Regeneration of primary afferent neurons containing substance P-like immunoreactivity

We compared changes in levels of substance P-like immunoreactivity (SPLI) in L4-6 dorsal root ganglia (DRG), L4-6 dorsal roots, sciatic nerve, tibial nerve and hind foot skin in rats following resection or crush injury of the sciatic nerve. The initial depletion of SPLI, which occurred in all areas sampled, was similar after either type of lesion. In DRG and dorsal roots, recovery to control values occurred in SPLI levels 35-45 days after sciatic crush, but not after resection. In sciatic nerve proximal to the injury, a partial recovery in SPLI content to about 60% of control occurred following crush injury, but not following resection. Distal to the injury, tibial nerve levels recovered rapidly following crush injury, consistent with the previously observed rapid regeneration of SPLI-containing axons. After resection, no recovery was observed until after 35 days, when it appeared that some axons succeeded in crossing the resection zone and regaining the distal nerve stump. Delayed and poor recovery of SPLI levels was observed in foot skin, even after crush injury. This correlated with the poor recovery of the plasma extravasation reaction, a functional index of SP-innervation of skin. In contrast, reinnervation by high-threshold mechanoreceptors was more rapid and complete, in agreement with a previous study. We conclude that although SPLI-containing axons regenerate rapidly, they appear to reinnervate skin less successfully than other afferents. Axon regeneration is associated with a recovery of SPLI levels which fell after axotomy: no recovery occurs if regeneration is prevented. Recovery was almost complete in DRG and roots, but incomplete in sciatic nerve. This peptide transmitter in afferent neurons thus behaves in a similar fashion to previously studied low-molecular weight transmitters and related materials in efferent neurons. Since recovery of SPLI levels begins before there is evidence for target reinnervation, it seems that axon regeneration is a sufficient condition for reversal of some axotomy-induced changes in these neurons. Further studies on substance P synthesis and on the response of individual DRG neurons to axotomy and regeneration will be required to explain fully the discrepancy between partial recovery of SPLI levels in sciatic nerve and full recovery in DRG and dorsal roots.     

Effects of methionine-enkephalin and substance P on the chemosensory discharge of the cat carotid body

The effects of methionine enkephalin (ME) and substance P (SP) were tested on the chemosensory discharge of the cat carotid body-nerve preparation in vitro. ME superfused in concentrations of 10(-8) to 10(-5) M depressed the sensory discharge, an effect followed by receptor excitation (rebound). Bolus applications of ME (30 ng to 3.0 microgram) induced variable effects (excitation or depression) on the discharge, excitation being more pronounced with the smaller doses. Superfusions with SP (10(-8) to 10(-5) M) either excited or depressed the discharge, excitation being more pronounced with higher SP concentrations (i.e. 10(-6) M). Bolus applications of SP (43 ng to 0.5 micrograms) also excited or depressed the sensory discharge. These variations may be dose-dependent. Superfused ME (10(-6) M) significantly depressed the chemoreceptor response to hypoxia (100% N2) and hypercapnia (6% CO2, pH 7.43). The responses to NaCN and acidity (pH 6.0) were marginally depressed. Superfused SP (10(-6) M) clearly depressed the responses to hypoxia, those to hypercapnia and NaCN were marginally affected but the effects of acidity were not altered. When the peptides were tested against the receptor responses to exogenously applied putative neurotransmitters (ACh, dopamine--DA), it was found that ME tended to depress both the ACh and DA actions whereas SP (10(-6) M) tended to increase their effects. Superfusions with naloxone (10(-6) M) increased the basal chemosensory discharge and this enkephalin blocker partially relieved the depressant effect of ME on the ACh-induced response. It is concluded that carotid body chemoreceptors have excitatory and inhibitory reactive sites to both ME and SP although their precise location is still unknown.     

