve fibres containing various neuroactive substances. This indicates a level of plasticity of the various enteric nerve types and may reflect the degree of importance of the different neurotransmitters in the physiological activities of the specific sphincter. Second, in all three sphincters of aged rats, the density of nerve fibres containing the markers for the excitatory neurotransmitters noradrenaline and substance P (although the precise role of substance P in ileocaecal sphincter is not known) was increased, while the density of nerve fibres containing the inhibitory neurotransmitters calcitonin gene-related peptide in the lower oesophageal and possibly ileocaecal sphincter, and vasoactive intestinal polypeptide in the pylorus were decreased. This is likely to underlie the physiological activity of the sphincters and may be associated with malfunction in old age. Investigations to determine the functional implications of these changes would be of great interest.     

VIP-, enkephalin-, substance P- and somatostatin-like immunoreactivity in neurons intrinsic to the intestine: immunohistochemical evidence from organotypic tissue cultures.

Small intestine from 18-day fetal mice grown for 3 weeks in organotypic tissue culture was found to contain numerous VIP, enkephalin, substance P and some somatostatin immunoreactive nerve fibers. Since these cultures should be devoid of all afferent or other extrinsic neuronal inputs, it is concluded that there are VIP, enkephalin, substance P and somatostatin containing neurons intrinsic to the intestinal wall. However, all 4 peptides may also be present in neurons originating outside the gastrointestinal tract as well as in the intrinsic neurons.     

Peptide-containing nerve fibres in guinea-pig coronary arteries: immunohistochemistry, ultrastructure and vasomotility

The peptidergic innervation of guinea-pig coronary arteries was investigated by means of immunohistochemical, ultrastructural and in vitro pharmacological techniques. A network of nerves was demonstrated in all major epicardial arteries by means of an antiserum to the neuronal marker protein gene product 9.5. The majority of nerve fibres possessed neuropeptide Y (NPY) and tyrosine hydroxylase (TH) immunoreactivity, the number and distribution of nerves immunoreactive for NPY being similar to that of nerves containing TH immunoreactivity. Numerous nerve fibres displaying immunoreactivity for substance P, neuropeptide K and calcitonin gene-related peptide (CGRP) were also found. In double-stained preparations substance P immunoreactivity was co-localized with CGRP and with neuropeptide K immunoreactivities in the same varicose nerve fibres. Ultrastructural studies revealed the presence of numerous axon varicosities at the adventitial-medial border. NPY immunoreactivity was localized in large granular vesicles in nerve varicosities which also contained numerous small granular vesicles. Large granular vesicle-containing nerves also displayed immunoreactivity for dopamine-beta-hydroxylase. With an in vitro method, the vasomotor responses to perivascular peptides were characterized in epicardial and intramyocardial arteries. In epicardial arteries neither noradrenaline nor NPY elicited a contractile response. Only in some intramyocardial arteries was an NPY-mediated contraction demonstrated. No potentiating effect of noradrenaline and NPY was observed in either epicardial or intramyocardial arterial segments. In contrast, CGRP, substance P and vasoactive intestinal peptide (VIP) all produced a concentration-dependent relaxation of both epicardial and intramyocardial arteries. These results suggest that peptide-containing nerves associated with guinea-pig coronary arteries may predominantly be involved in mediating vasodilation.     

Developmental timing of hair follicle and dorsal skin innervation in mice

The innervation of hair follicles offers an intriguing, yet hardly studied model for the dissection of the stepwise innervation during cutaneous morphogenesis. We have used immunofluorescence and a panel of neuronal markers to characterize the developmental choreography of C57BL/6 mouse backskin innervation. The development of murine skin innervation occurs in successive waves. The first cutaneous nerve fibers appeared before any morphological evidence of hair follicle development at embryonic day 15 (E15). Stage 1 and 2 developing hair follicles were already associated with nerve fibers at E16. These fibers approached a location where later in development the follicular (neural) network A (FNA) is located on fully developed pelage hair follicles. Prior to birth (E18), some nerve fibers had penetrated the epidermis, and an additional set of perifollicular nerve fibers arranged itself around the isthmus and bulge region of stage 5 hair follicles, to develop into the follicular (neural) network B (FNB). By the day of birth (P1), the neuropeptides substance P and calcitonin gene-related peptide became detectable in subcutaneous and dermal nerve fibers first. Newly formed hair follicles on E18 and P1 displayed the same innervation pattern seen in the first wave of hair follicle development. Just prior to epidermal penetration of hair shafts (P5), peptide histidine methionine-IR nerve fibers became detectable and epidermal innervation peaked; such innervation decreased after penetration (P7- P17). Last, tyrosine hydroxylase-IR and neuropeptide Y-IR became readily detectable. This sequence of developing innervation consistently correlates with hair follicle development, indicating a close interdependence of neuronal and epithelial morphogenesis.     

