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Substance P (SP) is a sensory neuropeptide that is expressed by the neurons innervating bone. There is considerable evidence that SP can regulate bone cell function in vitro, but it is unclear whether SP modulates bone modeling or remodeling in vivo. To answer this question we characterized the bone phenotype of mice with deletion of the Tac1 gene expressing SP. The phenotypes of 2-month-old and 5-month-old SP deficient mice and their wildtype controls were characterized by using μCT imaging, static and dynamic bone histomorphometry, and urinary deoxypyridinoline cross-links (DPD) measurement. No differences in bone phenotypes were observed between the 2 strains at 2 months of age. By 5 months both the wildtype and SP deficient mice had developed cancellous osteopenia, but relative to the wild-type mice the SP deficient mice had significantly greater cancellous bone loss. The SP deficient mice also exhibited decreased bone formation, increased osteoclast number, and increased urinary DPD levels. Cortical defect early repair was delayed in 5-month-old mice lacking SP. Collectively, these findings indicate that SP signaling is not required for bone modeling, but SP signaling reduces age-related osteopenia and accelerates cortical defect reparation, data supporting the hypothesis that SP is an anabolic physiologic regulator of bone metabolism., (Published by Elsevier Inc.)

The antinociceptive action of botulinum toxin type A (BoNT/A) has been demonstrated in behavioral animal studies and clinical settings. It was shown that this effect is associated with toxin activity in CNS, however, the mechanism is not fully understood. Substance P (SP) is one of the dominant neurotransmitters in primary afferent neurons transmitting pain and itch. Thus, here we examined association of SP-mediated transmission and BoNT/A antinociceptive action by employing gene knockouts. Antinociceptive activity of intraplantarly (i.pl.) injected BoNT/A was examined in mice lacking the gene encoding for SP/neurokinin A (tac1 -/- ) or SP-preferred receptor neurokinin 1 (tac1r -/- ), compared to control C57Bl/6J wild type animals. BoNT/A action was assessed in inflammatory pain induced by formalin and CFA, and neuropathic pain induced by partial sciatic nerve ligation. BoNT/A activity in CNS was examined by c-Fos and BoNT/A-cleaved SNAP-25 immunohistochemistry. In wild type mice, acute (formalin-evoked) and chronic pain (neuropathic and inflammatory) was reduced by peripherally injected BoNT/A. In tac1 -/- and tac1r -/- knockout mice, BoNT/A exerted no analgesic effect. In control animals BoNT/A reduced the formalin-evoked c-Fos expression in lumbar dorsal horn, while in knockout mice the c-Fos expression was not reduced. After peripheral toxin injection, cleaved SNAP-25 occurred in lumbar dorsal horn in all animal genotypes. BoNT/A antinociceptive activity is absent in animals lacking the SP and neurokinin 1 receptor encoding genes, in spite of presence of toxin's enzymatic activity in central sensory regions. Thus, we conclude that the integrity of SP-ergic system is necessary for the antinociceptive activity of BoNT/A., (Copyright © 2017. Published by Elsevier Ltd.)

Complete control of emesis caused by cyclophosphamide (CPA) is of immense interest to both patients and physicians. Serotonin 5-HT3- and tachykinin NK1-receptor antagonists are widely used antiemetics in clinic, but they fail to completely control CPA-induced emesis. New antiemetic targets for the full control of CPA-induced vomiting are lacking. We therefore examined the effects of CPA on emetic targets downstream of 5-HT3- and NK1- receptors in an attempt to better understand the molecular bases of CPA-induced emesis. Acute CPA (200 mg/kg, i.p.) administration in the least shrew caused a biphasic pattern of emesis over a 40 h observation period, with maximal peak vomit frequency during the 1st hour of treatment (acute phase), followed by a delayed-phase which peaks at 27th hour. The NK1 receptor mRNA levels increased significantly at 8 h post-CPA treatment in the brainstem, and at 28 h in the whole intestine. Substance P mRNA levels tended to increase both in the brainstem and intestine at most time-points post-CPA injection, however due to large variability, they failed to attain significance. Likewise, protein expression profiles of both NK1- and 5-HT3 -receptors in the brainstem were unchanged at any time-point. However, phosphorylation levels of protein kinase A (PKA), but not of extracellular signal-regulated protein kinase 1/2 (ERK1/2), were increased at 2, 8, 22, 28, and 33 h time-points after the treatment with CPA. Moreover, brainstem but not frontal cortex cAMP tissue levels tended to be elevated at most time-points, but significant increases occurred only at 1 and 2 h post-CPA treatment. The phosphodiesterase inhibitor, rolipram, caused significant increases in shrew brainstem cAMP levels which were associated with its capacity to produce vomiting, while pretreatment with SQ22536, an inhibitor of adenylyl cyclase, prevented rolipram-induced emesis. The results demonstrate that accumulation of cAMP and subsequent activation of PKA in the brainstem may help to initiate and sustain emesis induced by CPA in the least shrew. Our findings suggest that suppression of the cAMP/PKA cascade may have antiemetic potential in the management of CPA-induced emesis.