Axonal transport of substance P-like immunoreactivity in regenerating rat sciatic nerve

Anterograde axonal transport of substance P-like immunoreactivity (SPLI) decreases after injury (crush or resection) to rat sciatic nerve. If the axons regenerate a partial recovery of transport occurs. If regeneration is impeded the decrease in transport is more severe and prolonged. No changes in the proportion of mobile SPLI (31%) or transport velocity (10.0 mm/h) occur. The decrease in SPLI transport largely accounts for the decline in SPLI content which occurs in nerve following injury and probably reflects decreased cell body synthesis.     

Effect of spinal ganglionectomy on substance P-like immunoreactivity in the gastroduodenal tract of cats

Bilateral ligation of the splanchnic nerves and spinal ganglionectomy from T5 to L2 both produced a profound decrease of substance P (SP)-like immunoreactivity in Auerbach's plexuses of the duodenum and the antrum of the stomach in the cats. As unilateral dorsal rhizotomy from T5 to L2 produced no such changes, SP is probably synthesized in the spinal ganglion and transported through the splanchnic nerve to the gastroduodenal wall.     

Chemical characterization of substance P-like immunoreactivity in primary afferent neurones

Substance P-like immunoreactivity (SPLI) in rat dorsal root ganglia and dorsal spinal cord was characterized by high-performance liquid chromatography followed by radioimmunoassay. In spinal cord and ganglia, respectively, 87% and 64% of SPLI eluted with authentic SP. The remainder of the SPLI in ganglia eluted as a single peak which did not represent the sulphoxide of SP or any of its C-terminal fragments.     

Reduced substance P-like immunoreactivity in hereditary sensory neuropathy of Pointer dogs

Summary Two unrelated Pointer dogs, each from a breeding of normal parents which produced three affected pups in a litter of nine, began to bite their paws at 3 and 5 months of age. Insensitivity to painful stimuli was marked in the distal parts of the limbs and receded proximally. The affected dogs were euthanatized at 5 and 20 months because of acral mutilation and infection. Changes affecting the primary sensory neurons included: small spinal ganglia with reduced numbers of cell bodies, degeneration of unmyelinated and myelinated fibers in dorsal roots and peripheral nerves, and reduced fiber density in the dorsolateral fasciculus (dlf).Since nociceptive loss was the salient deficit in a neuropathy affecting primary sensory neurons, immunohistochemical studies focused on substance P, the undecapeptide imputed to mediate nociception at the first synapse in the spinal cord and brain. The localization and density of substance P-like (SPL) immunoreactivity was studied in three control dogs and the two Pointers by the indirect antibody peroxidase-antiperoxidase method. The spinal intumescences of the control dogs contained dense SPL-immuno-reactivity in fibers of the dlf and the superficial laminae of the dorsal horn (i.e., laminae I, II, and the dorsal part of III). Immunoreactive fascicles on the lateral aspect of the dorsal horn and in the reticular process sent contributions medially to a plexiform fiber arrangement in lamina V. Medially, SPL-immunoreactive fibers were more loosely arranged in the internal third of laminae VI and VII and in lamina X. In the Pointers, the loss of primary sensory neurons was associated with notable reduction of SPL staining in the dlf and superficial laminae of the dorsal horn. In the lumbar intumescence of the older Pointer greater preservation of SPL staining in the lateral third of laminae I and II was consistent with somatotopic termination of trunkal afferents in this region.In both Pointers there was no detectable decrease in trigeminal sensitivity and the SPL immunoreactivity in the spinal nucleus of the trigeminal nerve of the younger Pointer and the corresponding control dog appeared equivalent. In the older Pointer, however, the immunoreactivity in this nucleus was decreased relative to the control. This decrease and appearance of scattered fiber degeneration in the dorsal columns of the mature Pointer suggested that fiber degeneration progresses slowly with age to include sensory systems not affected in early postnatal life.The findings in the Pointers were compared with those made in immunohistochemical studies of SP in familial dysautonomia and the mutilated foot rat.Supported in part by a grant from the Rockland County Kennel Club     

