Electrophysiological and behavioral effects of neurotensin in rat globus pallidus: an in vivo study

The globus pallidus plays a critical role in movement regulation. Morphological study has indicated that the globus pallidus receives neurotensinergic innervation from the striatum. The present study investigated the effects of activating neurotensin receptor in globus pallidus. In vivo single unit electrophysiological recordings showed that micro-pressure ejection of neurotensin into the globus pallidus increased spontaneous firing of pallidal neurons. The excitatory effect of neurotensin could be mimicked by the C-terminal fragment, neurotensin (8-13), but not by the N-terminal fragment, neurotensin (1-8). Local administration of both the non-selective neurotensin receptor antagonist, SR142948A, and the selective neurotensin type-1 receptor antagonist, SR48692, blocked the excitatory effect induced by neurotensin. In the behaving rats, we observed the postural effects of neurotensin in the globus pallidus. Unilateral microinjection of neurotensin into the globus pallidus induced a SR48692-sensitive contralateral dystonic posturing in the presence of systemic haloperidol administration, which could be accounted for by the electrophysiological effect of neurotensin in increasing the firing rate of pallidal neurons. Our in vivo electrophysiological and behavioral findings suggest that pallidal neurotensin receptor plays a role in the basal ganglia motor circuit by mediating an excitation of spontaneous activity in the globus pallidus.     

Binding sites for [3H][Sar9, Met(O2)11]substance P in rat brain and guinea pig ileum.

[3H][Sar9,Met(O2)11]substance P (SP) with high specific activity (32 Ci/mmol) was used to study neurokinin-1 (NK-1) binding sites on rat brain and smooth muscle membranes of the guinea pig ileum. The specific binding of [3H][Sar9,Met(O2)11]SP was shown to be saturable, reversible and increased in parallel with the protein concentration. Scatchard analyses of equilibrium binding experiments revealed that [3H][Sar9,Met(O2)11]SP binds to a class of non-interacting binding sites in rat brain membranes (Kd = 2 nM, Bmax = 56 fmol/mg of protein) and ileum muscle membranes (Kd = 2 nM, Bmax = 194 fmol/mg of protein). Competition of [3H][Sar9,Met(O2)11]SP, 125I-BH[Sar9,Met(O2)11]SP and 125I-BH.SP with different tachykinin-related peptides gave the following rank order of potencies: SP greater than physalaemin greater than [Sar9,Met(O2)11]SP greater than N-Ac[Arg6,Sar9,Met(O2)11]SP(6-11) greater than neurokinin A (NKA) greater than or equal to eledoisin greater than or equal to neurokinin B (NKB) greater than [MePhe7]NKB (4-10) greater than [beta-Ala8]NKA(4-10). A very similar pattern was observed on ileum muscle membranes. [3H][Sar9,Met(O2)11]SP was found to be highly selective for NK-1 binding sites in rat brain and in the intestinal tissue. Binding showed good correlation with the biological activity of tachykinins and related peptides. From these data it can be suggested that (a) the NK-1 receptor characterized in the central nervous system is identical to the one in the periphery, (b) the NK-1 binding site of the muscle membranes appears to be similar to the contractile receptor of the guinea pig ileum and (c) the functional site mediating relaxation of the dog carotid artery is similar to the contractile receptor of the guinea pig ileum.     

Autoradiographic distribution of brain neurokinin-1/substance P receptors using a highly selective ligand [3H]-[Sar9,Met(O2)11]-substance P.

The autoradiographic distribution of neurokinin (NK)-1 receptors was visualized in the rat brain using the highly selective ligand, [3H]-[Sar9,Met(O2)11]-substance P. This ligand apparently binds to a single class of high affinity (Kd = 1.4 +/- 0.5 nM), low capacity (Bmax = 160 +/- 3.0 fmol/mg protein) sites in rat brain membrane preparations. The ligand selectivity profile reveals that substance P (SP) and unlabeled [Sar9,Met(O2)11]-SP are potent competitors of [3H]-[Sar9,Met(O2)11]-SP binding while NK-2 and NK-3 analogues are virtually inactive demonstrating the selectivity of this radioligand for the NK-1 receptor class. Autoradiographic data show that [3H]-[Sar9,Met(O2)11]-SP binding sites are broadly but discretely distributed in rat brain, the highest densities of sites being located in the external plexiform layer of the olfactory bulb, striatum, olfactory tubercule, amygdala-hippocampal area, endopiriform and entorhinal cortices, superior colliculus, locus coeruleus and substantia gelatinosa of the spinal cord. This distribution is similar, but not identical, to that previously reported for NK-1 sites using less selective ligands such as [125I]Bolton-Hunter SP. For example, some difference in labelling patterns are observed in the hippocampal formation. This could be explained by the existence of NK-1 receptor subtypes, only one of them being recognized by [3H]-[Sar9,Met(O2)11]-SP or by the greater selectivity of this radioligand for NK-1 over NK-2 and NK-3 receptor classes.     