Autoimmune disease and innervation

In the decades before 1987, most of the research devoted to neuronal innervation was carried out in primary and secondary lymphoid organs at very different locations. This was an important period in order to understand hard-wiring of immune organs in physiology. Between 1988 and 1997, with the appearance of specific antibodies against neuronal markers, innervation was studied in inflamed tissue of patients and of animals with autoimmune diseases. This period clearly revealed that nerve fibers of, both, the sympathetic and sensory nervous system are altered, but only small amounts of tissue have been investigated by qualitative but not quantitative techniques. Between 1998 and 2007, with the understanding that sympathetic and sensory neurotransmitters might play opposite roles in inflammation, nerve fibers of the different nervous systems have been studied in parallel using quantitative techniques. These studies have been carried out in a large number of patients with long-standing autoimmune diseases. It turned out that sympathetic nerve fibers are lost in chronically inflamed tissue, while substance P-positive nerve fibers sprout into the inflamed area. This might be important because high concentrations of sympathetic neurotransmitters are antiinflammatory whereas substance P has a proinflammatory role. The first challenge for future research is the determination of innervation in the early human autoimmune disease. The second challenge is the identification of reasons for the differential loss of sympathetic in relation to sensory nerve fibers. It might well be that nerve repellent factors specific for the sympathetic nerve fiber might play an important role for the observed differential loss. Whether, or not, a therapy can be based on these findings remains to be established.     

Fall in density, but not number of myenteric neurons and circular muscle nerve fibres in guinea-pig colon with ageing

Abstract  In guinea-pig ileum, ageing has been associated with a decrease in enteric neurons. This study examined guinea-pig colon and measured changes in gut dimensions, neuron size, density and ganglionic area. Changes in motor nerve fibres in the circular muscle were also measured. Myenteric neurons in whole-mount preparations of mid-colon from 2-week, 6-month, and 2-year-old guinea-pigs were labelled immunohistochemically with the neuronal marker human neuronal protein HuC/HuD, and numbers of neurons mm⁻², neuronal size, ganglionic area mm⁻², gut length, circumference and muscle thickness were measured. Corrected numbers of neurons mm⁻² and ganglionic area mm⁻² accounting for growth of the colon were calculated. Additionally, nerve fibres in circular muscle cross-sections were labelled with antibodies against nitric oxide synthase (NOS) and substance P (SP) and the density of nerve fibres in circular muscle was measured. The numbers of neurons mm⁻² decreased by 56% (from 2 weeks to 2 years) with no change in neuron size. Total neuron numbers decreased by 19% ( P  = 0.14) when adjusted for changes in length and circumference with age. The percentage area of NOS- and SP-immunoreactive (IR) nerve fibres in the circular muscle decreased ( P  < 0.001), but the total area of NOS and SP-IR nerve fibres increased ( P  < 0.01) due to an age-related increase in muscle thickness. The density of myenteric neurons in guinea-pig mid-colon halved from 2 weeks to 2 years, but when the increase in colon dimensions was considered, the number of neurons decreased by only 19%. The percentage area of motor nerve fibres in the circular muscle decreased with no change in total volume of nerve fibres.     

Distribution of substance P-immunoreactive elements in the preoptic area and the hypothalamus of the rat.

