Interactions between serotonin and substance P in the spinal regulation of nociception

Interactions between 5-hydroxytryptamine (5-HT) and substance P (SP) in the mouse spinal cord were investigated using the tail-flick test and the behavioral response evoked by intrathecal (i.th.) SP or i.th. 5-HT. I.th. injection of 5-HT (20 μg) or the 5-HT₁ receptor agonists(+)-8-hydroxy-2-(di-n-propylamino)tetralin ((+)-8-OH-DPAT) (20 μg) or 5-methoxy-3(1,2,3,6-tetrahydropyridine-4-yl)-1H-indole (RU 24969) (20 μg) markedly inhibited the tail-flick reflex. The effect of these compounds was reduced when SP (5 μg) was given i.th. 55 min, or 55 and 45 min before the agonists. The tail-flick latencies recorded 5 min before injection of a 5-HT agonist were similar in animals treated with SP or vehicle. The changes in the tail-flick test were not due to changes in tail skin temperature since only minimal differences in the skin temperature were recorded between the groups injected with SP or vehicle. I.th. injection of SP (10 ng) or 5-HT (2 μg) produced a similar behavioral response consisting of biting, licking and scratching of the caudal part of the body, indicative of nociceptive stimulation. The responses both to i.th. SP and 5-HT were reduced after i.th. application of SP receptor antagonist [d-Arg¹,d-Trp^7,9,Leu¹¹]-SP (Spantide) (5 μg), as well as 5 min after i.th. injection of the 5-HT receptor antagonist metergoline (4 μg). The data may indicate functional interactions between SP and 5-HT in the mouse spinal cord, which may take place in neurons involved in the processing of nociception.     

Antinociceptive involvement of substance P in the spinal cord of mice: dose effects of substance P on the behavior elicited by intrathecally administered NMDA

The functional interaction between substance P (SP) and N-methyl-d-aspartate (NMDA) was studied to clarify the diversity of the roles of SP in nociceptive processes at the spinal level in mice. Behavioral responses elicited by intrathecal co-administration of NMDA (0.25 nmol) with various doses of SP (0.3–12 pmol) were observed for 1 min. The high dose of SP (12 pmol) potentiated NMDA-induced responses, which consisted of caudally directed licking and biting, while the low dose of SP (1 pmol) significantly reduced the responses by 40% compared to control mice administered NMDA alone. The antinociceptive effect of the low dose of SP was negated by co-administration of the opioid receptor antagonist naloxone. Furthermore, the antinociception produced by SP was present in mice pre-treated with systemic administration of capsaicin during the neonatal period. These results suggest that one of the roles SP plays at the spinal level is an involvement in antinociception. The activities of excitatory dorsal horn neurons are considered to be inhibited by endogenous opioid peptides released from inhibitory dorsal horn neurons directly stimulated by SP.     

Differential roles of spinal cholera toxin- and pertussis toxin-sensitive G proteins in nociceptive responses caused by formalin, capsaicin, and substance P in mice

The aim of the present study is to characterize the roles of spinal cholera toxin (CTX)- and pertussis toxin (PTX)-sensitive G proteins in the regulation of various nociceptive responses. The effects of intrathecal (i.t.) pretreatments with CTX and PTX on the formalin (subcutaneous)-, capsaicin (i.t.)-, and substance P (SP; i.t.)-induced nociceptive behaviours were examined in mice. Pretreatment with CTX (i.t.; 24 h before) significantly and dose-dependently (0.05-0.5 microg) suppressed both the first and second phases of the formalin-induced nociceptive behaviour. On the other hand, pretreatment with PTX (i.t., 6 days before) at the same doses (0.05-N0.5 microg) did not affect the formalin-induced response. Capsaicin (i.t., 0.5 microg)- and SP (i.t., 0.7 microg)-induced nociceptive behaviours were attenuated by the pretreatment with CTX. In addition, SP-induced nociceptive response was also attenuated by the pretreatment with PTX. However, the capsaicin-induced nociceptive response was not influenced by PTX pretreatment. These findings suggest that, at the spinal cord level, CTX-sensitive G-proteins are involved in the formalin-, capsaicin-, and SP-induced nociceptive behavioural responses, whereas PTX-sensitive G proteins are involved in SP-induced nociceptive response.     