Distribution of substance P-like immunoreactivity in the goldfish brain

Immunohistochemical methods were used to study the distribution of SP-like immunoreactivity (SPLI) in the brain of the common goldfish (Carassius auratus). SPLI cell bodies and fibers were seen in distant areas and nuclei throughout the brain. In the telencephalon, SPL1 was found in the area dorsalis telencephali pars centralis and pars lateralis, areas that have been compared to the basal ganglia of land vertebrates. In the diencephalon, SPLI somata were seen in the hypothalamus. Four primary visual centers contained SPLI fibers: the nucleus of the posterior commissure, the area pretectalis, the nucleus dorsolateralis thalami and the optic tectum; the origin of these fibers could not be determined. SPLI cell bodies were seen in the oculomotor nucleus; the possibility that this may be the Edinger-Westphal nucleus is discussed. A high density of immunoreactive fibers was seen in the tractus retroflexus and the interpeduncular nucleus, though the habenula showed a few SP-like immunopositive fibers. In the hindbrain, SPLI cell bodies and fibers were seen in the nuclei ambiguus and commissuralis of Cajal and in the dorsal motornucleus of the vagus nuclei; even though these nuclei are known to belong to the visceral and sensory motor columns, the exact role SP plays in processing this information is at present unknown.     

Regional distribution of substance P-like immunoreactivity in the lower brainstem of the rat

A radioimmunoassay was used to quantify substance P-like immunoreactivity in the lower brainstem of the rat. The peptide was detectable in all brainstem nuclei investigated. A very high concentration was found in the substantia nigra, and high concentrations in the interpeduncular nucleus and central gray matter. Levels in the pons, the parabrachial nuclei; in the medulla, the nucleus tractus spinalis of the Vth nerve and the nucleus of the solitary tract were higher than the brain average.     

Distribution of substance P-like immunoreactivity in guinea pig central nervous system

The distribution of substance P-like immunoreactivity (SP-LI) in the guinea pig brain has been studied by immunohistochemistry and the results compared with the distribution in similar regions in the rat brain. In both species, dense SP-LI staining was found in the median eminence, arcuate hypothalamic nucleus, substantia nigra, dorsal raphe and dorsal tegmental nuclei, nucleus of the solitary tract, substantia gelatinosa of the spinal trigeminal nucleus, and spinal cord. Less dense staining was found in the caudate putamen, globus pallidus, nucleus accumbens, habenula, hypothalamic areas, and central grey. SP-LI cell bodies were found in areas previously described for the rat brain including several hypothalamic areas, limbic areas, central grey, and dorsal raphe and solitary tract nuclei. The major difference between the two species was found in the cortex and hippocampus. The guinea pig cortex contained many more SP-LI cells and fibres, distributed in layers II-VI, than the rat cortex. The guinea pig hippocampus contained marked staining, particularly in the pyramidal cell layer of CA1-3 fields of Ammon's horn and in the granular layer of the dentate gyrus, and SP-LI cells in the hilus of the dentate gyrus, whereas rat hippocampus contained few cells and no regions of dense staining. It is concluded that because the guinea pig brain has an extensive distribution of SP-LI in the cortex and hippocampus it resembles the primate brain more closely than does the rat brain.     

Distribution of transient receptor potential channels in the rat carotid chemosensory pathway