Permissive role of neurokinin NK(3) receptors in NK(1) receptor-mediated activation of the locus coeruleus revealed by SR 142801.

The present experiments investigated the role of neurokinin-1 (NK(1)) and neurokinin-3 (NK(3)) receptors on the activity of the locus coeruleus (LC)-noradrenergic system by using a dual probe microdialysis technique in anesthetized guinea pigs. The local application in the LC of the selective NK(1) receptor agonists [SAR(9),Met(O(2))(11)]-SP (10 microM) and septide (1 microM) as well as the selective NK(3) receptor agonist senktide (1 microM), enhanced the extracellular norepinephrine (NE) levels in the prefrontal cortex. The enhancing effect of [SAR(9),Met(O(2))(11)]-SP was completely blocked by the peripheral administration of the selective non peptide NK(1) and NK(3) receptor antagonists, GR 205171 (1 mg/kg, i.p.) and SR 142801 (0.1 mg/kg, i.p.), respectively, whereas SR 142806 (0.1 mg/kg, i.p.) the inactive enantiomer of SR 142801 had no effect. Moreover, the [SAR(9),Met(O(2))(11)]-SP-induced increase in LC DOPAC concentrations, is only antagonized by GR 205171. In contrast, only SR 142801 (0.3 mg/kg, i.p.) could block stereoselectively the senktide-evoked increase in NE levels. Both [SAR(9),Met(O(2))(11)]-SP and senktide effects were blocked by local infusion into the LC of SR 142801 (10(-9) M). These results demonstrate that stimulation of NK(1) and NK(3) receptors located in the LC area modulates the activity of the LC-NE system, and that the excitatory effects of NK(1) receptor agonists require NKB/NK(3) receptor activation in the LC.     

A selective and extremely potent antagonist of the neurokinin-1 receptor

Sendide [Tyr6,D-Phe7,D-His9]-substance P(6-11) has been examined by measurements of ligand binding to crude membrane fractions and by functional tests on the spinally mediated behavioral response. Sendide potently displaced [3H]-labeled substance P (SP) binding to mouse spinal cord membranes in a competitive manner. In vivo, sendide, intrathecally co-injected with SP, competitively antagonized SP-induced scratching, biting and licking. The behaviors elicited by physalaemin, septide and [Sar9, Met(O2)11]-SP were also reduced by co-administration of sendide. Large doses of sendide were needed to reduce the action of neurokinin A, D-septide, neurokinin B and eledoisin. The in vitro and in vivo pharmacological profile of sendide demonstrated that it is a selective and extremely potent antagonist of the neurokinin-1 receptor.     

Contractile effect of tachykinins on Suncus murinus (house musk shrew) isolated ileum

Recent studies used Suncus murinus to investigate the anti-emetic potential of NK(1) tachykinin receptor antagonists. However, the pharmacology of tachykinin receptors in this species has not been fully characterized. In the present studies, therefore, we examined a range of tachykinin receptor agonists for a capacity to induce contractions of the isolated ileum. The tachykinin NK1 receptor preferring agonists substance P, septide and [Sar9Met(O2)11] substance P, and the tachykinin NK2 preferring agonists neurokinin A and GR 64349 (Lys-Asp-Ser-Phe-Val-Gly-R-gamma-lactam-Leu-Met-NH2) caused concentration dependent contractions with EC50 values in the nanomolar range. However, the tachykinin NK3 preferring agonists neurokinin B and senktide (1nM-1microM) induced only weak contractions. The action of senktide, but not [Sar9Met(O2)11] substance P, septide, or GR 64349, was antagonized significantly by atropine (P<0.05); tetrodotoxin and hexamethonium were inactive. The tachykinin NK1 receptor antagonist CP-99,994 ((+)-[(2S,3S)-3-(2-methoxy-benzyl-amino)-2-phenylpiperidine]) (10-100nM) inhibited substance P- and septide-induced contractions non-competitively. The pA2 value estimated for CP-99,994 against septide was 7.3+/-0.1. It also non-competitively antagonized the contractile responses induced by [Sar9Met(O2)11] substance P with a pA2 of 7.4+/-0.1. CP-99,994 also had a slight inhibitory action on neurokinin A-induced contractions, but did not modify the action of GR 64349. Conversely, the tachykinin NK2 receptor antagonist, saredutant, competitively antagonized GR 64349-induced contractions with a pA2 of 7.34+/-0.02. On the other hand, the presence of both CP-99,994 and saredutant competitively antagonized substance P-induced contraction. The present studies indicate that tachykininNK1 and NK2 receptors exist in the ileum of S. murinus and are involved in mediating contractions directly on smooth muscle, whereas tachykinin NK3 receptors may play a minor role involving a release of acetylcholine.     