Zebrafish has emerged as an important vertebrate animal model for the study of human diseases and for developmental studies in mammals. Since there are few studies of the tachykinin 1 gene (TAC1), precursor of substance P (SP), in relation to embryonic development, we aimed to study the expression of SP transcript (mRNA) and determine the influence of cocaine and opioid receptors on the expression of this neuropeptide. In order to analyse the spatial and temporal SP mRNA expression in zebrafish, we cloned - based on human TAC1 sequence - the sequence that originates SP. Phylogenetic analyses of the precursor of SP, revealed an alignment in the fish cluster, with a clear distinction from other species (amphibians, birds and mammals). Real time PCR (qPCR) results showed that SP mRNA was expressed in several stages of embryonic development, where it increased progressively from gastrula-8hpf (hour post-fertilisation) to the end of the embryogenesis-72hpf. SP mRNA was expressed mainly in the spinal cord in embryos at 20-30hpf, whereas at 36, 42 and 48hpf embryos SP mRNA was expressed mainly in the CNS telencephalon, diencephalon, hypothalamus, rhombomeres, epiphysis and in peripheral areas (heart and somites). Exposure of embryos to 1.5μM cocaine altered the SP mRNA expression at 24 (increasing) and 48hpf (decreasing). We also report that knockdown of μ-opioid receptor induced an increase of SP mRNA expression while the knockdown of the two delta opioid receptors did not produce changes in SP mRNA expression. In conclusion, SP mRNA in zebrafish is expressed during embryonic development in the CNS and peripherally, suggesting that SP would play a critical role during embryogenesis. Furthermore, cocaine exposure and the knockdown of μ-opioid receptor affect the SP mRNA expression. These observations can be important in the pain and addiction field where SP is involved., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

Mechanical stress plays a vital role in maintaining bone architecture. The process by which osteogenic cells convert the mechanical signal into a biochemical response governing bone modeling is not clear, however. In this study, we investigated how Atlantic salmon (Salmo salar) vertebra responds to exercise-induced mechanical loading. Bone formation in the vertebrae was favored through increased expression of genes involved in osteoid production. Fourier transform infrared spectroscopy (FT-IR) showed that bone matrix secreted both before and during sustained swimming had different properties after increased load compared to control, suggesting that both new and old bones are affected. Concomitantly, both osteoblasts and osteocytes in exercised salmon showed increased expression of the receptor nk-1 and its ligand substance P (SP), both known to be involved in osteogenesis. Moreover, in situ hybridization disclosed SP mRNA in osteoblasts and osteocytes, supporting an autocrine function. The functional role of SP was investigated in vitro using osteoblasts depleted for SP. The cells showed severely reduced transcription of genes involved in mineralization, demonstrating a regulatory role for SP in salmon osteoblasts. Investigation of α-tubulin stained osteocytes revealed cilia-like structures. Together with SP, cilia may link mechanical responses to osteogenic processes in the absence of a canaliculi network. Our results imply that salmon vertebral bone responds to mechanical load through a highly interconnected and complex signal and detection system, with SP as a key factor for initializing mechanically-induced bone formation in bone lacking the canaliculi system., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Scant information is available regarding the effects of cisplatin on the expression profile of tachykinin NK(1) receptors and downstream signaling during cisplatin-induced emesis. Cisplatin causes peak early- and delayed-phase emesis in the least shrew at 1-2 and 33 h post-injection. To investigate the expression profile of NK(1) receptor during both emetic phases, we cloned the cDNA corresponding to a ~700 base pairs of mRNA flanked by two stretches of nucleotides conserved among different species and demonstrated that the shrew NK(1) receptor nucleotide sequence shares ~90% sequence identity with the human NK(1) receptor. Of the 12 time-points tested, significant increases in expression levels of NK(1) receptor mRNA in the shrew brainstem occurred at 2 and 28 h post-cisplatin injection, whereas intestinal NK(1) receptor mRNA was increased at 28 h. Shrew brainstem and intestinal substance P mRNA levels also tended to increase during the two phases. Furthermore, expression levels of NK(1) receptor protein were significantly increased in the brainstem at 2, 8, and 33 h post-cisplatin. No change in brainstem 5-HT(3) receptor protein expression was observed. The temporal enhancements in NK(1) receptor protein expression were mirrored by significant increases in the phosphorylation status of the brainstem ERK1/2 at 2, 8, and 33 h post-cisplatin. Phosphorylation of PKA significantly increased at 33rd and 40th hour. Our results indicate associations between cisplatin's peak immediate- and delayed-phase vomiting frequency with increased: (1) expression levels of NK(1) receptor mRNA and its protein level, and (2) downstream NK(1) receptor-mediated phosphorylation of ERK1/2 and PKA signaling., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A satisfactory management to ensure a full restoration of peripheral nerve after trauma is not yet available. Using an experimental protocol, in which crush injury was applied 1 cm above the bifurcation of the rat sciatic nerve for 20 s, we here demonstrate that the levels of neuroactive steroids, such as pregnenolone and progesterone (P) metabolites (i.e. dihydroprogesterone, DHP, and tetrahydroprogesterone, THP) present in injured sciatic nerve were significantly decreased. On this basis, we have focused our attention on DHP and its direct precursor, P, analyzing whether these two neuroactive steroids may have neuroprotective effects on biochemical, functional and morphological alterations occurring during crush-induced degeneration-regeneration. We demonstrate that DHP and/or P counteract biochemical alterations (i.e. myelin proteins and Na(+),K(+)-ATPase pump) and stimulate reelin gene expression. These two neuroactive steroids also counteract nociception impairment, and DHP treatment significantly decreases the up-regulation of myelinated fibers' density occurring in crushed animals. Altogether, these observations suggest that DHP and P (i.e. two neuroactive steroids interacting with progesterone receptor) may be considered protective agents in case of nerve crush injury.

