Preprotachykinin mRNA Research Articles

(601) Preprotachykinin (PPT) mRNA and Substance P Receptor (NK1) mRNA Are Co-Localized in Rat Dorsal Horn Neurons

(601) Preprotachykinin (PPT) mRNA and Substance P Receptor (NK1) mRNA Are Co-Localized in Rat Dorsal Horn Neurons

Cyclic AMP regulates substance P expression in developing and mature spinal sensory neurons.

The tachykinin, substance P, has long been associated with transmission of noxious stimuli. However, relatively little is known about signal transduction pathways subserving peptidergic regulation in sensory neurons. To investigate whether cyclic AMP (cAMP) could be a potential second messenger subserving substance P expression, dorsal root ganglion neurons were grown in culture in the presence of agents that increase content of cAMP. In developing neurons, forskolin increased substance P content and survival almost threefold. Anti-nerve growth factor (NGF) blocked the effect of NGF but not forskolin, suggesting that increased cAMP acts directly and not via increased secretion of NGF from Schwann cells and fibroblasts. In adult neurons, which do not require supplemental trophic factors for survival, NGF and forskolin had similar effects on substance P levels. Neither agent had any effect on somatostatin content of either developing or mature sensory neurons. 8-bromo cAMP and isobutyl methylxanthine duplicated the action of forskolin. Further, all three agents increased expression of preprotachykinin mRNA. Forskolin appeared to increase both total and neuron-specific expression of message as well as the number of neurons expressing mRNA. Our results suggest that cAMP directly regulates substance P content in sensory neurons from adult and neonatal rats.

Alterations in preproenkephalin and adenosine-2a receptor mRNA, but not preprotachykinin mRNA correlate with occurrence of dyskinesia in normal monkeys chronically treated with L-DOPA.

Chronic treatment with L-DOPA induces dyskinesia in patients with Parkinson's disease (PD) and 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP)-treated monkeys, but is not thought to do so in normal humans or primates. However, we have shown that chronic oral high dose L-DOPA administration, with the peripheral decarboxylase inhibitor, carbidopa and with or without the peripherally acting catechol-O-methyl transferase (COMT) inhibitor, entacapone, to normal macaque monkeys for 13 weeks induced dyskinesia in a proportion of animals. In the present study, in situ hybridization histochemistry was used to investigate the effect of chronic L-DOPA administration on the activity of the direct and indirect striatal output pathways by measuring striatal preprotachykinin (PPT), preproenkephalin-A (PPE-A) and adenosine-2a (A2a) receptor gene expression in these monkeys. Overall there was no significant difference in striatal PPT, PPE-A and A2a receptor mRNA levels between normal animals and all L-DOPA (plus carbidopa and/or entacapone)-treated animals irrespective of whether or not dyskinesia occurred. However, when the level of PPE-A and A2a receptor mRNA was analysed in eight monkeys displaying marked dyskinesias as a result of L-DOPA (plus carbidopa with or without entacapone) treatment, there was a significant increase in PPE-A and A2a receptor mRNA message levels in the striatum compared with animals receiving identical treatment, but displaying few or no involuntary movements, and compared with normal controls. There was no difference in striatal PPT mRNA levels in monkeys exhibiting severe dyskinesia compared with those showing little or no dyskinesia after L-DOPA treatment or to normal controls. These results suggest that prolonged L-DOPA treatment alone has no consistent effect on either the direct or indirect pathways, as judged by striatal PPT, PPE-A or A2a receptor mRNA levels in normal monkeys. However, in monkeys exhibiting marked dyskinesia resulting from chronic L-DOPA treatment, abnormal activity is detected in the indirect striato-pallidal output pathway, as judged by striatal PPE-A and A2a receptor mRNA levels, indicating an imbalance between the direct and indirect striatal pathway which may explain the emergence of dyskinesia in these animals.

Serotonin 2A and 2C receptor biosynthesis in the rodent striatum during postnatal development: mRNA expression and functional linkage to neuropeptide gene regulation.