125I-BH[Sar9, Met(O2)11]-SP, a new selective ligand for the NK-1 receptor in the central nervous system.

The selective agonist [Sar9,Met(O2)11]-SP was radioiodinated with 125I-Bolton Hunter in order to study its binding to rat brain membranes and for further comparison with 125I-BH.SP. Specific binding of 125I-BH[Sar9,Met(O2)11]-SP was temperature-dependent, saturable and reversible. In brain homogenates, 125I-BH[Sar9,Met(O2)11]-SP interacted with a single class of high affinity (kd = 1.0 nM) non-interacting binding sites (Bmax of 15 fmol/mg protein). In the central nervous system, 125I-BH-[Sar9,Met(O2)11]-SP apparently labeled the same number of binding sites as 125I-BH.SP (19 fmol/mg proteins). Competition studies with tachykinins, neurokinins and selective neurokinin agonists indicated that the pharmacological profile of the site labeled by 125I-BH[Sar9,Met(O2)11]-SP is identical with that of NK-1 receptors. In dose-displacement studies made with radiolabeled SP and [Sar9,Met(O2)11)]-SP, an excellent correlation (r = 0.96) was found for the Ki values of the different compounds tested; these findings suggest that both radioligands recognize the same receptor in rat brain. The affinity (Ki) of various neurokinin-related peptides for the brain site were compared with their biological activities on various isolated organs (dog carotid artery, guinea-pig ileum, rat portal vein). NK-1 binding sites characterized in rat brain homogenates appear to be identical with those present on the dog carotid artery, a preparation known to possess exclusively the NK-1 receptor type.     

Orexin‐A increases the activity of globus pallidus neurons in both normal and parkinsonian rats

Orexin is a member of neuropeptides which was first identified in the hypothalamus. The globus pallidus is a key structure in the basal ganglia, which is involved in both normal motor function and movement disorders. Morphological studies have shown the expression of both OX₁ and OX₂ receptors in the globus pallidus. Employing single unit extracellular recordings and behavioural tests, the direct in vivo electrophysiological and behavioural effects of orexin‐A in the globus pallidus were studied. Micro‐pressure administration of orexin‐A significantly increased the spontaneous firing rate of pallidal neurons. Correlation analysis revealed a negative correlation between orexin‐A induced excitation and the basal firing rate. Furthermore, application of the specific OX₁ receptor antagonist, SB‐334867, decreased the firing rate of pallidal neurons, suggesting that endogenous orexinergic systems modulate the firing activity of pallidal neurons. Orexin‐A increased the excitability of pallidal neurons through both OX₁ and OX₂ receptors. In 6‐hydroxydopamine parkinsonian rats, orexin‐A‐induced increase in firing rate of pallidal neurons was stronger than that in normal rats. Immunostaining revealed positive OX₁ receptor expression in the globus pallidus of both normal and parkinsonian rats. Finally, postural test showed that unilateral microinjection of orexin‐A led to contralateral deflection in the presence of systemic haloperidol administration. Further elevated body swing test revealed that pallidal orexin‐A and SB‐334867 induced contralateral‐biased swing and ipsilateral‐biased swing respectively. Based on the electrophysiological and behavioural findings of orexin‐A in the globus pallidus, the present findings may provide a rationale for the pathogenesis and treatment of Parkinson's disease.     