Glomus cells in the carotid body respond to decreases in oxygen tension of the blood and transmit this sensory information in the carotid sinus nerve to the brain via neurons in the petrosal ganglion. G-protein-coupled membrane receptors linked to phospholipase C may play an important role in this response through the activation of the cation channels formed by the transient receptor potential (TRP) proteins. In the present study, expression of TRPC proteins in the rat carotid body and petrosal ganglion was examined using immunohistochemical techniques. TRPC3, TRPC4, TRPC5, TRPC6, and TRPC7 were present in neurons throughout the ganglion. TRPC1 was expressed in only 28% of petrosal neurons, and of this population, 45% were tyrosine hydroxylase (TH)-positive, accounting for essentially all the TH-expressing neurons in the ganglion. Because TH-positive neurons project to the carotid body, this result suggests that TRPC1 is selectively associated with the chemosensory pathway. Confocal images through the carotid body showed that TRPC1/3/4/5/6 proteins localize to the carotid sinus nerve fibers, some of which were immunoreactive to an anti-neurofilament (NF) antibody cocktail. TRPC1 and TRPC3 were present in both NF-positive and NF-negative fibers, whereas TPRC4, TRPC5, and TRPC6 expression was primarily localized to NF-negative fibers. Only TRPC1 and TRPC4 were localized in the afferent nerve terminals that encircle individual glomus cells. TRPC7 was not expressed in sensory fibers. All the TRPC proteins studied were present in type I glomus cells. Although their role as receptor-activated cation channels in the chemosensory pathway is yet to be established, the presence of TRPC channels in glomus cells and sensory nerves of the carotid body suggests a role in facilitating and/or sustaining the hypoxic response.     

Decrease of substance P-like immunoreactivity in the substantia nigra and pallidum of parkinsonian brains

Substance P-like immunoreactivity was measured in the basal ganglia of controls and parkinsonian patients. A significant decrease in substance P-like immunoreactivity was observed within the substantia nigra and the external segment of the globus pallidus from parkinsonian brains. This suggests an alteration of the striatonigral substance P-containing neurons in Parkinson's disease.     

Development of substance P-like immunoreactivity in Xenopus embryos

The development of substance P-like immunoreactivity (SPLI) was studied in the Xenopus embryonic nervous system in order to determine in which neuronal populations and at what developmental times SPLI is expressed. Although Rohon-Beard neurons initially were thought to be the only substance P-immunoreactive cells in the embryonic frog spinal cord, we have demonstrated that several neuronal phenotypes are immunoreactive. The earliest evidence of SPLI was seen at stage 28 (Nieuwkoop and Faber, '67), at which time only some trigeminal ganglion cells, their axons in the ophthalmic nerve, and axons in the lateral tracts of the hindbrain showed SPLI. In the embryonic brain at stages 29/30, 37/38, and 42, SPLI was seen in the hypothalamus, trigeminal ganglion cells and their peripheral axons, the sensory roots of cranial nerve IX/X, and axons in the hindbrain lateral tracts. At premetamorphic stages, SPLI was found in several populations that are immunoreactive in adult amphibia. In the embryonic spinal cord, Rohon-Beard neurons were labeled consistently with reaction product; there was a rostrocaudal time gradient of immunoreactivity with increasing development. The Rohon-Beard neurons were not immunoreactive at developmental stages in which axonal outgrowth was beginning (stage 21), but were strongly immunoreactive at stages in which target cells had been contacted (stage 29). Several types of interneurons in the spinal cord (as classified by Roberts and Clarke, '82) showed SPLI during embryonic stages. At premetamorphic stages the Rohon-Beard neurons began to disappear and the immunoreactive interneurons were distributed similarly to those reported in the adult. Dorsal root ganglia differentiated during these stages, and at this time some of the neurons belonging to these ganglia exhibited substance P-like immunoreactivity.     

The co-occurrence of substance P-like immunoreactivity and dynorphin-like immunoreactivity in striatopallidal and striatonigral projection neurons in birds and reptiles

Using an immunofluorescence procedure that allows the simultaneous labeling of tissue for two different antigens, substance P-like immunoreactivity (SPLI) and dynorphin-like immunoreactivity (DLI) were observed to co-occur extensively in striatal neurons of the avian and reptilian basal ganglia and in fibers and terminals in the projection targets of the avian and reptilian striata. Thus, SPLI and DLI apparently co-occur extensively in striatopallidal and striatonigral projection neurons of the avian and reptilian basal ganglia. Since basal ganglia organization is fundamentally similar among amniotes, the present results suggest that SPLI and DLI may also co-occur extensively in striatal neurons in mammals.     