The localization and morphology of neurons, processes, and neuronal groups in the rat preoptic area and hypothalamus containing substance P-like immunoreactivity were studied with a highly selective antiserum raised against synthetic substance P. The antiserum was thoroughly characterized by immunoblotting; only substance P was recognized by the antiserum. Absorption of the antiserum with synthetic substance P abolished immunostaining while addition of other hypothalamic neuropeptides had no effect on the immunostaining. The specificity of the observed immunohistochemical staining pattern was further confirmed with a monoclonal substance P antiserum. The distribution of substance P immunoreactive perikarya was investigated in colchicine-treated animals, whereas the distribution of immunoreactive nerve fibers and terminals was described in brains from untreated animals. In colchicine-treated rats, immunoreactive cells were reliably detected throughout the preoptic area and the hypothalamus. In the preoptic region, labeled cells were found in the anteroventral periventricular and the anteroventral preoptic nuclei and the medial and lateral preoptic areas. Within the hypothalamus, immunoreactive cells were found in the suprachiasmatic, paraventricular, supraoptic, ventromedial, dorsomedial, supramammillary, and premammillary nuclei, the retrochiasmatic, medial hypothalamic, and lateral hypothalamic areas, and the tuber cinereum. The immunoreactive cell groups were usually continuous with adjacent cell groups. Because of the highly variable effect of the colchicine treatment, it was not possible to determine the actual number of immunoreactive cells. Mean soma size varied considerably from one cell group to another. Cells in the magnocellular subnuclei of the paraventricular and supraoptic nuclei were among the largest, with a diameter of about 25 microns, while cells in the supramammillary and suprachiasmatic nuclei were among the smallest, with a diameter of about 12 microns. Immunoreactive nerve fibers were found in all areas of the preoptic area and the hypothalamus. The morphology, size, density, and number of terminals varied considerably from region to region. Thus, some areas contained single immunoreactive fibers, while others were innervated with such a density that individual nerve fibers were hardly discernible. During the last decade, knowledge about neural organization of rodent hypothalamic areas and mammalian tachykinin biochemistry has increased substantially. In the light of these new insights, the present study gives comprehensive morphological evidence that substance P may be centrally involved in a wide variety of hypothalamic functions. Among these could be sexual behavior, pituitary hormone release, and water homeostasis.     

Circadian variations in substance P, luliberin (LH-RH) and thyroliberin (TRH) contents in hypothalamic and extra hypothalamic brain nuclei of adult male rats

Using both the ‘punch’ microdissection and radioimmunological techniques, circadian variations in substance P, luliberin (LH-RH) and thyroliberin (TRH) concentrations can be statistically validated in some discrete brain regions of the (Wistar CFY) male rat investigated in May. Animals were synchronized with light from 06.00 to 18.00 h and darkness. Water and food were available ad libitum. Very well marked circadian rhythms were in evidence in brain areas where the neuronal peptides investigated are mostly present in nerve terminals in high concentrations: medial basal hypothalamus for luliberin and thyroliberin and substantia nigra for substance P. On the contrary, no significant rhythms were detected in a number of areas where luliberin (preoptic area) or substance P (preoptic area, central gray matter, amygdala, globus pallidus) synthetizing perikarya are present. This suggests that at least luliberin and substance P are not secreted according to a circadian rhythmicity.The most striking finding was that crest time locations were situated at only two different times of the day: 14.00 h for thyroliberin and 20.00 h for substance P and luliberin. This suggests that circadian variations are not solely dependent on the activity of the endocrine system.     

Substance P: Depletion in the dorsal horn of rat spinal cord after section of the peripheral processes of primary sensory neurons

The substance P content, glutamic acid decarboxylase and choline acetyltransferase activities and the level of [3H]diprenorphine binding were measured in various regions of the lumbar spinal cord of rats after unilateral section of the sciatic nerve or after dorsal rhizotomy. Sciatic nerve section produced a 75--80% depletion of substance P in the dorsal horn but did not change the substance P content of the ventral horn. The onset of substance P depletion occurred within 7 days and was maintained for 2 months. The substance P content of the dorsal root ganglia and both the peripheral and central branches of primary sensory neurons was also reduced after sciatic nerve section. Glutamic acid decarboxylase and choline acetyltransferase activity were unchanged; however, a small decrease in opiate receptor binding occurred 1 month after nerve section. Dorsal rhizotomy produced an 80% depletion of substance P in the dorsal horn. In addition, the substance P content of the ventral horn was significantly reduced. Glutamic acid decarboxylase activity in the dorsal horn was unaffected by dorsal rhizotomy whereas opiate receptor binding was reduced by 40%. From these studies it appears that peripheral nerve injury results in the degeneration of primary sensory neurons which contain and release substance P as neurotransmitter.     