Altered tachykinergic influence on gastric mechanical activity in mdx mice

This study investigated whether alterations in gastric activity in dystrophic mdx mouse can be attributed to dysfunctions of tachykinins. Endoluminal pressure was recorded and the expression of neuronal nitric oxide synthase (nNOS), NK1 and NK2 neurokinin receptors was investigated by immunohistochemistry. SR48968, NK2 receptor antagonist, but not SR140333, NK1 receptor antagonist, decreased the tone only in mdx gastric preparations. In the presence of N(omega)-nitro-l-arginine methyl ester (l-NAME), inhibitor of NOS, SR48968 reduced the tone also in normal stomach. [Sar(9), Met(O(2))(11)]-SP, agonist of NK1 receptors, caused tetrodotoxin-sensitive relaxations, antagonized by SR140333 or l-NAME, with no difference in the potency or efficacy between normal and mdx preparations. [beta-Ala(8)]-NKA(4-10), an NK2 receptor agonist, induced SR48968-sensitive contractions in both types of preparations, although the maximal response of mdx tissues was significantly lower than normal preparations. Immunohistochemistry demonstrated a consistent reduction of nNOS and NK2 receptor expression in mdx stomach smooth muscle cells and no change in nNOS and NK1 receptor expression in neurones. In conclusion, in mdx stomach the activation of NK2 receptors plays a role in the development of the tone, associated with a reduced NO production by muscular nNOS. The hypo-responsiveness to NK2 receptors could depend on the reduced expression of these receptors.     

Characterization of NK-1 receptors in guinea pig and rat brain membranes with NK-1 peptides and a non-peptide antagonist.

The major finding of the present investigation is the demonstration of different NK-1 receptors in rat and guinea pig brain membranes with CP 96345 (non-peptide NK-1 antagonist) and R-544 (NK-1 peptide antagonist). We used [3H][Sar9,Met(O2)11]SP, the highly selective ligand for NK-1 receptor to compare NK-1 binding sites in rat and guinea pig brain membranes. Scatchard analysis revealed the existence of a single population of [3H][Sar9,Met(O2)11]SP binding sites in both preparations. The affinity and the maximal number of binding sites were found closely similar in rat (Kd 2 nM, Bmax = 37 fmol/mg protein) and guinea pig brain membranes (Kd = 3 nM, Bmax = 25 fmol/mg of protein). The order of potency of neurokinins to inhibit [3H][Sar9,Met(O2)11]SP binding from rat brain (SP > NKA > NKB) was found different of that observed on guinea pig brain (SP > NKB > NKA). Results obtained with [Sar9,Met(O2)11]SP, [beta Ala8]NKA(4-10) and [MePhe7]NKB suggest that selective agonists cannot discriminate between NK-1 receptors of different species. Using the non-peptide antagonist CP 96345 and the tripeptide R-544, we found that these two NK-1 antagonists discriminate between rat and guinea pig [3H][Sar9,Met(O2)11]SP binding sites.     

Pharmacological characterization of mucin secretion from CHO-K1-hNK(1)R cells

Numerous respiratory diseases increase mucin secretion from human airways. Several investigators hypothesize that mucin secretion from airway epithelium is NK(1)-receptor mediated. We have developed a mucin secretion assay using CHO-K1 cells transfected with the human NK(1)receptor (CHO-K1-hNK(1)R) that respond to NK(1)-specific agonists. Cells were labeled with [(3)H]-glucosamine and stimulated with agonists including Ac-[Arg(6), Sar(9), Met(O(2))(11)] Substance P(6-11) (ASMSP; NK(1)-specific), [beta-Ala(8)]-Neurokinin A(4-10) (BANK; NK(2)-specific), or human neutrophil elastase (HNE). Basal mucin secretion from CHO-K1-hNK(1)R and non-transfected cells was similar. Stimulation of CHO-K1-hNK(1)R, but not CHO-K1, with ASMSP or BANK concentration-dependently increased mucin secretion (pD(2)value[Emax] = 8.9(1)+/-0.1(3)[175%] and 7.56+/-0.05[100%], respectively). SR140333 (NK(1)antagonist), but not SR48968 (NK(2)antagonist), decreased ASMSP- and BANK-induced mucin release from CHO-K1-hNK(1)R. In these cells, endothelin-1, angiotensin II, serotonin, phenylephrine, senktide, and methacholine showed negligible effects on mucin secretion. A similar lack of effect of these agonists was observed in non-transfected CHO-K1 cells. HNE increased mucin release four to five fold in both cell types. These studies demonstrate that stimulation of CHO- K1-hNK(1)R with ASMSP and BANK causes robust and NK(1)-selective mucin release.     