Intrathecal substance P elicits a caudally-directed biting and scratching behavior in mice

Intrathecal injection of substance P in mice elicited a dose-related biting and scratching response. Systemic but not intrathecal morphine sulfate antagonized this response. We interpret these observations to define a novel nociceptive response to intrathecal substance P.     

Involvement of microglial P2X7 receptors and downstream signaling pathways in long-term potentiation of spinal nociceptive responses

Tetanic stimulation of the sciatic nerve (TSS) produces long-term potentiation (LTP) of C-fiber-evoked field potentials in the spinal cord. This potentiation is considered to be a substrate for long-lasting sensitization in the spinal pain pathway. Because microglia have previously been shown to regulate the induction of spinal LTP, we hypothesize that P2X7 receptors (P2X7R), which are predominantly expressed in microglia and participate in the communication between microglia and neurons, may play a role in this induction. This study investigated the potential roles of P2X7Rs in spinal LTP and persistent pain induced by TSS in rats. OxATP or BBG, a P2X7R antagonist, prevented the induction of spinal LTP both in vivo and in spinal cord slices in vitro and alleviated mechanical allodynia. Down-regulation of P2X7Rs with P2X7-siRNA blocked the induction of spinal LTP and inhibited mechanical allodynia. Double immunofluorescence showed colocalization of P2X7Rs with the microglial marker OX-42, but not with the astrocytic marker GFAP or the neuronal marker NeuN. Intrathecal injection of BBG suppressed the up-regulation of microglial P2X7Rs and increased expression of Fos in the spinal superficial dorsal horn. Further, pre-administration of BBG inhibited increased expression of the microglial marker Iba-1, phosphorylated p38 (p-p38), interleukin 1β (IL-1β) and GluR1 following TSS. Pre-administration of the IL-1 receptor antagonist (IL-1ra) blocked both the induction of spinal LTP and the up-regulation of GluR1. These results suggest that microglial P2X7Rs and its downstream signaling pathways play a pivotal role in the induction of spinal LTP and persistent pain induced by TSS.     

Concurrent measurement of substance P and serotonin in spinal superfusates: failure of capsaicin and p-chloroamphetamine to co-release

This study examined the efflux of endogenous serotonin and substance P-like immunoreactive material into in vivo superfusates of the rat spinal cord under quiescent conditions and during the addition of either capsaicin or p-chloroamphetamine to the superfusate. Using a method which permitted concurrent measurement of serotonin and substance P-like immunoreactive material in the same sample of superfusate, it was found that capsaicin increased the efflux of substance P-like immunoreactive material from the spinal cord but did not alter the efflux of serotonin. Conversely, the addition of p-chloroamphetamine caused the efflux of serotonin from the spinal cord to increase, but did not affect the efflux of substance P-like immunoreactive material.     

Involvement of nitric oxide in long-term potentiation of spinal nociceptive responses in rats

Nitric oxide plays an important role in spinal nociception. The present study explored the effects of nitric oxide on the spinal long-term potentiation associated with nociception. (1) Nitric oxide synthase inhibitor L-NAME (1 mM, 20 microl) and the nitric oxide scavenger hemoglobin (2 mg/ml, 20 mul) strikingly blocked the induction of tetanic sciatic stimulation-induced spinal long-term potentiation of C-fiber-evoked field potentials. L-arginine, a substrate of nitric oxide synthase, completely reversed L-NAME-induced inhibition. However, D-NAME (1 mM, 20 microl), an inactive form of L-NAME, had little effect on the spinal LTP. (2) The same tetanic sciatic stimulation induced long-term thermal hyperalgesia, which was blocked by pre-application of L-NAME. These results suggest the involvement of nitric oxide in the spinal long-term potentiation of C-fiber-evoked field potentials and related behavior changes.     

