Dynorphin A content in the rat brain and spinal cord after a localized trauma to the spinal cord and its modification with p-chlorophenylalanine. An experimental study using radioimmunoassay technique.

The distribution of dynorphin A in the spinal cord and brain of normal rats and of rats subjected to a focal injury of the spinal cord was examined in a rat model using a radioimmunoassay (RIA) technique. The validity of RIA was checked by high performance liquid chromatography (HPLC). Furthermore, the possibility that the peptide is somehow functionally related with endogenous 5-hydroxytryptamine (5-HT, serotonin), was also evaluated using a pharmacological approach. In normal animals, the peptide content was very similar in the spinal cord segments (T9, T10-11, and T12) examined whereas, the dynorphin content of the whole brain was about two-fold higher compared with that in the spinal cord. A focal injury to the spinal cord in the right dorsal horn (about 1.5 mm deep, 2.5 mm long and 1.5 mm to the right of the midline) of the lower thoracic cord (T10-11) in urethane anaesthetised animals significantly altered the peptide content in the whole brain as well as in the spinal cord. Thus, a decrease in the peptide level in whole brain, T10-11 and in the T12 segments of the spinal cord was observed 1 and 2 h after trauma. At 5 h, the peptide had accumulated markedly in the T9 segment (about a two-fold increase) as compared with the controls. At this time, the peptide content had been restored in the T10-11 and T12 segments. On the other hand, the whole brain dynorphin level continued to remain low (by 55%) as compared to the control group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neural actions of several substance P antagonists in the rat spinal cord

Four substance P (SP) antagonists were tested on anaesthetized rats, by injecting 8 microgram amounts into the spinal cord (T8-T9), and by observing their effects on the hypothalamo-neurohypophysial responses to a presumably painful stimulus, the superfusion of the hepatic portal vein with 1 microM bradykinin. Only two antagonists, the new D-Pro4,D-Trp7,9,10,Val8-SP4-11 and D-Pro4,D-Trp7,9,10-SP4-11 were capable of inhibiting the responses by 50-60%, the former compound having 3 times less agonistic activity. The results suggest that substitution of the aromatic phenylalanine by the non-polar valine in position 8 may significantly improve the overall characteristics of neurally active SP antagonists.     

Morphine and substance P release in the spinal cord

Summary In anaesthetised cats, antibody microprobes were used to measure the release of immunoreactive substance P (irSP) in the lumbar dorsal horn during noxious cutaneous stimulation or high-intensity electrical stimulation of a hind limb nerve. The major region of irSP release detected was centred on the substantia gelatinosa, with lesser release at the dorsal cord surface. Release at these sites was unchanged by systemic administration of morphine, or of morphine followed by naloxone. During superfusion of the dorsal cord surface with high concentrations of morphine, irSP release in the substantia gelatinosa region was slightly reduced and surface release was not observed, effects not reversed by systemic naloxone administration. The results suggest that the analgesic action of morphine does not involve reduced release of SP in the spinal cord.     

Edinger-Westphal neurons that project to spinal cord contain substance P

Combined retrograde transport and immunocytochemical methods were used to determine whether Edinger-Westphal neurons projecting to spinal cord also demonstrate substance P-like immunoreactivity (SPLI). Large injections of horseradish peroxidase (HRP) into cervical and lumbar enlargements retrogradely labeled cells throughout the length of the Edinger-Westphal complex (EW). Nearly all HRP-labeled EW neurons also stained for SPLI, evidence that EW is the origin of a direct substance P pathway linking rostral mesencephalon with spinal cord.     

Effects of substance P on neurones and glial cells in cultured rat spinal cord

Substance P caused a depolarization of cultured spinal neurones which was often accompanied by a decrease in membrane conductance. The peptide usually had no effect on the membrane potential and resistance of glial cells. In contrast to the depolarization by glutamate, which is associated with an influx of Na+ and an efflux of K+, the action of substance P may be due to a decrease of the K+-conductance.     