The present study was designed to determine if there are region-specific differences in serotonin (5-HT) neurotransmission and 5-HT receptor expression that may limit the stimulatory effects of the 5-HT releaser p-chloroamphetamine (pCA) on striatal neuropeptide gene expression to the posterior striatum (P-STR) during postnatal maturation. Sprague-Dawley rat brains from postnatal days (PND) 1-35 were processed for 5-HT(2A) and 5-HT(2C) receptor mRNA expression by in situ hybridization and monoamine analysis by HPLC. Within the P-STR, 5-HT(2A) receptor mRNA expression reached young adult (PND 35) levels by PND 3, while levels in the A-STR were significantly less (range: 1.43 +/- 0.219-6. 36 +/- 0.478) than P-STR (5.36 +/- 0.854-12.11 +/- 1.08) at each respective age throughout the time course. 5-HT(2C) receptor mRNA expression reached young adult levels at PND 7 in the A-STR and by PND 3 in the P-STR. At each PND age 5-HT(2C) receptor mRNA levels within the P-STR were significantly less (6.23 +/- 1.02-12.32 +/- 0.427) than the A-STR (7.31 +/- 1.65-26.84 +/- 2.24). 5-HT content increased across the developmental time course within the P-STR (5.01 +/- 0.327-15.7 +/- 1.03 ng/mg protein) and A-STR (2.97 +/- 0. 223-11.2 +/- 0.701 ng/mg protein). Four hours following injection (i. p.) of pCA (10 mg/kg), preprotachykinin (PPT) mRNA levels increased 89% in the P-STR but not the anterior (A-STR) striatum of the 3-week-old rat, which were prevented by preinjection (30 min, i.p.) of the 5-HT(2) receptor antagonist ritanserin (1 mg/kg). Together, these data suggest that faster maturity of 5-HT(2A) receptor expression in the P-STR may be sufficient to convey the region-specific acute stimulatory effects of pCA on PPT mRNA transcription in the developing rodent striatum. These results provide further evidence that the influence of 5-HT on neuropeptide gene expression is far stronger in caudal vs. rostral striatal regions during postnatal development.

Quantification of substance P mRNA in human mononuclear phagocytes and lymphocytes using a mimic-based RT-PCR

We have recently demonstrated that human monocytes and lymphocytes express the substance P (SP) gene at both the mRNA and protein level [Ho, W.Z., Lai, J.P., Zhu, X.H., Uvaydova, M., Douglas S.D., 1997. Human monocytes and macrophages express substance P and neurokinin-1 receptor. Journal of Immunology, 159, p. 5654; Lai, J.P., Douglas, S.D., Ho, W.Z., 1998. Human lymphocytes express substance P and its receptor. Journal of Neuroimmunology, 86, p. 80; Lai, J.-P., Douglas, S.D., Rappaport, E., Wu, J., Ho, W.-Z., 1998. Identification of a delta isoform of preprotachykinin mRNA in human mononuclear phagocytes and lymphocytes. Journal of Neuroimmunology, 91, p. 121]. Using RT-PCR assay with several specific human SP primer pairs, we were able to differentiate four isoforms of preprotachykinin (PPT-A, the SP precursor) mRNA transcripts on ethidium bromide-stained agarose gels and clone the PCR amplified cDNA of the four isoforms (α, β, γ, and δ) of the PPT-A gene. In an effort to quantitatively measure PPT-A mRNA levels, we have developed a mimic-based RT-PCR assay to analyze total PPT-A mRNA levels in human monocytes and lymphocytes. We designed a specific human SP primer pair (HSP4/HSP3) to amplify a single fragment of cDNA derived from all four isoforms of PPT-A mRNA transcripts, with a sensitivity of 120 molecules per reaction. Thus the PPT-A mRNA transcripts in an unknown sample can be quantitatively analyzed using the mimic-based RT-PCR. The accuracy and reproducibility of this assay were confirmed by the plasmids containing α, β, γ and δ cDNA inserts and by in vitro synthesized mRNA from a plasmid containing β isoform cDNA insert. Our data indicate that the SP mimic-based RT-PCR assay has potential advantages in studies of SP levels in a variety of human cells as well as in clinical specimens.

Synergistic interaction between serotonin-2 receptor and dopamine D1 receptor stimulation on striatal preprotachykinin mRNA expression in the 6-hydroxydopamine lesioned rat