Thyrotropin-releasing hormone in spinal cord: coexistence with serotonin and with substance P in fibers and terminals apposing identified preganglionic sympathetic neurons

In this study we utilized the technique of simultaneous immunofluorescent double-labeling to investigate possible coexistence of the putative neurotransmitter thyrotropin-releasing hormone (TRH) with serotonin (5-HT) and with substance P (SP) in the intermediolateral cell column (IML) of rat spinal cord. We observed fibers and terminals immunoreactive for both TRH and 5-HT or TRH and SP in IML. In addition, this technique was used in animals in which we retrogradely labeled, with fluorescent tracer dyes, preganglionic sympathetic neurons within IML from either the adrenal medulla or the proximal cut end of the cervical sympathetic trunk. In these animals, fibers and terminals containing these combinations of neurotransmitters appeared to oppose identified preganglionic sympathetic neurons in IML. These data represent the first direct immunohistochemical demonstration of fibers and terminals in spinal cord which display coexistence of TRH- with either 5-HT- or SP-immunoreactivity. In addition, the proximity of TRH-immunoreactive fibers and terminals to sympathetic preganglionic neurons in IML support a role for TRH in the regulation of central sympathetic outflow.     

Evidence for a differential innervation of the spinal motor columns by substance P and serotonin in the fetal monkey

In the late-term fetal monkey, serotonin (5HT) and substance P (SP) immunoreactivities were localized to two distinct neural networks in the ventral horn of the spinal cord. In contrast, the adult monkey has SP and 5HT colocalized to the same terminals.     

Antidepressant-anxiolytic interactions: involvement of the benzodiazepine-GABA and serotonin systems.

1. Recent studies have demonstrated that antidepressant drugs are actually more effective than BZ's in the treatment of anxiety states. The role of two major neurochemical substrates that may be implicated in the anxiolytic activity of antidepressants, the benzodiazepine (BZ)-GABA receptor chloride ionophore complex and central serotonergic pathways, are focused on in this review. 2. A wide range of antidepressants elicit a reduction in BZ receptors and display anxiolytic effects within a conflict paradigm. 3. The anxiolytic activity of antidepressants, however, does not appear to be mediated via the BZ receptor, but possibly via another component of the complex such as the chloride channel-associated with the GABAA receptor. 4. Additionally, as possible candidates for the mechanism of anxiolytic activity of these compounds, results of pharmacological, behavioral and clinical studies point to the importance of serotonin (5-HT)1A receptors and 5-HT transporter sites as targets for the action of antidepressants, triazolobenzodiazepines and anxioselective piperazine derivatives.     

Involvement of group III metabotropic glutamate receptors in the modulation of spinal nociceptive signals

BACKGROUND:Previous morphological studies have demonstrated that group III metabotropic glutamate receptors (mGluRs) are commonly found in nociceptive pathways,particularly in the terminals of primary afferent fibers in the spinal dorsal horn.OBJECTIVE:To investigate the role of group III mGluRs in a rat model of spinal nociception by intrathecal administration of a selective agonist,L-Serine-O-phosphate (L-SOP).DESIGN,TIME AND SETTING:A randomized,controlled,animal experiment.The study was performed at the Department of Physiology and Neurobiology,Shanxi Medical University,between March 2007 and May 2008.MATERIALS:L-SOP of group III mGluRs (Tocris Cookson Ltd,UK),formalin (Sigma,USA),rabbit anti-c-Fos polyclonal antibody and biotin-labeled goat anti-rabbit IgG (Cell Signaling Technology,USA) were used in this study.METHODS:A total of 26 healthy Wistar rats,aged 1 month and weighing 100-120 g,were subjected to intrathecal catheter implantation.After 5-8 days,10 rats were selected according to experimental requirements.L-SOP 250 nmol in 10 μL,or the equivalent volume of normal saline,was administered by intrathecal injection into the L3-5 region of the spinal cord in the experimental and control groups,respectively.After 15 minutes,formalin (5%,50 μL) was subcutaneously injected into the plantar of the left hindpaw of each rat to establish formalin-induced pain models.MAIN OUTCOME MEASURES:Nociceptive behavioral responses and immunohistochemical examination of Fos expression.RESULTS:Intrathecal injection of L-SOP significantly attenuated the second phase nociceptive response compared with the control group (P<0.05),and Fos expression in the spinal dorsal horn was significantly decreased along with the number of Fos-like immunoreactive neurons (P<0.05).CONCLUSION:Group III mGluRs are involved in the modulation of nociceptive signals,and their activation suppresses the transmission of nociceptive signals.     