Effects of selective tachykinin-receptor antagonists on tachykinin-induced airway mucus secretion in the rat

Tachykinins like substance P (SP), neurokinin A (NKA), neurokinin B (NKB) differentially stimulate airway mucus secretion with the following rank order of potency in rat trachea: SP>NKA>NKB. These differential actions are most likely due to different affinities to the tachykinin receptors, termed neurokinin (NK)(1), NK(2)and NK(3). In this study we characterized the receptor subtype responsible for the differential secretagogue effects in rat trachea by means of selective receptor antagonists and receptor agonists.SR 140333 [NK(1)-antagonist] completely inhibited SP action (283,29+/-21, 12%-->84,53+/-4, 09%; P<0,01) and significantly reduced the effects of NKA (179,08+/-17,34%-->118,86+/-6,7%; P<0,01) and NKB (171,89+/-5, 75%-->109,5+/-4,11%; P<0,01). SR 48968 [NK(2)-antagonist] did not affect SP action, but reduced the effects of NKA and NKB. SR 142801 [NK(3)-antagonist] did not change any effect of SP, NKA or NKB. [Sar(9)]SP (NK(1)-agonist) caused strong dose-dependent secretagogue effects similar to SP, [betaAla(8)]NKA (NK(2)-agonist) showed only slight and [Pro(7)]NKB (NK(3)-agonist) no effects.The present data suggest that the secretagogue effects elicited by tachykinins in rat trachea are mediated via NK(1)receptors.     

Dopamine and adenosine mediate substance P-induced depression of evoked IPSCs in the rat nucleus accumbens in vitro

The major projection cells of the nucleus accumbens (NAc) are under a strong inhibitory influence from GABAergic afferents and depend on afferent excitation to produce their output. We have earlier reported that substance P (SP), a peptide which is colocalized with GABA in these neurons, depresses excitatory synaptic transmission in this nucleus (Kombian, S.B., Ananthalakshmi, K.V.V., Parvathy, S.S. & Matowe, W.C. (2003) J. Neurophysiol., 89, 728-738). In order to better understand the role of this peptide in the synaptic physiology of the NAc, it is important to determine its effects on inhibitory synaptic responses. Using whole-cell recording in rat forebrain slices, we show here that SP also depresses evoked inhibitory postsynaptic currents (IPSCs) in the NAc via intermediate neuromodulators. SP caused a partially reversible, dose-dependent decrease in evoked IPSC amplitude. This effect was present without measurable changes in the holding current, input resistance of recorded cells or decay rate (tau) of IPSCs. It was mimicked by a neurokinin-1 (NK1) receptor-selective agonist, [Sar9, Met (O2)11]-SP, and blocked by an NK1 receptor-selective antagonist, L 732 138. The SP-induced IPSC depression was prevented by SCH23390, a dopamine D1-like receptor antagonist and by 8-cyclopentyltheophylline, an adenosine A1 receptor blocker. Furthermore, the SP effect was also markedly attenuated by exogenous adenosine, dipyridamole, rolipram and barium. These data show that SP, acting on NK1 receptors, depresses inhibitory synaptic transmission indirectly by enhancing extracellular dopamine and adenosine levels. SP therefore acts in the NAc to modulate both excitatory and inhibitory afferent inputs using the same mechanism(s).     

Inhibition of neurokinin-1-substance P receptor and prostanoid activity prevents and reverses the development of morphine tolerance in vivo and the morphine-induced increase in CGRP expression in cultured dorsal root ganglion neurons