The painful sensation during acute myocardial ischemia or infarction is a common symptom and results from neural activity in humans. Little is known about the role of neuropeptides in this effect of myocardial ischemia. The aim of the current study was to investigate the role of substance P in mediating the noxious neural signals in spinal cord in acute myocardial ischemia by exploring the change in substance P and its mRNA in thoracic dorsal root ganglia and spinal dorsal horn (T1-T5) after coronary artery occlusion. The experiment was performed with immunohistochemistry, enzyme immunoassay and real time reverse transcription-polymerase chain reaction techniques on rats' hearts. In acute myocardial ischemia (<6 h), substance P and preprotachykinin mRNA were up-regulated in the neurons of the dorsal root ganglia and spinal dorsal horn. The increase in the density of immunoreactive material was mainly observed in small-diameter neurons of the dorsal root ganglia and the superficial laminae (I and II) of the spinal cord. The increase in the expressions was statistically significant compared with the control and the sham surgery groups (P<0.05). The results suggest that substance P is involved in the mediation of the noxious neural signals of acute myocardial ischemia in spinal cord. The pathophysiological role and significance need to be investigated.

Neuropeptide Y has been implicated in pain modulation and is substantially up-regulated in dorsal root ganglia after peripheral nerve injury. To identify the role of neuropeptide Y after axotomy, we investigated the behavioral and neurochemical phenotype of neuropeptide Y Y1 receptor knockout mice with focus on dorsal root ganglion neurons and spinal cord. Using a specific antibody Y1 receptor immunoreactivity was found in dorsal root ganglia and in dorsal horn neurons of wild-type, but not knockout mice. The Y1 receptor knockout mice exhibited a pronounced mechanical hypersensitivity. After sciatic nerve axotomy, the deletion of Y1 receptor protected knockout mice from the axotomy-induced loss of dorsal root ganglion neurons seen in wild-type mice. Lower levels of calcitonin gene-related peptide and substance P were identified by immunohistochemistry in dorsal root ganglia and dorsal horn of knockout mice, and the axotomy-induced down-regulation of both calcitonin gene-related peptide and substance P was accentuated in Y1 receptor knockout. However, the transcript levels for calcitonin gene-related peptide and substance P were significantly higher in knockout than in wild-type dorsal root ganglia ipsilateral to the axotomy, while more calcitonin gene-related peptide- and substance P-like immunoreactivity accumulated proximal and distal to a crush of the sciatic nerve. These results indicate that the deletion of the Y1 receptor causes increased release and compensatory increased synthesis of calcitonin gene-related peptide and substance P in dorsal root ganglion neurons. Together, these findings suggest that, after peripheral nerve injury, neuropeptide Y, via its Y1 receptor receptor, plays a key role in cell survival as well as in transport and synthesis of the excitatory dorsal horn messengers calcitonin gene-related peptide and substance P and thus may contribute to pain hypersensitivity.

To complete a series of studies on the expression of substance P and neurokinin receptors in mammalian retinas, we investigated the occurrence of these molecules in developing mouse retinas and in retinas of mice with genetic deletion of the neurokinin 1 receptor, the preferred substance P receptor. Using semi-quantitative reverse transcription-polymerase chain reaction, we measured detectable levels of the gamma isoform of preprotachykinin A (a substance P precursor) mRNA at postnatal day 4. Neurokinin 1 receptor and neurokinin 3 receptor mRNAs were also detected at postnatal day 4. While gamma preprotachykinin A and neurokinin 1 receptor mRNA levels significantly increased up to eye opening (postnatal day 11), neurokinin 3 receptor mRNA levels remained constant throughout development. Substance P, neurokinin 1 receptor and neurokinin 3 receptor immunoreactivities were present at postnatal day 5. Substance P was in amacrine cells, neurokinin 1 receptor in developing amacrine and bipolar cells and neurokinin 3 receptor in OFF-type cone bipolar cells. Interestingly, a transient increase in the density of neurokinin 1 receptor immunoreactive processes was observed at eye opening in lamina 3 of the inner plexiform layer, suggesting a role of substance P and neurokinin 1 receptor in this developmental phase. However, in neurokinin 1 receptor knockout retinas, besides a significant increase of the gamma preprotachykinin A mRNA levels, no major changes were detected: neurokinin 3 receptor mRNA levels as well as substance P and neurokinin 3 receptor immunostainings were similar to wild types. Together with previous studies, these observations indicate that there are major differences in neurokinin 1 receptor expression patterns among developing mammalian retinas. The observations in neurokinin 1 receptor knockout mice may not be applicable to rats or rabbits, and substance P and neurokinin 1 receptor may play different developmental roles in different species.