Interactions between serotonin, thyrotropin-releasing hormone, and substance P in the CNS regulation of adrenocortical secretion

Double-labeling immunohistochemical studies were performed to discern the morphological relationships between corticotropin-releasing factor-immunoreactive (CRF-ir) perikarya and afferent innervation in the hypothalamic paraventricular nucleus (PVN) of the rat. Attention was focussed on the local innervation by serotonin (5-hydroxytryptamine, 5-HT), thyrotropin-releasing hormone (TRH) and substance P (SP)-ir nerve terminal fibers. 5-HT-ir and SP-ir fibers were present in moderate numbers, in close apposition with CRF-ir perikarya. Sparse TRH-ir fibers were observed, but a population of TRH-ir perikarya was found in proximity with the CRF-ir cell bodies. TRH-ir perikarya in the PVN were surrounded by both 5-HT- and SP-ir fibers. Neuroendocrine studies were performed to investigate the interactions between 5-HT, TRH and SP in the regulation of hypothalamo-pituitary-adrenocortical (HPA) secretion. Male rats were prepared bearing cannulae for intracerebroventricular (ICV) or intra-PVN administration of drugs. 5-HT, at all doses tested (0.1, 40, or 80 nmol, ICV), caused increases in plasma corticosterone (CS) concentrations in tail-vein blood collected 20 min after injection. ICV injections of TRH caused dose-dependent increases in plasma CS, but did not further increase HPA responses when injected together with 5-HT. SP alone had little effect, although a significant reduction in plasma CS concentrations was observed in several individual experiments. However, SP (0.1 nmol) significantly attenuated CS responses following high doses of 5-HT (40 and 80 nmol, ICV), although the response to 0.1 nmol 5-HT was not affected. Combined injection of SP with TRH resulted in HPA responses not different from those following TRH alone. Similarly, SP did not reduce the HPA response observed with TRH and 40 nmol 5-HT in combination. Intra-PVN injections of 5-HT (0.1 or 40 nmol) and TRH also increased plasma CS concentrations. Intra-PVN injections of SP had little effect on plasma CS concentrations although a tendency toward a decrease in plasma CS was observed, as with the ICV injections. Combined intra-PVN injection of 5-HT (0.1 nmol) with TRH (0.1 nmol) did not significantly alter the response compared with that observed following TRH alone, although plasma CS concentrations were greater than with 0.1 nmol 5-HT. Combined intra-PVN injections of SP (0.1 nmol) with 5-HT (0.1 nmol) resulted in a significant decrease in plasma CS concentration compared with that following 5-HT alone, but SP did not prevent the CS response to a higher dose of 5-HT (40 nmol). Similar to the results observed following ICV administration, SP in combination with TRH did not affect the HPA response observed with TRH, or with 5-HT and TRH in combination. The results of these studies demonstrate that 5-HT and TRH serve to stimulate adrenocortical secretion, both at the level of the PVN and at extrahypothalamic structures, but that SP exerts an inhibitory influence that is capable of overcoming some of the stimulatory effects of 5-HT.     