Chronic treatment with opioid drugs such as morphine leads to the development of tolerance, which manifests as a loss of drug potency. The mechanisms underlying this phenomenon are poorly understood, but recent evidence suggests that increased activity of nociceptive sensory transmitters [calcitonin gene-related peptide (CGRP) and substance P] and other signalling messengers (prostaglandins) contribute to its development. Chronic intrathecal morphine administration to rats for 7 days produced analgesic tolerance. Co-administration of SR140333, a selective substance P receptor (neurokinin-1) antagonist, or nimesulide, a cyclooxygenase-2-selective inhibitor, augmented the acute effects of morphine, prevented morphine tolerance and reversed established tolerance. In cultured adult dorsal root ganglion neurons, exposure to morphine for 5 days increased the number of neurons expressing CGRP immunoreactivity. Co-exposure with the peptide CGRP receptor antagonist CGRP8-37, SR140333 or nimesulide prevented the morphine-induced increase in the expression of CGRP immunoreactivity. Additionally, BIBN4096BS, a nonpeptide CGRP receptor antagonist, stereoselectively produced similar effects. In summary, this investigation demonstrates that activity of CGRP and substance P contributes to both the induction and expression of opioid analgesic tolerance. Additionally, it highlights the involvement of prostaglandins generated by spinal cyclooxygenase-2 activity in the genesis of opioid tolerance. The neuropeptide and prostanoid activity contributing to tolerance is expressed at the level of the primary afferents terminating in the spinal cord. The combination of opioids with agents that block this activity may represent a useful strategy for the prevention as well as the reversal of clinical opioid tolerance.     

Neurokinin-1 receptor expression in dorsal root ganglion neurons of young rats

The expression of neurokinin-1 receptors was studied in the fourth lumbar dorsal root ganglia of young rats using immunohistochemical and electrophysiological techniques. Use of a specific immunoserum raised against the C-terminal fragment of rat neurokinin-1 receptor revealed immunoreactivity in 32 +/- 1.5% of dorsal root ganglion neurons. The diameter of the majority of the neurokinin-1 receptor immunostained neurons was smaller than 30 microm. Double immunohistochemical labelling using neurokinin-1 receptor and substance P antibodies revealed that about 1/3 of the neurokinin-1 receptor expressing neuron contains substance P. Likewise, about 1/3 of the substance P producing DRG cells expressed the neurokinin-1 receptor. Superfusion of substance P (1 microM) to an in vitro preparation of the fourth lumbar dorsal root ganglion induced a reversible long-lasting depolarization as measured by extracellular suction electrodes attached to the dorsal roots. This response to substance P was only partially antagonized by the selective neurokinin-1 receptor antagonist RP 67580 (1 microM). Intracellular recordings distinguished between Aalpha/beta-, Adelta- and C-sub-types of ganglion neurons. Superfusion of substance P (1 microM) evoked excitatory responses in Adelta- and C-type neurons. These results demonstrate the expression of functional neurokinin-1 receptors on a subpopulation of Adelta- and C-type sensory ganglion neurons. Our data suggest the possible physiological importance of peripheral neurokinin-1 receptors located on dorsal root ganglion neurons.     

Involvement of tachykinin NK2 receptors in the modulation of spontaneous motility in rat proximal colon

The role of endogenous tachykinins and the mechanisms whereby they act on NK2 receptors, modulating spontaneous motility, were investigated in rat isolated proximal colon. The mechanical activity was detected as changes in intraluminal pressure. The NK2 receptor antagonist, MEN 10627, produced a concentration-dependent reduction of the contraction amplitude. [beta-Ala8]-neurokinin A(4-10), an NK2 receptor agonist, and [Sar9, Met(O2)11]-Substance P ([Sar9, Met(O2)11]-SP), an NK1 receptor agonist, induced a concentration-dependent contractile response, characterized by an increase in basal tone with superimposed phasic contractions. MEN 10627 antagonized the response to [beta-Ala8]-neurokinin A(4-10), without affecting that to [Sar9, Met(O2)11]-SP. Tetrodotoxin (TTX), hexamethonium and Nomega-nitro-L-arginine methyl ester (L-NAME) significantly reduced the response to MEN 10627. The NK3 receptor agonist, senktide, was able to activate the nitrergic inhibitory pathway, as it induced a TTX-and L-NAME-sensitive inhibitory effect. [beta-Ala8]-neurokinin A(4-10) was able to antagonize the inhibitory response to senktide. These findings suggest that tachykinins acting on NK2 receptors play a role in the modulation of the spontaneous mechanical activity. The mechanism of this action would be, in part, acting directly on the smooth muscle cells, and, in part neurogenic, sustained by nicotinic inputs, and possibly due to inhibition of NO tonic release.     