Corticostriatal circuits participate in limbic, attentional, motor and other networks, and are implicated in psychostimulant addiction. The psychostimulant methylphenidate is used in the treatment of attention-deficit hyperactivity disorder and for recreational purposes. Recent studies indicate that methylphenidate alters gene expression in striatal neurons. We investigated whether methylphenidate affects gene regulation in specific corticostriatal circuits, by comparing drug-induced molecular changes in different functional domains of the striatum with changes in their cortical input regions. In order to assess the potential functional significance of methylphenidate-induced molecular changes, we examined members of two different classes of plasticity-related molecules, the transcription factor zif 268 and the synaptic plasticity factor Homer 1a. Acute methylphenidate administration in adult rats increased the expression of Homer 1a and zif 268 in both cortex and striatum in a dose-dependent and regionally selective manner. These changes in gene expression occurred after doses of 2 mg/kg (i.p.) and higher, and were highly correlated between cortical regions and their striatal targets. In the cortex, increases were maximal in the medial agranular (premotor) and cingulate cortex, followed by motor and somatosensory cortex, and were minimal in the insular cortex. Correspondingly, in the striatum, increases were most robust in sensorimotor sectors that receive medial agranular input, and were weaker or absent in ventral sectors. The methylphenidate-induced increases in cortical Homer 1a and zif 268 expression were also correlated with increases in striatal substance P and dynorphin expression (direct pathway). Overall, the regional distribution of methylphenidate-induced molecular changes in the striatum was similar to that of changes induced by psychostimulants such as cocaine. These findings demonstrate that methylphenidate affects transcription and synaptic plasticity regulatory proteins in specific corticostriatal circuits, including those implicated in attentional functions and psychostimulant addiction. Such methylphenidate-induced gene regulation may contribute to the therapeutic effects and/or abuse liability of this psychostimulant.

Human dorsal root ganglia (DRGs) were obtained during various procedures and processed for single and double in situ hybridisation using oligonucleotide probes complementary to three peptide mRNAs. Some postmortem ganglia were also analysed. In donor (unlesioned) DRGs 12.5% of the neuron profiles (NPs) were galanin mRNA-positive (mRNA(+)), 47.5% calcitonin gene-related peptide (CGRP) mRNA(+) and 32.7% substance P mRNA(+). The corresponding percentages for cervical/thoracic DRGs from patients suffering from severe brachial plexus injury were 32.8%, 57.4% and 34.5%, respectively. In these DRGs a high proportion of the galanin mRNA(+) NPs contained CGRP mRNA and substance P mRNA. In DRGs from a patient with migraine-like pain a comparatively small proportion expressed galanin, whereas in DRGs from a herpes zoster patient galanin mRNA(+) NPs were comparatively more frequent. The results from human postmortem DRGs revealed only weak peptide mRNA signals. The present results demonstrate that galanin is expressed in DRGs not only in a number of animal species including monkey as previously shown, but also in a considerable proportion of human DRG neurons, often together with CGRP and substance P, and mostly in small neurons. Thus, galanin may play a role in processing of sensory information, especially pain, in human DRGs and dorsal horn. However, to what extent a similarly dramatic upregulation of galanin expression can be seen after peripheral nerve lesion in man, as has been reported for rat, mouse and monkey, remains to be analysed.

Metabotropic glutamate receptor 1 (mGluR1) is a G-protein-coupled receptor and is expressed in the medium spiny projection neurons of mouse striatum. To define the role of mGluR1 in actions of psychostimulant, we compared both motor behavior and striatal neuropeptide mRNA expression between mGluR1 mutant and wild-type control mice after a single injection of amphetamine. We found that acute amphetamine injection increased motor activity in both mutant and control mice in a dose-dependent manner (1, 4, and 12 mg/kg, i.p.). However, the overall motor responses of mGluR1 -/- mice to all three doses of amphetamine were significantly greater than those of wild-type +/+ mice. Amphetamine also induced a dose-dependent elevation of preprodynorphin mRNA in the dorsal and ventral striatum of mutant and wild-type mice as revealed by quantitative in situ hybridization. In contrast to behavioral responses, the induction of dynorphin mRNA in both the dorsal and ventral striatum of mutant mice was significantly less than that of wild-type mice in response to the two higher doses of amphetamine. In addition, amphetamine elevated basal levels of substance P mRNA in the dorsal and ventral striatum of mGluR1 mutant mice to a similar level as that of wild-type mice. There were no differences in basal levels and distribution patterns of the two mRNAs between the two genotypes of mice treated with saline. These results demonstrate a clear augmented behavioral response of mGluR1 knockout mice to acute amphetamine exposure that is closely correlated with reduced dynorphin mRNA induction in the same mice. It appears that an intact mGluR1 is specifically critical for full dynorphin induction, and impaired mobilization of inhibitory dynorphin system as a result of lacking mGluR1 may contribute to an augmentation of motor stimulation in response to acute administration of psychostimulant.

The type of trigeminal ganglion cells that express 5-HT1B receptors has not been well characterized, despite the fact that these receptors are important targets for anti-migraine drugs. We have therefore used combined in situ hybridization and immunofluorescence to examine the expression of 5-HT1B receptor messenger RNA in identified subpopulations of rat trigeminal ganglion cells. 5-HT1B-expressing cells accounted for 15% of all trigeminal ganglion cells, were medium sized, and showed immunoreactivity for either 200,000 mol. wt neurofilament, calcitonin gene-related peptide, or nerve growth factor receptor (trkA). In contrast few 5-HT1B cells showed immunoreactivity for substance P or binding of the lectin Griffonia simplicifolia IB4. Our results are consistent with 5-HT1B receptors acting to control the release of calcitonin gene-related peptide from trigeminal neurons with finely myelinated axons. 5-HT1B receptor agonists may reduce neurogenic vasodilation by activating such receptors. However many nociceptive trigeminal neurons, including the substance P and IB4-binding populations, do not express the 5-HT1B receptor.