Ultrastructural study of substance P receptors in the dorsal horn of the rat spinal cord using monoclonal anti-complementary peptide antibody

A monoclonal antibody directed against a peptide (PS5) specified by RNA complementary to the mRNA coding for substance P (SP), was used to label SP receptors in the rat spinal cord as demonstrated by light and electron microscopy. An immunocytochemical method (avidin-biotin-peroxidase) was used on vibratome sections from rats perfused with paraformaldehyde. Immunoreactivity was observed principally in the two superficial layers of the dorsal horn, in lamina X and the region of motoneurons. The labeling was absent when the antibody was preincubated with the complementary peptide (PS5) used as immunogen. Competition between the anti-complementary peptide antibody and different ligands was tested by preincubation of tissue sections with the ligand in the presence of peptidase inhibitors before addition of the antibody. A specific agonist (SP) or antagonist (spantide, RP 67580) at 10⁻⁶M led to total absence of labeling. These results indicate that under our experimental conditions, the anti-complementary peptide antibody recognizes a SP binding site in the rat spinal cord. Electron microscopic study of the two superficial laminae of the dorsal horn showed that immunolabeling was mainly localized extracellularly at apposing neuronal plasma membranes. It was mostly associated with axodendritic or axosomatic appositions. Occasionally labeling was observed between two axon terminals. In all cases, these appositions were non junctional. Generally, neuronal processes involved in these appositions did not contain large granular vesicles. These observations suggest that SP may act in a diffuse, nonsynaptic manner probably on targets distant from SP release sites.     

Preganglionic neurons in the sacral spinal cord of the rat: an HRP study.

The pelvic nerve in the rat was exposed to HRP in order to localize the cells of origin of preganglionic fibers coursing in this nerve. Labeled cells were located in spinal segments L₆-S₁, primarily in the lateral intermediate gray. Occassionally, an HRP-filled cell was observed in the ventral horn in association with somatic motoneurons.     

Primary afferent fibers that contribute to increased substance P receptor internalization in the spinal cord after injury

Upon noxious stimulation, substance P (SP) is released from primary afferent fibers into the spinal cord where it interacts with the SP receptor (SPR). The SPR is located throughout the dorsal horn and undergoes endocytosis after agonist binding, which provides a spatial image of SPR-containing neurons that undergo agonist interaction. Under normal conditions, SPR internalization occurs only in SPR+ cell bodies and dendrites in the superficial dorsal horn after noxious stimulation. After nerve transection and inflammation, SPR immunoreactivity increases, and both noxious as well as nonnoxious stimulation produces SPR internalization in the superficial and deep dorsal horn. We investigated the primary afferent fibers that contribute to enhanced SPR internalization in the spinal cord after nerve transection and inflammation. Internalization evoked by electrical stimulation of the sciatic nerve was examined in untreated animals, at 14 days after sciatic nerve transection or sham surgery and at 3 days after hindpaw inflammation. Electrical stimulation was delivered at intensities to excite Abeta fibers only, Abeta and Adelta fibers or A and C fibers as determined by the compound action potential recorded from the tibial nerve. Electrical stimuli were delivered at a constant rate of 10 Hz for a duration of 5 min. Transection of the sciatic nerve and inflammation produced a 33.7 and 32.5% increase in SPR and immunoreactivity in lamina I, respectively. Under normal conditions, stimulation of Adelta or C fibers evoked internalization that was confined to the superficial dorsal horn. After transection or inflammation, there was a 20-24% increase in the proportion of SPR+ lamina I neurons that exhibited internalization evoked by stimulation of Adelta fibers. The proportion of lamina I SPR+ neurons that exhibited internalization after stimulation of C-fibers was not altered by transection or inflammation because this was nearly maximal under normal conditions. Moreover, electrical stimulation sufficient to excite C fibers evoked SPR internalization in 22% of SPR+ lamina III neurons after nerve transection and in 32-36% of SPR+ neurons in lamina III and IV after inflammation. Stimulation of Abeta fibers alone never evoked internalization in the superficial or deep dorsal horn. These results indicate that activation of small-caliber afferent fibers contributes to the enhanced SPR internalization in the spinal cord after nerve transection and inflammation and suggest that recruitment of neurons that possess the SPR contributes to hyperalgesia.     

Binding sites for [3H]substance P on neurons of cultured rat spinal cord and brain stem: an autoradiographic study

Binding of substance P (SP) was studied in organotypic cultures of rat central nervous system by means of autoradiography. In spinal cord cultures, binding sites for [3H]SP were observed on many interneurons located in the dorsal horn, whereas in the ventral horn, mainly large neurons, probably, motoneurons, were labelled. Almost no binding was detected on neurons of attached dorsal root ganglia. Binding of [3H]SP was also found on a relatively great number of brain stem neurons of various sizes. In contrast, glial cells did not reveal binding sites for [3H]SP. These binding studies together with electrophysiological investigations provide strong evidence for the existence of SP receptors on spinal and brain stem neurons but not on glial cells.     