Substance P depolarizes nerve terminals in an autonomic ganglion

The effect of substance P on presynaptic nerve terminals was examined by intracellular impalements of calyciform terminals within the chick ciliary ganglion. Substance P produced a slow depolarization of the nerve terminals which was associated with an increase in input resistance. The postsynaptic ciliary neurons were unaffected by exposure to substance P, indicating that at this synapse the physiological effects of substance P may be largely presynaptic in nature.     

Neuropeptide and noradrenaline distributions in rat interscapular brown fat and in its intact and obstructed nerves of supply

Following demonstration of noradrenergic and peptidergic nerves in rat interscapular brown adipose tissue, neuronal courses were investigated with reference to both the segmentally derived nerve traversing the fat pad and to paravascular projections, employing respectively, sucrose potassium glyoxylic acid and indirect immunofluorescence methodologies. Following ligation of the segmental nerve before its pad entry and distal to the pad, accumulations of noradrenaline, neuropeptide-Y, calcitonin gene-related peptide and substance P were observed immediately proximal to the ligature in all nerve components (T1-5). Two to three days after unilateral nerve resection (proximal to the pad), with or without simultaneous contralateral padectomy, there was, in some sections of the denervated pads either total absence of, or a very sparse distribution of both noradrenergic and peptidergic labelled nerves, thus suggesting minimal paravascular projection of these nerves and a major projection via the defineable segmental nerve route.     

The visualisation of cardiovascular innervation in the guinea pig using an antiserum to protein gene product 9.5 (PGP 9.5)

The various subpopulations of autonomic and sensory nerves supplying the mammalian cardiovascular system may be demonstrated using specific immunocytochemical and histochemical techniques, but no single marker has previously been available for the visualisation of the entire innervation. Protein gene product (PGP) 9.5 was first identified in extracts of human brain and found to represent a major protein component of the neuronal cytoplasm. We have demonstrated that PGP 9.5 immunoreactivity occurs in the guinea pig cardiovascular innervation and is present in more individual nerve fibres than other general neuronal markers (neuron-specific enolase and neurofilaments). PGP 9.5 immunoreactivity was localized to both intrinsic neurones and nerve fibres in the guinea pig heart. In the vascular system PGP 9.5-immunoreactivity occurred in an extensive plexus of fine perivascular nerve fibres and fascicles running around and along both arteries and veins, mainly at the adventitial-medial border. At the ultrastructural level, this immunoreactive material was localized to the axonal cytoplasm and did not appear to be associated with cytoskeletal elements or secretory vesicles. 6-Hydroxydopamine (6-OHDA) pretreatment resulted in the degeneration of noradrenergic axon terminals containing PGP 9.5, tyrosine hydroxylase (TH) and neuropeptide tyrosine (NPY) immunoreactivities. Most of the perivascular nerve fibres which remained displayed substance P- and calcitonin gene-related peptide (CGRP) immunoreactivity, as well as PGP 9.5 immunoreactivity. Capsaicin pretreatment resulted in a depletion of both substance P and CGRP immunoreactivity, but had no apparent effect on PGP 9.5 immunostaining. In the heart PGP 9.5 immunoreactivity also appeared to be present in presumed postganglionic cholinergic nerves. PGP 9.5 may be a useful marker when examining regional variations in cardiovascular innervation and for determining the relative proportions of nerve subpopulations.     

Loss of substance P and Enkephalin immunoreactivity in the human substantia nigra after striato-pallidal infarction

Post-mortem neuropathological material from 3 patients with striato-pallidal infarction provided the first immunohistochemical evidence for substance P- and enkephalin-containing nerve fibre projections from the striato-pallidum to the substantia nigra in the human. The nigra corresponding to the normal side showed abundant substance P and enkephalin immunoreactivity whose patterns of immunostaining were notably similar. In contrast, the substantia nigra ipsilateral to the striato-pallidal infarction showed a decrease in substance P and enkephalin immunoreactivity which was proportional to the extent of the infarction. This suggests that much of the substance P and enkephalin immunoreactivity in the nigra is present in nerve fibres projecting from the striato-pallidum. Furthermore, the similar distribution of remaining substance P and enkephalin immunoreactivity in corresponding areas of the nigra of the infarcted side indicates that the origins and/or projections of nerve fibres containing these two neuropeptides may be closely approximated anatomically. Depletion of substance P immunoreactivity in the pars reticulata of the substantia nigra in a fourth patient with anterior striatal infarction suggests a topographic projection for substance P immunoreactive fibres from the striatum. There is some evidence in one patient with cognitive and behavioural abnormalities to support the suggestion that the basal ganglia may be involved in non-motor functions.     