Evidence for differential storage of calcitonin gene-related peptide, substance P and serotonin in synaptosomal vesicles of rat spinal cord

Homogenates of rat ventral and dorsal spinal cords were subjected to differential and gradient centrifugation on linear sucrose gradients following lysis of a synaptosomal fraction. The distribution of calcitonin gene-related peptide (CGRP) and substance P (SP) in dorsal spinal cord as well as the distribution of serotonin (5-HT) and SP in ventral spinal cord was determined, using radioimmunoassay (RIA) for CGRP and SP and high-performance liquid chromatography for 5-HT. In dorsal spinal cord. CGRP and SP had an almost identical distribution, with one peak in an intermediate density fraction which according to electron microscopy contained i.a. large dense-cored vesicles, and a second peak in a heavy fraction enriched in synaptosome-like structures. In ventral spinal cord, 5-HT and SP had different distribution patterns; 5-HT peaked in a light fraction containing many small synaptic vesicles, whereas SP peaked in an intermediate density fraction similar to the one in dorsal spinal cord. Both 5-HT and SP had second peaks in high density fractions containing synaptosome-like structures. The study demonstrates that the peptides CGRP and SP, partly coexisting in dorsal spinal cord, both seem to be stored in large vesicles inside synaptosomes, whereas the amine 5-HT and the peptide SP, coexisting in ventral spinal cord, appear to have a differential storage with 5-HT mainly in small vesicles and SP in large vesicles, both inside synaptosomes.     

Coexistence of immunoreactive substance P and serotonin in neurones of the gut

Serotonin (5-HT) and substance P (SP) were visualized by indirect immunofluorescence using two fluorochromes applied sequentially on the same preparation of myenteric plexus from the guinea pig gut. Three groups of labelled neurones were found: (i) neurones with 5-HT-like immunoreactivity only; (ii) neurones with SP-like immunoreactivity only; (iii) neurones with both 5-HT- and SP-like immunoreactivity, which represented approximately one-third of the 5-HT-like immunoreactive neurones.     

Impact of substance P receptor antagonism on the serotonin and norepinephrine systems: relevance to the antidepressant/anxiolytic response

Substance P (neurokinin-1 [NK1]) receptor antagonists appear to be effective antidepressant and anxiolytic agents, as indicated in 3 double-blind clinical trials. In laboratory animals, they promptly attenuate the responsiveness of serotonin (5-hydroxytryptamine [5-HT]) and norepinephrine (NE) neurons to agonists of their cell-body autoreceptors, as is the case for some antidepressant drugs that are currently in clinical use. Long-term, but not subacute, antagonism of NK1 receptors in rats increases 5-HT transmission in the hippocampus, a property common to all antidepressant treatments tested thus far. This enhancement seems to be mediated by a time-dependent increase in the firing rate of 5-HT neurons. Mice with the NK1 receptor deleted from their genetic code also have an increased firing rate of 5-HT neurons. Taken together, these observations strongly suggest that NK1 antagonists could become a new class of antidepressant and anxiolytic agents.     

Effects of serotonin and substance P on bulbar respiratory neurons in vivo

We studied the effects of separate or co-applications by microiontophoresis of serotonin (5-HT) and substance P(4–11) onto brainstem respiratory neurons in anesthetized or decerebrate cats. 5-HT either produced an excitation (36%,n = 10) or an inhibition (43%,n = 12) or had no effect (21%,n = 6). SP(4–11) had predominantly an excitatory effect (84%,n = 26) or no effect. Fifteen respiratory neurons responded to both 5-HT and SP(4–11). Test applications of 5-HT were made during a long application of SP(4–11). We obtained ‘additive effects’ when the inhibitory effect of 5-HT was superimposed to the excitation of SP(4–11) with slight modification (n = 1) or without any modification (n = 2). In other cases, called ‘non-additive effects’, we observed a great modification of the responsiveness of the neuron to the inhibitory effect of 5-HT (n = 2) or a complete blockade of the excitatory effect of 5-HT (n = 2) during co-application. The remaining results presented a potentiation of 5-HT effect by SP(4–11) or a biphasic response to 5-HT during SP(4–11) application. The results indicate that both 5-HT and SP receptors coexist on the membrane of the same respiratory-related neurons in the brainstem of cat and suggest an interaction between both substances in vivo in the central respiratory system.     