Common marmosets (Callithrix jacchus) with near-complete unilateral 6-hydroxydopamine denervation of the dopaminergic input received a single injection of saline or L-DOPA (15mg/kg plus 6.25mg/kg benserazide). Using in situ hybridization, the effects of these treatments on c-fos messenger RNA expression in the cerebral cortex, the striatal complex and the external layer of the pallidum were studied. Moreover, receptor autoradiography was used to determine the levels of dopamine D(1) and D(2) receptors in these areas. In the cerebral cortex, animals treated with L-DOPA displayed a high expression of c-fos messenger RNA restricted to the dopamine-denervated hemisphere. No changes in the levels of cortical D(1) and D(2) receptors were found in the dopamine-denervated hemisphere. L-DOPA treatment also induced a strong expression of c-fos messenger RNA in the striatal complex in the dopamine-denervated hemisphere. The levels of striatal D(2), but not D(1), receptors were increased in the dopamine-denervated hemisphere. In the external pallidum, the major terminal region for D(2) dopamine receptor-containing striatal projection neurons, L-DOPA treatment induced c-fos messenger RNA expression in both the intact and the dopamine-denervated hemispheres.Thus, using c-fos messenger RNA as a biochemical marker of postsynaptic neuronal activation, these results provide evidence that near-complete dopamine depletion causes a profound supersensitization to L-DOPA treatment in the cerebral cortex and in the striatal complex, but not in the external layer of the pallidum, of the primate brain. The cortical response may be unique to the primate brain, but c-fos messenger RNA activation within the striatum has also been reported in the rodent. The effects of L-DOPA probably depend both on a direct activation of supersensitized dopamine receptors by dopamine produced in the few remaining, but hyperactive, dopaminergic nerve terminals and in serotonergic nerve terminals, as well as on indirect actions of L-DOPA related to activation of circuitries connecting cerebral cortex and basal ganglia structures. These results provide novel information on the mechanisms underlying L-DOPA's action in the cerebral cortex, striatum and external pallidum in a primate model of Parkinson's disease.

Detailed analysis of a novel transgenic model of basal ganglia disease has been undertaken. In this model the expression of an attenuated form of the diphtheria toxin gene was tightly controlled by D1 dopamine receptor regulatory domains. The behavioral and both direct toxin-mediated and transneuronal effects observed in pups in the first postnatal week have been described. Although younger pups are bradykinetic, older pups have a hyperkinetic syndrome with gait abnormality, postural instability and myoclonic jerks typical of human basal ganglia diseases such as Huntington's disease. As expected, striatal D1 dopamine receptor, dynorphin and substance P transcripts were not detected by in situ hybridization but there was a 27% increase in striatal D2 dopamine receptor messenger RNA and a 65% increase in enkephalin messenger RNA expression. Receptor autoradiographic studies confirmed the lack of D1-class binding in the mutant striatum and in contrast to young pups, a substantial increase in striatal D2-class binding. Autoradiographic quantitation also showed a 30% increase in striatal dopamine transporter binding. In addition to the changes described in the striatopallidal and nigrostriatal pathways, up-regulated dynorphin and substance P messenger RNA expression was also seen in the cortex. The capacity of the developing brain for neurochemical adaptation following injury is dramatic. The results show that primary loss of D1 dopamine receptor-positive striatonigral pathway neurons is sufficient to generate a hyperkinetic phenotype.

Recent pathophysiological models of basal ganglia function in Parkinson's disease predict that specific neurochemical changes in the indirect pathway would follow the lack of stimulation of D(2) dopamine receptors. Post mortem studies of the basal ganglia in genetically modified mice lacking functional copies of the D(2) dopamine receptor gene allowed us to test these predictions. When compared with their congenic N(5) wild-type siblings, mice lacking D(2) receptors show an increased expression of enkephalin messenger RNA in the striatum, and an increased activity and expression of cytochrome oxidase I in the subthalamic nucleus, as expected. In addition, D(2) receptor-deficient mice display a reduced expression of glutamate decarboxylase-67 messenger RNA in the globus pallidus, as the basal ganglia model predicts. This reduction contrasts with the lack of change or increase in glutamate decarboxylase-67 messenger RNA expression found in animals depleted of dopamine after lesions of the mesostriatal dopaminergic system. Furthermore, D(2) receptor-deficient mice show a significant decrease in substance P messenger RNA expression in the striatonigral neurons which form the direct pathway. Finally, glutamate decarboxylase-67 messenger RNA expression in the basal ganglia output nuclei was not affected by mutations in the D(2) receptor gene, a fact that could probably be related to the absence of a parkinsonian locomotor phenotype in D(2) receptor-deficient mice. In summary, these findings provide compelling evidence demonstrating that the lack of endogenous stimulation of D(2) receptors is sufficient to produce subthalamic nucleus hyperactivity, as assessed by cytochrome oxidase I histochemistry and messenger RNA expression, and strongly suggest the existence of interactions between the basal ganglia direct and indirect pathways.