Distribution of input synapses from processes exhibiting GABA- or glutamate-like immunoreactivity onto terminals of prosternal filiform afferents in the locust

The locust prosternum carries a population of long filiform hairs that are very sensitive to air currents. The sensory afferent neurons that innervate the hairs make strong monosynaptic connections with an identified intersegmental interneuron (A4I1) which is known to contact motor neurons that supply muscles controlling wing angle during flight. In order discover how the synapse between the afferents and interneuron A4I1 might be modulated, the afferents were labelled intracellularly by backfilling with horseradish peroxidase to reveal their central terminals which lie in the prothoracic ganglion. A postembedding immunogold method was used to make a quantitative assessment of the prevalence of immunoreactivity for GABA and glutamate in processes presynaptic to the afferent terminals. In one afferent neuron, where 77 synapses were examined, 40 (52%) of the presynaptic processes were immunoreactive for GABA. When adjacent sections through the same terminal branches were labelled with the two antibodies, it was demonstrated that GABA- and glutamate-like immunoreactivity was present in different populations of presynaptic processes. A series of 110 ultrathin sections was cut through one set of afferent terminal branches and alternate grids were stained with GABA and glutamate antibodies. From these sections, the terminals were reconstructed and the position of 35 input and 21 output synapses mapped. Of the 35 input synapses, 18 (51%) were immunoreactive for GABA, 14 (40%) were immunoreactive for glutamate and 3 (9%) were unlabelled by either antibody. On these terminals, the different classes of input synapses appeared to be intermingled at random with the output synapses made by the afferent, and no pattern govering synapse distribution could be discerned. © 1994 Wiley-Liss, Inc.     

Regional distribution of methionine-enkephalin and substance P-like immunoreactivity in normal human brain and in Huntington''s disease

The regional distributions of substance P and Methionine-enkephalin (Met-enkephalin) were determined in normal human brains and in Huntington's disease using sensitive radioimmunoassays. Model experiments showed that both Met-enkephalin- and substance P-like immunoreactivities were stable for up to 72 h post-mortem in mouse brain. The results of high pressure liquid chromatography (HPLC) analyses indicated that the majority of the immunoreactivity detected in human globus pallidus corresponded to the native peptides, substance P or Met-enkephalin. In Huntington's disease the present results confirm that there is a substantial drop (> 80%) in the substance P content of the globus pallidus (both medial and lateral segments) and substantia nigra, and there was also a reduction (> 50%) in the Met-enkephalin content of these areas. This result suggests the loss of striato-pallidal and striato-nigral substance P and enkephalin-containing projections in Huntington's disease.     

Immunoreactivity in Limulus: III. Morphological and biochemical studies of FMRFamide-like immunoreactivity and colocalized substance P-like immunoreactivity in the brain and lateral eye

FMRFamide-like immunoreactivity (FLI) and the colocalization of FMRFamide and substance P-like (SPLI) immunoreactivities were examined in the brain and lateral eye of the horseshoe crab with FITC- and TRITC-labeled secondary antibody techniques. In the brain, fibers with FLI were localized in the neuropils of the lamina, medulla, central body, corpus pedunculatum, optic tract, circumesophageal connective, and central neuropil. An extensive network of reactive fibers innervatives the brain's vascular sheath. Somata with FLI were found in the dorsal medial group, dorsal lateral posterior groups #1 and #2, and ventral posterior lateral groups #1 and #2. Several distinct subgroups of reactive somata were noted in both the medullar and ventral medial groups. The distribution of fibers in the brain with colocalized FLI and SPLI includes those which innervate the vascular sheath and widespread populations of small-diameter beaded fibers in the central neuropil and circumesophageal connective. Somata with colocalized FLI and SPLI constitute minority populations in the medullar and dorsal medial groups but form the majority population of a subgroup in the ventral medial group. Overall localization of SPLI was reevaluated and is reported here according to the nomenclature of the new Chamberlain and Wyse brain atlas. In addition to those previously reported, somata with SPLI were found in the dorsal lateral posterior groups #1 and #2, the ventral lateral posterior groups #1 and #2, and several distinct subgroups of the medial and ventral medial groups. In the retina of the lateral eye, fibers with both FLI and SPLI ramify in the lateral plexus and ultimately innervate the corneal epidermis. Brain homogenates were examined for immunoreactive (ir) FMRFamide and ir-substance P with radioimmunoassay techniques. Ir-FMRFamide and ir-substance P eluted in different fractions from both gel filtration chromatography and HPLC. Furthermore, the binding curves for both substances were similar to those of the corresponding synthetic compounds. Brain homogenates were also bioassayed on the lateral eye. Three gel filtration fractions mimic natural circadian activity by increasing the sensitivity of the lateral eye, but they were not coincident with ir-FMRFamide or ir-substance P. Although it is not completely resolved what the active molecules in these fractions are, it is clear that neither ir-FMRFamide nor ir-substance P is a possible candidate.     