Intracerebral substance P in mice: behavioral effects and narcotic agents

Acute intracerebral injection of the undecapeptide, substance P, in mice induced a unique reciprocal hindlimb scratching response whose intensity was dose-related. Similar intracerebral dose-response curves were obtained by the structurally related undecapeptides, physalaemin and eledoisin, but not by several unrelated peptides (TRH, neurotensin, bradykinin, somatostatin), prostaglandins E2 and F2a, dibutyryl cyclic AMP or dibutyrylcyclic GMP. Analgesic narcotic agents with predominant agonist activity administered i.p. prevented the reciprocal hindlimb scratching response induced by intracerebral substance P (0.625 microgram/mouse = ED 95). In this in vivo assay their action was stereospecific and exhibited a rank order of potency similar to that reported for analgesic activity and binding to opiate receptors in vitro. Narcotic agents with mixed agonist-antagonist activity were inactive while the narcotic antagonist, naloxone, completely reversed the action of morphine. Higher doses of naloxone alone potentiated substance P-induced reciprocal hindlimb scratching which may explain why partial narcotic agonists failed to abolish the response. There is now considerable evidence in support of a sensory neurotransmitter/modulator role for substance P within the central nervous system, and one of its actions may be associated with nociception. This concept is supported by observations in the present study which indicate that the substance P-induced reciprocal hindlimb scratching response involves nociceptive pathways within the central nervous system.     

A comparison of the effects of serotonin, substance P and thyrotropin-releasing hormone on excitability of rat spinal motoneurons in vivo

Lumbar spinal motoneurons of urethane-anesthetized rats were driven at stable low firing rates by automatically cycled iontophoretic applications of glutamate or aspartate. The effects of iontophoretically applied serotonin, substance P or thyrotropin-releasing hormone (TRH) on glutamate or aspartate-evoked activity were then tested. All 3 substances were found to enhance both glutamate- and aspartate-induced excitation of the motoneurons. This enhancement of excitability was usually preceded by a brief period of inhibition at current onset. Although the effects of serotonin and substance P were qualitatively remarkably similar, TRH differed in that TRH occasionally inhibited motoneuron excitability without subsequent facilitation, and tachyphylaxis developed for the facilitatory effects of TRH. After TRH desensitization, serotonin could still enhance spinal motoneuron excitability.     

Immunohistochemical distribution of substance P, serotonin, and methionine enkephalin in sexually dimorphic nuclei of the rat lumbar spinal cord

The purpose of the present study was to identify chemically some potential inputs to lumbar motoneurons of the rat in the spinal nucleus of the bulbocavernosus, ventral motor pool, dorsolateral nucleus, and retrodorsolateral nucleus. Substance P-like immunoreactivity and serotonin-like immunoreactivity were found in all four motor nuclei, with dense immunoreactive profiles surrounding motoneurons and their processes. Enkephalin-like immunoreactivity was restricted to the sexually dimorphic nuclei, the spinal nucleus of the bulbocavernosus, and the dorsolateral nucleus. Within the spinal nucleus of the bulbocavernosus, enkephalin-like immunoreactive profiles were apposed to the processes of motoneurons but not their somata. In contrast, enkephalin-like immunoreactivity surrounded motoneuron somata in the medial part but not the lateral part of the dorsolateral nucleus, in the location of motoneurons projecting to the ischiocavernosus muscle. Moreover, the density of serotonin-like immunoreactivity was also greater in the medial part of the dorsolateral nucleus. On the basis of the chemo-architecture and the connections of the dorsolateral nucleus, we suggest the division of this motor column into a medial part composed of ischiocavernosus motoneurons surrounded by enkephalin- and serotonin-like immunoreactivity and a lateral part that contains neurons that project to the sphincter urethrae muscle. Total spinal transection severely depleted both serotonin-like and substance P-like material in the lumbar ventral horn. No changes in the distribution of enkephalin-like immunoreactivity were observed following this lesion. It is therefore suggested that in the ventral horn, substance P- and serotonin-like material are derived from supraspinal tracts, whereas enkephalin-like material is derived from intrinsic nerve cell bodies of the spinal cord.