Intramural neurons of the guinea-pig urinary bladder: histochemical localization of putative neurotransmitters in cultures and newborn animals

Histochemical methods have been used to study the distribution of putative neurotransmitters in the urinary bladder of newborn guinea-pigs and in cultures of intramural ganglia. Following the nicotinamide adenine dinucleotide (NADH)-diaphorase reaction which specifically labels nerve cell bodies, up to 66 ganglia were observed in stretch preparations of the newborn urinary bladder. Each ganglion contained 2-50 nerve cell bodies. Vasoactive intestinal polypeptide was localized in a few nerve cell bodies of intramural ganglia both in in situ and culture preparations. In the in situ preparations it was widely distributed in nerve fibres to the muscle, being most dense at the base of the bladder, and in some mucosal epithelial cells. Somatostatin was contained in numerous neuronal cell bodies in the detrusor muscle both in situ and in culture. Extensively distributed varicose fibres were found in culture and in the muscle, submucous and mucosal layers in situ. Substance P immunofluorescence was demonstrated in a few neuronal cell bodies in ganglia both in situ and in vitro, particularly in those of the mucosa at the base of the bladder. In the in situ preparations varicose nerve fibres containing substance P were seen in the muscle coats with greatest density in the bladder base. Met-enkephalin-immunoreactive nerve cell bodies were not seen either in situ or in culture. Nerve fibres in in situ preparations were found largely enveloping neuronal cell bodies within the ganglia. Neither serotonin-immunoreactive nor catecholamine-containing neuronal cell bodies were seen in the in situ bladder preparation. However, some nerve cell bodies in culture showed positive staining, possibly as a result of selective uptake of serotonin and catecholamine known to be contained in foetal calf serum in the culture medium or possibly as the result of increased synthetic activity in certain neurones in the culture situation. In whole-mount stretch preparations, no serotonin-immunoreactive nerve fibres were seen, but catecholamine-containing small intensely fluorescent cells and nerve fibres were observed. Acetylcholinesterase-positive nerve cell bodies and nerve fibres were observed both in in situ and culture preparations of the bladder. Quinacrine-positive nerve cell bodies (as an indicator of purinergic neurones) were found in numerous intramural neurones examined. in situ; however, under the culture conditions used, non-selective staining of all cell types occurred.     

Expression of substance P and vanilloid receptor (VR1) in trigeminal sensory neurons projecting to the mouse nasal mucosa

Substance P and neurokinin A (NKA) have potent pro-inflammatory effects in the airways. The release of these neuropeptides from primary afferent (sensory) nerve endings to various stimuli is considered to be induced by activation of the capsaicin (vanilloid) receptor (VR1). In this study, retrograde neuronal tracing studies were combined with immunohistochemistry for VR1 and substance P to investigate the occurrence and distribution of substance P and VR1 receptor expression in mouse trigeminal neurons that were identified by retrograde labeling with Fast blue dye from the nasal mucosa. Fast blue signaling was observed in mucosa layers of the right nasal cavity and in sensory trigeminal neurons close to the division of the ophthalmic and maxillary nerve. Expression patterns of VR1 and substance P were found with different frequencies: 11.3+/-1.2% (mean+/-SEM) were immunoreactive for VR1, 4.9+/-1.1% for VR1 and SP, and 6.4+/-1.3% only for VR1 but not for SP. These VR1-positive neurons were partly binding to lectin I-B4, indicating VR1-expression in non-peptidergic upper airway C-fibers. In conclusion, based on the extent of SP and VR1 co-localization in nasal afferent neurons, the present study suggests that, following a peripheral activation of the VR1 receptor on SP afferents, there could be a triggering of SP-mediated phenomena, including those related to inflammation, such as plasma extravasation.     