Relationship between capsaicin-evoked substance P release and neurokinin 1 receptor internalization in the rat spinal cord

The relationship between substance P release and the activation of its receptor in the spinal cord remains unclear. Substance P release is usually measured by radioimmunoassay, whereas the internalization of the neurokinin 1 (NK1) receptor has been used to assess its activation by noxious stimuli. Our objective was to compare substance P release and NK1 receptor internalization produced by capsaicin in rat spinal cord slices. Superfusion of the slices with capsaicin for 3 min produced a gradual increase in substance P release that peaked 3-7 min afterward, and then decreased to baseline levels. The concentration-response curve for capsaicin was biphasic, with concentrations above 10 microM producing significantly less release. The effective concentration for 50% of response (EC(50)) for capsaicin, calculated from its stimulatory phase, was 2.3 microM. However, the potency of capsaicin to elicit NK1 receptor internalization in the same slices was one order of magnitude higher (EC(50)=0.37 microM) in lamina I, probably because NK1 receptors become saturated at relatively low concentrations of substance P. The potency of capsaicin to produce internalization was progressively lower in lamina III (EC(50)=1.9 microM) and lamina IV (EC(50)=14.5 microM), suggesting that neurokinins released in laminae I-II become diluted as they diffuse to the inner dorsal horn. To study the correlation between these two measures, we plotted substance P release against NK1 receptor internalization and fitted a saturation binding function to the points. The correlation was good for laminae I (R(2)=0.82) and III (R(2)=0.78), but it was poor (R(2)=0.35) for lamina IV because NK1 receptor internalization kept on increasing at high concentrations of capsaicin, whereas substance P release decreased. In conclusion, amounts of substance P able to activate NK1 receptors may fall under the threshold of detection of radioimmunoassay. Conversely, radioimmunoassay often detects levels of substance P release well over those required to saturate NK1 receptors in the superficial dorsal horn, but that may be able to activate these receptors in nearby regions of the spinal cord.     

Antinociceptive and neurotoxic actions of substance P analogues in the rat's spinal cord after intrathecal administration

Intrathecal administration of both (D-Pro2,D-Trp7,9)-substance P (DPDT) and (D-Arg1,D-Trp7,9,Leu11)-substance P (DADTL) elicited antinociception in hot-plate and tail-flick test, with DADTL as the most potent. The animals injected with 2.0 micrograms DADTL, and several animals administered with DPDT at the same dose, developed bilateral motor blockade of the hind-legs, persisting for up to 3 days after DADTL. The effect of DPDT appeared to be reversible at this dose. On histopathological examination it was found that these animals with persistent paralysis had widespread neuronal necrosis in the lumbar region of the spinal cord. It is concluded that the peptides have antinociceptive effects after the intrathecal administration in rats, but that there is a small margin between the dose producing this effect and that causing irreversible toxic reactions in the spinal cord.     

Upregulation of an opioid-mediated antinociceptive mechanism in transgenic mice over-expressing substance P in the spinal cord

In transgenic mice expressing ectopic substance P fibres in the spinal white matter, a normally innocuous mechanical stimulus induces hyperalgesia and allodynia which are reversed by substance P and N-methyl-D-aspartate receptor antagonists. This period of enhanced excitation is followed by a rebound overshoot in these animals. As previous evidence indicates opioid mechanisms in a similar rebound in normal animals, the present study was done to determine the effects of systemic administration of morphine and the opiate receptor antagonist, naloxone, on the stimulus-induced responses in the tail withdrawal reflex. Once baseline reaction times had been taken, different combinations of saline, naloxone and morphine were administered intraperitoneally to transgenic and control mice of either sex. A mechanical conditioning stimulus of 450g was then applied to the tip of the tail for 2s. This stimulus was innocuous in control mice given saline or naloxone, but provoked a nociceptive response in transgenic mice given these compounds. In control and transgenic mice, following morphine administration there was an antinociceptive effect. In control mice the subsequent mechanical stimulus had no effect. However, in transgenic mice the mechanical stimulus produced a further antinociception. Naloxone blocked the effect of morphine and the subsequent conditioning stimulus in both control and transgenic mice.The results indicate that while morphine is equally effective on the withdrawal reflex in both types of animal, in the transgenic mice morphine reveals an intrinsic, naloxone-sensitive antinociceptive mechanism. The data are interpreted to suggest that over-expression of substance P or some other factor in the spinal cord of transgenic mice is associated with the up-regulation or facilitation of an opiate-mediated intrinsic antinociceptive mechanism. This is a novel observation because the genetic manipulation in this transgenic mouse results in a transient over-expression of nerve growth factor during development that leads to the formation of ectopic primary afferent fibres in the spinal cord containing substance P. These fibres persist indefinitely after the nerve growth factor levels return to normal. Opioid mechanisms, which are likely of dorsal horn origin, do not fall under the direct influence of nerve growth factor mechanisms and therefore the intriguing possibility is raised that opioid mechanisms in the spinal cord are regulated at least in part by substance P-related mechanisms.     