The regulation of striatal preprotachykinin (PPT) mRNA expression can be mediated through both dopamine (DA) D 1 and serotonin (5-HT) 5-HT 2A/2C receptors. In the present study, we used in situ hybridization to examine possible synergistic interactions between 5-HT 2A/2C and D 1 receptor-mediated regulation of striatal PPT mRNA levels in the rat depleted of DA with 6-hydroxydopamine. Acute administration of the 5-HT 2A/2C receptor agonist DOI (2 mg/kg) significantly increased (+75%) PPT mRNA levels in the dorsal striatum. Acute administration of the D 1 receptor agonist SKF-38393 (2 mg/kg) did not significantly alter PPT mRNA levels in the dorsal striatum. However, the co-administration of SKF-38393 and DOI produced a significant increase (+300%) in striatal PPT mRNA expression restricted to the periventricular region of the dorsal–medial striatum. This synergistic interaction was not observed in the remaining aspect of the dorsal striatum where DOI alone increased PPT mRNA expression. These data show that 5-HT 2A/2C and D 1 receptors can act in a synergistic manner to regulate striatal PPT mRNA in a subregion of the DA-depleted striatum.

Acute p-chloroamphetamine increases striatal preprotachykinin mRNA: role of the serotonin 2A/2C receptor

Acute administration of p-chloroamphetamine ( pCA) significantly increased (+90%) preprotachykinin (PPT) mRNA levels in the rat striatum. Administration of the serotonin 2A/2C receptor antagonist, ritanserin, blocked the pCA-induced increase in PPT mRNA levels. α-Methyl- p-tyrosine pretreatment (α-MT, to reduce dopamine transmission) inhibited the pCA-induced increase in PPT mRNA levels. These results indicate that the pCA-induced increase in striatal PPT mRNA expression is mediated by serotonin 2A/2C receptors but also requires dopamine tone.

Differential regulation of striatal preproenkephalin and preprotachykinin mRNA levels in MPTP-lesioned monkeys chronically treated with dopamine D1 or D2 receptor agonists.

Studies in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned monkeys and in parkinsonian patients show elevated preproenkephalin (PPE) mRNA levels, unaltered by chronic L-DOPA therapy, whereas preprotachykinin (PPT) mRNA levels are decreased by the lesion and corrected by L-DOPA. The relative contributions of the dopamine D1 and D2 receptors for PPE mRNA regulation were investigated in the present study and compared with those for PPT mRNA. In situ hybridization was used to measure peptide mRNA levels in the striatum of MPTP cynomolgus monkeys after chronic 1-month treatment with the D1 agonist SKF-82958, administered subcutaneously in pulsatile or continuous mode, compared with the long-acting D2 agonist cabergoline. Normal as well as untreated MPTP animals were also studied. PPE mRNA levels were elevated in the caudate nucleus and putamen of untreated MPTP monkeys compared with control animals with a more pronounced increase in the lateral as compared with the medial part of both structures. PPT mRNA levels showed a rostrocaudal gradient, with higher values in the middle of the caudate-putamen and more so in the medial versus the lateral parts. PPT mRNA levels were decreased in the caudate and putamen of untreated MPTP monkeys compared with control animals, and this was observed in the middle and posterior parts of these brain areas. Elevated PPE and decreased PPT mRNA levels observed after MPTP exposure were corrected after treatment with cabergoline (0.25 mg/kg, every other day), a dose that had antiparkinsonian effects and did not give sustained dyskinesia. In contrast, elevated PPE mRNA levels observed in untreated MPTP monkeys were markedly increased by pulsatile administration of SKF-82958 (1 mg/kg, three times daily) in two monkeys in which the parkinsonian symptoms were improved and dyskinesias developed, whereas it remained close to control values in a third one that did not display dyskinesias despite a sustained improvement in disability; a shorter duration of motor benefit (wearing off) over time was observed in these three animals. By contrast, pulsatile administration of SKF-82958 corrected the decreased PPT level observed in untreated MPTP monkeys. Continuous treatment with SKF-82958 (equivalent daily dose) produced no clear antiparkinsonian and dyskinetic responses and did not alter the denervation-induced elevation of PPE or decrease of PPT mRNA levels. The present data suggest an opposite contribution of the dopamine D1 receptors (stimulatory) as compared with the dopamine D2 receptors (inhibitory) on PPE mRNA, whereas a similar stimulatory contribution of D1 or D2 receptors is observed for PPT mRNA. An increase in PPE expression could be involved in the induction of dyskinesias and wearing off, whereas our data do not support this link for PPT. The antiparkinsonian response was associated with a correction of the lesion-induced decrease of PPT.