Substance P and calcitonin gene-related peptide expression in dorsal root ganglia in sciatic nerve injury rats

The neuropeptides, substance P and calcitonin gene-related peptide, have been shown to be involved in pain transmission and repair of sciatic nerve injury. A model of sciatic nerve defect was prepared by dissecting the sciatic nerve at the middle, left femur in female Sprague Dawley rats. The two ends of the nerve were encased in a silica gel tube. L5 dorsal root ganglia were harvested 7, 14 and 28 days post sciatic nerve injury for immunohistochemical staining. Results showed that substance P and cal- citonin gene-related peptide expression increased significantly in dorsal root ganglion of rats with sci- atic nerve injury. This increase peaked at 7 days, declined at 14 days, and reduced to normal levels by 28 days post injury. The findings indicate that the neuropeptides, substance P and calcitonin gene- related peptide, mainly increased in the early stages after sciatic nerve injury.     

The effects of decentralization on substance P-immunoreactivity in the anterior major pelvic ganglion of the male guinea pig.

The anterior major pelvic ganglion (AMPG) of the male guinea pig possesses a substance P (SP)-immunoreactive peri-neuronal plexus. Selective nerve transections involving the principal inputs of the AMPG, the hypogastric and pelvic nerves, indicate that the SP-immunoreactive peri-neuronal plexus is derived from multiple sources: an extrinsic source involving both the hypogastric and pelvic nerves, and another source (possibly the projections of small intensely fluorescent cells). SP-immunoreactivity (IR) is not normally present in the neuronal perikarya of the AMPG. Evidence is presented that suggests the absence of SP-IR is due to an active suppression of SP-synthesis. This seems to be achieved by a trans-synaptic mechanism involving the hypogastric nerve which, after transection, leads to the appearance of neuronal perikarya exhibiting SP-IR (less than 1% of the total neuronal population of the AMPG). Up to 65% of the neuronal perikarya of the AMPG have the ability to synthesize SP, as demonstrated by SP-IR after 24 h in vitro. A more potent factor in the down-regulation of SP synthesis seems to be exerted by the pelvic genito-urinary organs, especially the prostate.     

Release of substance P from peripheral nerve terminals following electrical stimulation of the sciatic nerve

Substance P is a putative mediator of neurogenic inflammation, where it is postulated to be released from nerve terminals in the skin in response to noxious and electrical stimulation. To demonstrate release of substance P from cutaneous nerve terminals a blister model has been established. Electrical stimulation of the sciatic nerve for 20 min at 50 V, 15 Hz, 0.5 ms, resulted in a significant increase in substance P-like immunoreactivity (SPLI) in blister fluid, which was abolished after neonatal pretreatment of rats with capsaicin. There was no increase of SPLI, following stimulation of the sciatic nerve at 4 V. There was a corresponding decrease in skin SPLI following nerve stimulation at 50 V. Characterization of blister fluid SPLI after stimulation at 50 V demonstrated a peak of immunoreactivity, which co-eluted with synthetic substance P and SPLI from rat skin. These results confirm the hypothesis that electrical stimulation results in release of substance P from nerve terminals in the skin, and therefore fulfils an important criterion of its role as a mediator of neurogenic inflammation.     

Neuronal phenotypes in mouse dorsal root ganglion cell cultures: Enrichment of substance P and calbindin D-28k expressing neurons in a defined medium

The primary sensory neurons in mouse dorsal root ganglia consist of diversified subpopulations which express distinct phenotypic characteristics such as substance P or calbindin D-28k. To determine whether neuronal phenotypes are altered or not in in vitro cultures carried out in a defined synthetic medium, dissociated dorsal root ganglion cells from newborn mice were grown in the alpha-modified minimum essential medium either supplemented with 10% fetal calf serum or serum-free. About 80% of the neurons survived after 5 days of culture in both media, but only 35% or 65% were rescued after 12 days in serum-free or fetal calf serum supplemented medium, respectively. The neuronal subpopulations expressing substance P or calbindin D-28k displayed similar morphological properties in both media and a higher resistance to culture conditions than the whole neuronal cell population, especially in serum-free medium. It is therefore concluded that a defined synthetic medium offers reproducible conditions to culture dorsal root ganglion cells for at least 5 days, stimulates the expression of substance P and enriches preferentially neuronal phenotypes expressing substance P or calbindin D-28k, for a longer period of culture.