A transgenic mouse has been developed which, during development, over-expresses nerve growth factor under the control of a myelin basic protein promoter. These animals display an ectopic network of substance P-containing sensory fibers in the white matter of the spinal cord. To study the functional significance of this model to nociception, these mice were studied in a test measuring the latency to tail withdrawal from a noxious radiant heat stimulus. Baseline reaction times were significantly less in transgenic mice, suggesting thermal allodynia. A mechanical stimulus was then applied to the tip of the tail at either 450 g or 1400 g for 2 s and tail withdrawal readings were taken for another 10 min. In control mice, the 450 g stimulus was without effect, suggesting that it is normally innocuous. In transgenic mice, this stimulus induced a transient decrease in withdrawal latency at 1 min. Thus, transgenic mice exhibited mechanical allodynia. The 1400 g stimulus decreased withdrawal latency in both transgenic and control mice. However, the response was greater in transgenic mice, indicating that they exhibited mechanical hyperalgesia. The neurokinin-1 receptor antagonist CP-96,345, but not the inactive stereoisomer CP-96,344, administered subcutaneously 30 min before the 450 g stimulus, blocked the stimulation-induced allodynia in transgenic mice, and revealed a transient antinociception in transgenic and control mice. Ketamine, an N-methyl-D-aspartate receptor antagonist, given intraperitoneally 10 min before 450 g stimulation, blocked the allodynia in transgenic mice. These results indicate that these transgenic mice display hyperalgesia and allodynia, and that these nociceptive responses are reversed by substance P and N-methyl-D-aspartate receptor antagonists.

The long-term effects of intrastriatal injections of the agonist of N-methyl-D-aspartate receptors, quinolinic acid, have been extensively characterized. Much less is known, however, about the early molecular and neurochemical changes which occur within a few hours of the toxin injection. In the present study, we have performed intrastriatal injections of low doses of quinolinic acid which induce DNA damage 10-12 h post-lesion, and selective death of striatal projection neurons two weeks later. We examined the time-course of alterations in the microtubule-associated protein 2, an early marker of cytoskeletal disruption, and enkephalin and substance P, two neuropeptides present in largely distinct subpopulations of striatal efferent neurons projecting to the globus pallidus and entopeduncular nucleus, respectively. Immunoreactivity for microtubule-associated protein 2 was decreased at the periphery of the lesion 10 h after quinolinate injection. Levels of enkephalin messenger RNA were markedly decreased as early as 6 h post-lesion; however, a significant decrease in enkephalin immunoreactivity was not observed in the globus pallidus (external pallidum) until 12 h post-injection. Levels of substance P messenger RNA were decreased 12 h post-injection in striatal neurons. However, in contrast to enkephalin immunoreactivity, immunolabeling for substance P was not significantly decreased at this time-point in the internal pallidum, a finding reminiscent of early grades of Huntington's disease. The results reveal the time-course of change in messenger RNA and peptide levels in striatal efferent neurons shortly after an excitotoxic insult. These data have implications for the interpretation of findings in post mortem brain and mouse models of Huntington's disease.

The selective M4 muscarinic receptor toxin, MT3, was used in vivo to evaluate the role of M4 receptors in cholinergic inhibition of neuropeptide mRNA expression in striatonigral neurons. Unilateral injection of the muscarinic toxin 3 (0.04-4 nmol) into the dorsal striatum of chronically-cannulated rats elevated basal levels of preprodynorphin, substance P and preproenkephalin mRNAs in the ipsilateral dorsal striatum as revealed by quantitative in situ hybridization. Pretreatment with muscarinic toxin 3 also augmented amphetamine (2.5 mg/kg, i.p.)-stimulated preprodynorphin and substance P expression in the dorsal striatum in a manner similar to that observed after the muscarinic antagonist, scopolamine. Since muscarinic toxin 3 has a much greater affinity for muscarinic M4 receptors than for other subtypes, it is possible that muscarinic toxin 3, by interacting with the muscarinic M4 subtype, regulates basal and/or dopamine-stimulated striatal neuropeptide gene expression.

This study investigated the effects of pharmacological blockade or stimulation of muscarinic receptors on constitutive and amphetamine-stimulated preprodynorphin, substance P and pre-proenkephalin gene expression in rat striatum. Acute administration of the non-selective muscarinic antagonist, scopolamine (2.5, 5 and 10 mg/kg, s.c.), caused a dose-dependent increase in preprodynorphin and substance P, but not preproenkephalin, messenger RNA expression in the dorsal and ventral striatum as revealed by quantitative in situ hybridization. In contrast, acute injection of the non-selective muscarinic receptor agonist, oxotremorine (0.125, 0.25 and 0.5 mg/kg, s.c.), caused a dose-dependent increase in basal levels of preproenkephalin messenger RNA in the dorsal striatum, without causing a significant effect on constitutive striatal preprodynorphin and substance P expression. Pretreatment with scopolamine (2.5 mg/kg, s.c.) significantly augmented striatal induction of preprodynorphin and substance P messenger RNA induced by acute injection of amphetamine (1.25 and 2.5 mg/kg, i.p.), whereas scopolamine blocked amphetamine-stimulated striatal preproenkephalin expression. Pretreatment with oxotremorine (0.25 mg/kg, s.c.) significantly attenuated amphetamine (1.25 and 2.5 mg/kg, i.p.)-stimulated striatal preprodynorphin and, to a lesser degree, substance P messenger RNA expression. Oxotremorine tended to increase amphetamine-stimulated preproenkephalin messenger RNA expression, but the effect did not reach statistical significance. In addition, scopolamine increased spontaneous, and enhanced amphetamine-stimulated, behavioral activity, whereas oxotremorine attenuated amphetamine-stimulated behaviors. These data support the concept that cholinergic transmission, via interaction with muscarinic receptors, inhibits basal and D1 receptor-stimulated striatonigral dynorphin/substance P gene expression and facilitates striatopallidal enkephalin gene expression.