Distribution of substance P-like immunoreactivity in the brain of the newt (Triturus cristatus)

The distribution of immunoreactive substance P (sP)-containing structures in the newt brain and spinal cord was explored with an indirect im-munofluorescence method. Five sP-positive elements were detected: perikarya, dots, fibers, pericellular appositions, and pipe-shaped structures. Perikarya were seen at the levels of the spinal ganglia, spinal cord, raphe nucleus, interpeduncular nucleus, mesencephalon, preoptic area, infundibulum, dorsocaudal part of the ventral hypothalamus, habenula, and corpus striatum. Pericellular terminals were observed in periventricular areas, known to be rich in catecholaminergic cells; pipe-shaped structures were ob-served from the corpus striatum to diencephalon, and in mesencephalon. The olfactory nerve and nuclei were devoid of sP-positive elements. Six sP-immunofluorescent pathways were detected. One of them is composed of axons with huge varicosities and extends from the lateral spinal cord area to the mesencephalon. This pathway has not been described as yet in other animals and could be peculiar to the newt.     

Distribution of substance P-like immunoreactivity in the lower brainstem of the human fetus: An immunohistochemical study

The regional distribution of substance P-like immunoreactive (SPI) structures in the lower brainstem of the human fetus was investigated using the indirect immunoflourescence technique. SPI cells were found in a number of areas including the inferior colliculus, central gray matter of the midbrain, n. laterodorsalis tegmenti, midbrain and medullary reticular formation, n. vestibularis inferior, and n. prepositus hypoglossi. An extensive network of SPI fibers of varying densities were identified throughout the lower brainstem.     

Quinolinic and kainic acids can enhance calcitonin gene-related peptide-like immunoreactivity in striatal neurons with substance P-like immunoreactivity

Quinolinic or kainic acid, when injected into the cat striatum, caused neurodegenerative changes in striatal neurons. In parallel with these changes, however, calcitonin gene-related peptide-like immunoreactivity became visible in perikarya of many striatal neurons which were distributed around the lesions produced by the quinolinic or kainic acid injections. The vast majority of these lesion-spared neurons showing calcitonin gene-related peptide-like immunoreactivity were striatal neurons with substance P-like immunoreactivity.     

Immunoelectron microscopic study of substance P-containing fibers in feline cerebral arteries

The ultrastructure of substance P-containing fibers in feline cerebral arteries was examined by combining substance P immunohistochemistry and electron microscopy. At the light and electron microscopic level, positive fibers were observed in the adventitia and at the border between the adventitia and media, but not within the media or the endothelium. The substance P-containing fibers were unmyelinated with diameters consistent with C-fiber caliber. Positive axons were in close contact with Schwann cell processes. Positive axons contained 24 nm microtubules, 10 nm neurofilaments, clear vesicles and scattered mitochondria. The number of mitochondria and organelles resembling vesicles appeared to increase in presumptive axon terminals. No synaptic membrane specializations were observed.