Cellular and subcellular localization of neurokinin-1 and neurokinin-3 receptors in primate globus pallidus

The primate globus pallidus receives massive innervations from GABAergic striatal neurons that co-release the neuropeptide substance P (SP). To expand our knowledge regarding SP interaction at pallidal level, we used single and double antigen retrieval methods to study the cellular and subcellular localization of SP and its high-affinity receptors neurokinin-1 (NK-1R) and neurokinin-3 (NK-3R) in the globus pallidus of the squirrel monkey (Saimiri sciureus). At the light microscopic level, a large number of neurons and fibers located in both the external (GPe) and internal (GPi) segments of the globus pallidus expressed NK-1R or NK-3R immunoreactivity. At the electron microscopic level, both NK-1R and NK-3R were mainly associated with intracellular sites or located at extrasynaptic positions on the plasma membrane. Presynaptic axon terminals forming symmetric and asymmetric synapses occasionally contained NK-1R and NK-3R. Neurokinin receptors were also observed in a proportion of SP-immunoreactive axon terminals, but these terminals preferentially expressed NK-3R. The pattern of distribution of NK-1R and NK-3R in GPe and GPi indicates that SP effects at pallidal level are mediated through postsynaptic receptor as well as presynaptic autoreceptors and heteroreceptors. These morphological data suggest that, either alone or in conjunction with GABA, SP could have a wide range of effects at pallidal level. This neuroactive peptide may influence in a significant manner the integration and treatment of neural information that flows through the basal ganglia.     

Multiple neurotransmitter receptors contribute to the spinal Fos expression

The aim of this study is to identify the receptors which could potentially mediate the activation of c-Fos. Therefore, the effects of neurotransmitter receptor agonists in the activation of c-Fos in spinal neurons were studied by intrathecal injection of excitatory amino acid (EAA) receptor agonists: N-Methyl-D-Aspartate (NMDA), (S)-alpha-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic acid (AMPA), 2-Carboxyl-3-carboxymethyl-4-isopropenylpyMidine (Kainic acid, KA), (1S-3R)-1-Aminocyclopentane-1, 3-dicarboxylacid (ACPD), and substance-P receptor (neurokinin-1) agonist, [Sar9, Met (O2)11] SP (SarMet-SP). All drugs tested activated the production of c-Fos in spinal dorsal horn neurons. AMPA was found as the most potent agonist tested producing market production of c-Fos particularly in neurons of lamina II at doses of 10 pM per 10-microl injection. At this dose, other agonists were relatively ineffective. At higher doses, AMPA significantly increased the activated cells. NMDA significantly increased c-Fos production to a marked extent only at doses above 10 nM per 10-microl injection. KA and ACPD were least potent of the excitatory amino acid agonists. Injection of SarMet-SP at doses of 1 nM activated Fos selectively in neurons of lamina I. A dose-dependent increase in number of c-Fos-positive cells was observed for AMPA, KA, ACPD, and SarMet-SP, whereas NMDA gave a very strong expression after a high dose with no dose dependency. These finding suggest that multiple neurotransmitter receptors lead to c-Fos production in spinal neurons.     

Profile of neuronal excitation following selective activation of the neurokinin-1 receptor in rat deep dorsal horn in vitro

The excitatory actions of the selective neurokinin-1 receptor (NK1R) agonist [Sar⁹,Met(O₂)¹¹]substance P (SP) were tested on a sample (n=50) of deep dorsal horn neurones in the isolated and hemisected young rat spinal cord. Superfusion of the NK1R agonist (2 μM) elicited a prolonged membrane depolarisation (6.6±0.5 mV) and an increase in action potential firing in 41/50 (82%) neurones. These [Sar⁹,Met(O₂)¹¹]SP-induced depolarisations were attenuated by the selective NK1R antagonist GR82334 (1 μM). An increased neuronal excitability after [Sar⁹,Met(O₂)¹¹]SP application was indicated by an augmented spike frequency generated in response to long duration, step depolarisations. In order to assess whether a direct excitatory action existed, [Sar⁹,Met(O₂)¹¹]SP was re-tested on a sample of TTX-treated neurones (n=14). The majority (9/14) retained agonist sensitivity although the amplitude of the depolarisation was reduced to 48% of the control value. A sample of neurones (n=7) that responded to the NK1R agonist were morphologically characterised after filling with the intracellular dye, biocytin. Dorsal dendrites that clearly penetrated lamina II and that could receive a direct C-afferent input, were identified in only 2/7 neurones. These electrophysiological and neuroanatomical data demonstrate that deep dorsal horn neurones possess functional NK1Rs. The implications of the existence of these NK1Rs in the context of spinal somatosensory systems and SP is considered.