The expression of enkephalin and substance P messenger RNAs was examined in the caudate-putamen of human post mortem tissue from control and Huntington's disease tissue using in situ hybridization techniques and human specific enkephalin and substance P [35S] oligonucleotides. Macroscopic and microscopic quantification of enkephalin and substance P gene expression was carried out using computer-assisted image analysis. Tissue was collected from six control cases with no sign of neurological disease and six Huntington's disease cases ranging from grades 0 to 3 as determined by neuropathological evaluation. The clinical and pathological diagnosis of Huntington's disease was confirmed unequivocally by genetic analysis of the CAG repeat length in both copies of IT15, the Huntington's disease gene. A marked reduction in both enkephalin and substance P messenger RNAs was detected in all regions of the caudate nucleus and putamen in Huntington's disease grades 2/3 when compared to controls; in the dorsal caudate few enkephalin or substance P messenger RNA-positive cells were detected. For the early grade (0/1) Huntington's disease cases, a heterogeneous reduction in both enkephalin and substance P messenger RNAs were noted; for enkephalin messenger RNA the striatal autoradiograms displayed a conspicuous patchy appearance. Detailed cellular analysis of the dorsal caudate revealed a striking reduction in the number of enkephalin and substance P messenger RNA-positive cells detected and in the intensity of hybridization signal/cell. These data suggest that both the "indirect" GABA/enkephalin and "direct" GABA/substance P pathways are perturbed very early in the course of the disease and that these early changes in chemical signalling may possibly underlie the onset of clinical symptoms.

Striatal dopamine depletion produces an increase in enkephalin and a decrease in substance P messenger RNAs. Subsequent systemic administration of either the D2 dopamine agonist, quinpirole, or the muscarinic antagonist, scopolamine, results in the reduction of the lesion-induced elevation in striatal enkephalin messenger RNA. These changes in enkephalin messenger RNA levels may be mediated solely within the striatum or through trans-synaptic circuits involving the striatum. To dissociate these possibilities, we have compared the effects of systemic and central administration of quinpirole and scopolamine on striatal enkephalin and substance P messenger RNAs using in situ hybridization histochemistry. Systemic administration of both quinpirole and scopolamine blocked the elevation of striatal enkephalin messenger RNA normally observed in 6-hydroxydopamine-lesioned rats. In addition, high doses of systemic scopolamine (25 and 50 mg/kg per day) prevented the lesion-induced decrease in striatal substance P messenger RNA levels. In order to determine whether the effects of these drugs are mediated directly within the striatum, central administration of quinpirole and scopolamine were compared. In contrast to systemic administration, intraventricular and intrastriatal infusion of quinpirole but not scopolamine prevented the lesion-induced change in striatal enkephalin messenger RNA. However, neither quinpirole nor scopolamine administered centrally affected the level of substance P messenger RNA in the striatum of 6-hydroxydopamine-induced lesioned animals. Together, these data suggest that changes in D2 receptor activation directly in the striatum are responsible for the effects of quinpirole on enkephalin messenger RNA. In contrast, the effect of systemic scopolamine on striatal enkephalin and substance P messenger RNAs may not be mediated within the striatum.

Changes in the levels of enkephalin and substance P messenger RNA expression were examined in the striatum following dopamine depletion resulting from unilateral injection of 6-hydroxydopamine into the substantia nigra. In response to striatal dopamine depletion, the levels of enkephalin messenger RNA were elevated, whereas substance P messenger RNA was decreased within all regions of the striatum. Examination of the striatal peptide messenger RNAs between one and 21 days after the injection of 6-hydroxydopamine revealed a temporal dissociation between changes in enkephalin and substance P messenger RNAs. Within one day of the 6-hydroxydopamine injection, substance P messenger RNA was significantly decreased by 30% at all levels of the striatum. This decrease was maintained for up to 21 days after the lesion. In contrast, striatal enkephalin messenger RNA was not significantly elevated until three days following the injection of 6-hydroxydopamine, after which there was a gradual increase up to 21 days. In order to correlate alterations in peptide messenger RNA expression with 6-hydroxydopamine-induced changes in striatal dopamine innervation, tissue punches from the striatum were examined for dopamine content at one, two, three and seven days after the lesion. One day after the lesion, striatal dopamine levels were significantly increased by 47%. In contrast, within two days tissue dopamine content was reduced by 77% compared to control levels. A further decrease of 90% or more was observed at three and seven days after the lesion. Taken together, these data demonstrate a temporal dissociation between changes in enkephalin and substance P messenger RNA levels following 6-hydroxydopamine-induced striatal dopamine depletions. This temporal dissociation may reflect a differential response of enkephalin and substance P messenger RNAs to alterations in dopamine release and subsequent receptor activation.