Immunoreactive calcitonin gene-related peptide and substance P coexist in sensory neurons to the spinal cord and interact in spinal behavioral responses of the rat

Using immunohistochemistry evidence was obtained for the coexistence of calcitonin gene-related peptide (CGRP)- and substance P (SP)-like immunoreactivity in spinal sensory neurons. Analysis of caudally directed biting and scratching (CBS) behavior was carried out after intrathecal administration of CGRP and SP alone or in combination. Thus, SP (up to 20 micrograms) alone caused CBS only for a few minutes after injection, whereas SP (10 micrograms) plus CGRP (20 micrograms) caused a response with a duration up to 40 min. CGRP (20 micrograms) alone had no effects in this model. These findings provide support for a possible interaction of the two peptides at synapses in the dorsal horn of the spinal cord.     

Studies on the cellular localization of spinal cord substance P receptors

Substance P-immunoreactivity and specific substance P binding sites are present in the spinal cord. Receptor autoradiography showed the discrete localization of substance P binding sites in both sensory and motor regions of the spinal cord and functional studies suggested an important role for substance P receptor activation in autonomic outflow, nociception, respiration and somatic motor function. In the current studies, we investigated the cellular localization of substance P binding sites in rat spinal cord using light microscopic autoradiography combined with several lesioning techniques. Unilateral injections of the suicide transport agent, ricin, into the superior cervical ganglion reduced substance P binding and cholinesterase-stained preganglionic sympathetic neurons in the intermediolateral cell column. However, unilateral electrolytic lesions of ventral medullary substance P neurons which project to the intermediolateral cell column did not alter the density of substance P binding in the intermediolateral cell column. Likewise, 6-hydroxydopamine and 5,7-dihydroxytryptamine, which destroy noradrenergic and serotonergic nerve terminals, did not reduce the substance P binding in the intermediolateral cell column. It appears, therefore, that the substance P binding sites are located postsynaptically on preganglionic sympathetic neurons rather than presynaptically on substance P-immunoreactive processes (i.e. as autoreceptors) or on monoamine nerve terminals. Unilateral injections of ricin into the phrenic nerve resulted in the unilateral destruction of phrenic motor neurons in the cervical spinal cord and caused a marked reduction in the substance P binding in the nucleus. Likewise, sciatic nerve injections of ricin caused a loss of associated motor neurons in the lateral portion of the ventral horn of the lumbar spinal cord and a reduction in the substance P binding. Sciatic nerve injections of ricin also destroyed afferent nerves of the associated dorsal root ganglia and increased the density of substance P binding in the dorsal horn. Capsaicin, which destroys small diameter primary sensory neurons, similarly increased the substance P binding in the dorsal horn. These studies show that the cellular localization of substance P binding sites can be determined by analysis of changes in substance P binding to discrete regions of spinal cord after selective lesions of specific groups of neurons. The data show the presence of substance P binding sites on preganglionic sympathetic neurons in the intermediolateral cell column and on somatic motor neurons in the ventral horn, including the phrenic motor nucleus.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential distribution of 5-hydroxytryptamine and substance P in synaptosomal vesicles of rat ventral spinal cord

The subcellular distribution of 5-hydroxytryptamine (5-HT) and substance P (SP) was investigated in rat ventral spinal cord using differential and density gradient centrifugation. 5-HT was determined by high performance liquid chromatography (HPLC) and SP by radioimmunoassay (RIA). Both substances were found to be stored within synaptosomes at high densities. After partial lysis of the synaptosomes, 5-HT was also recovered in low density fractions, while substance P was found also in a fraction of intermediate density. The results indicate that within synaptosomes from ventral spinal cord, most of the 5-HT is stored in small synaptic vesicles, while SP is only stored in large synaptic vesicles. A minor part of the 5-HT may in addition also be stored in large vesicles, as previously suggested by ultrastructural studies.