Male rats were treated i.p. with either 5 mg/kg amphetamine, 3 and 30 mg/kg cocaine or 100 mg/kg caffeine and killed after 30 min. Brains were sectioned and processed for radioactive in situ hybridization histochemistry for the labelling of either c-fos, enkephalin, substance P, neurokinin B, choline acetyltransferase, somatostatin or adenosine A2A receptor messenger RNA. The distribution of c-fos messenger RNA was investigated both at the regional level using film autoradiography, and at the cellular level using emulsion autoradiography. All drug treatments except 3 mg/kg cocaine induced an increased level of c-fos messenger RNA in cells that had a neuron-like morphology. The cells that contained the c-fos messenger RNA were identified by making pairs of 5-microns sections in which one section was processed for c-fos messenger RNA and the other was processed for one of the other messenger RNA species. After amphetamine treatment, only some 10% of the cells in the striatum were labelled, and to a variable extent. Instead there was prominent labelling of a band in the cortex that runs parallel to the cortical surface. There was also a moderate degree of labelling in the nucleus accumbens. c-fos-positive cells were substance P-positive and negative for enkephalin or A2A receptor messenger RNA. Cocaine (30 mg/kg) induced a modest labelling in the caudate-putamen, as well as in the accumbens. With cocaine treatment (30 mg/kg), about 30% of striatal neuron-like cells were c-fos labelled. Most c-fos-positive cells were substance P-positive, but none of the c-fos-positive cells were enkephalin-positive or A2A-receptor-positive. Cocaine (3 mg/kg) had no significant effect on c-fos. Caffeine gave rise to a strong hybridization signal in the caudate-putamen, particularly the dorsolateral part. No other region examined differed significantly from control. With caffeine treatment, about 73% of neuron-like cells were c-fos labelled in the lateral striatum, but labelling was much less pronounced in the medial part or in the accumbens. c-fos-labelled cells were found in enkephalin-positive and enkephalin-negative, substance P-positive and substance P-negative, neurokinin B-positive and neurokinin B-negative groups. No choline acetyltransferase-positive or somatostatin-positive cells were found that were also c-fos-positive with any of the treatments. We conclude that each of the different CNS stimulant drugs induces a highly specific pattern of c-fos messenger RNA.(ABSTRACT TRUNCATED AT 400 WORDS)

We have recently localized delta-9-tetrahydrocannabinol (THC) receptor in the projecting neurons of the striatum which are known to express the neuropeptides substance P and enkephalin. We now report for the first time, by quantitative in situ hybridization, that a 3-week treatment with THC significantly increases in the adult rat caudate-putamen the messenger RNAs levels for substance P and enkephalin. These data indicate that THC may regulate gene expression of neuropeptides in the brain.

The expression of tachykinin-like and opioid-like peptides was studied in medium-sized neurons of the caudate nucleus in tissue from adult cats pretreated with colchicine. Two methods, a serial thin-section peroxidase-antiperoxidase technique and a two-fluorochrome single-section technique, were applied. Quantitative estimates were made mainly with the peroxidase-antiperoxidase method. The numbers of neurons expressing substance P-like, dynorphin B-like, and enkephalin-like immunoreactivity were recorded in regions identified, respectively, as striosomes and extrastriosomal matrix. Striosomes were defined by the presence of clustered substance P-positive and dynorphin B-positive neurons and neuropil. Tests for the co-existence of enkephalin-like peptide and glutamate decarboxylase-like immunoreactivity were also made with the peroxidase-antiperoxidase method. Co-expression of substance P-like and dynorphin B-like immunoreactivities was the rule both in striosomes and in the matrix. In striosomes, substance P-like immunoreactivity was found in 96% of dynorphin B-immunoreactive neurons, and in the matrix 89% of dynorphin B-positive cells contained substance P-like immunoreactivity. Substance P/dynorphin B-positive neurons corresponded to over half (57%) of the neurons in striosomes but only 39% of the neurons in the matrix. Both in the matrix and in striosomes, about two-thirds of all neurons (63% and 65%, respectively) were identified as enkephalin-positive. Among all substance P/dynorphin B-positive medium-sized neurons, 76% also contained enkephalin-like antigen. The enkephalin-positive neurons characterized by triple peptide co-existence (enkephalin/substance P/dynorphin B) represented a mean of 63% of striosomal enkephalin-positive neurons (41% of all striosomal neurons) and 35% of matrical enkephalin-positive neurons (26% of all matrical neurons). Finally, nearly all enkephalin-positive neurons were immunoreactive for glutamate decarboxylase, and therefore probably GABAergic, but only about half the glutamate decarboxylase-positive population was enkephalin-immunoreactive. These findings suggest that neuropeptides from three distinct precursors may be co-localized in single medium-sized neurons in the striatum, and that the differential patterns of co-expression of substance P-like, dynorphin B-like, and enkephalin-like peptides may confer functional specializations upon subpopulations of GABAergic neurons giving rise to the efferent projections of the striatum. The linked expression of substance P-like and dynorphin B-like peptides in single neurons both in striosomes and matrix suggests that some regulatory mechanisms controlling peptide expression apply regardless of compartment.(ABSTRACT TRUNCATED AT 400 WORDS)