Effects of systemic resiniferatoxin treatment on substance P mRNA in rat dorsal root ganglia and substance P receptor mRNA in the spinal dorsal horn

Capsaicin depletes the sensory neuropeptide substance P (SP) in the rat due to a combination of neuron loss and decreased synthesis in the surviving cells. Resiniferatoxin (RTX) mimics most, but not all, capsaicin actions. In the present study, the effects of RTX (300 μg/kg, s.c.) were examined on mRNA levels for SP and its receptor in the adult rat. The percentage of dorsal root ganglia (DRG) neuronal profiles showing an in situ hybridization signal for preprotachykinin mRNAs encoding SP was not altered following RTX treatment (up to 8 weeks), though the signal became perceptibly weaker. In accord, 2 weeks after RTX administration a 60% decrease was observed in the steady-state levels of SP-encoding mRNAs using Northern blot analysis, leaving the ratio of β- and γ-preprotachykinin mRNAs unchanged. No change was, however, observed in mRNA levels encoding tachykinins NK-1 receptors in the dorsal horn, the spinal targets for SP. The present findings suggest that RTX does not kill SP-positive DRG neurons, though it suppresses the synthesis of SP. Since RTX treatment does not alter NK-1 receptor expression, this reduced SP synthesis is likely to play a central role in the analgesic actions of RTX.

Identification of a δ isoform of preprotachykinin mRNA in human mononuclear phagocytes and lymphocytes

We have characterized preprotachykinin (PPT-A) gene transcript splicing products and identified a fourth isoform of PPT-A mRNA transcript in human peripheral blood-isolated monocytes and PBL. Using RT-PCR, Southern blot analysis and nucleotide sequencing analysis, we have identified the four isoforms of PPT-A transcripts (α, β, γ and δ) in human peripheral blood-isolated monocytes and PBL. The δ-PPT transcript present in the immune cells lacks exons 4 and 6. The sequences of exons 3, 5 and 7 of δ-PPT transcript completely match those of β-PPT transcript. The α-PPT and β-PPT sequences in these cells are identical to those obtained by Tan and Too (GenBank accession number U37539) and Harmar et al. (Genbank accession number X54469), but differ by a single nucleotide from another entry by Chiwakata et al. (Genbank accession number M68906). In comparison to this latter sequence, there was a C→T change at amino acid position 87 (CCT→CTT) which may result in a Pro to Leu change. Identification of the new SP mRNA transcript in both human CNS and immune cells supports the concept of an important biological link between CNS and immune system.

Stimulation of nerve growth factor and substance P expression in the iris–trigeminal axis of diabetic rats—involvement of oxidative stress and effects of aldose reductase inhibition

In rats with streptozotocin-induced diabetes, we measured increased (by 61%; P<0.05) mRNA for nerve growth factor (NGF) in the iris together with increased (by 82%; P<0.05) mRNA for preprotachykinin (the substance P precursor) in the trigeminal ganglion, suggesting that increased NGF was driving increased substance P gene expression. In other diabetic rats, these changes were prevented by treatment with either an antioxidant (butylated hydroxytoluene; 1% by diet) or an aldose reductase inhibitor (ARI) (sorbinil; 25 mg/kg/day p.o.) and the sorbinil treatment was associated with significant inhibition of polyol pathway intermediates in both lens and sciatic nerve. This suggests that polyol pathway activity in the lens may translate to oxidative stress-driving stimulation of NGF gene expression in the iris. The change is selective for NGF, because expression of the analogous neurotrophin, neurotrophin-3 (NT-3), was unaltered in the same irises. These changes suggest that oxidative stress and/or inflammation can drive up NGF expression in diabetes—a mechanism that might participate in iritis.

L‐DOPA‐induced dyskinesia in the rat is associated with striatal overexpression of prodynorphin‐ and glutamic acid decarboxylase mRNA

Rats sustaining unilateral near-complete 6-hydroxydopamine lesions of the mesostriatal dopamine pathway received daily injections of 3, 4 dihydroxyphenyl-l-alanine (L-DOPA, 8 mg/kg plus 15 mg/kg benserazide) for 3 weeks. During this period, about 50% of the rats gradually developed abnormal involuntary movements, lasting for 2-3 h following each L-DOPA dose. Rats were killed 3 days after the last L-DOPA injection, and sections through the striatum were processed for in situ hybridization histochemistry. Within the L-DOPA-treated group, levels of preproenkephalin (PPE) mRNA, glutamic acid decarboxylase (GAD67) mRNA, and prodynorphin (PDyn) mRNA in the dopamine-denervated caudate-putamen, as well as GAD67 mRNA expression in the globus pallidus ipsilateral to the 6-hydroxydopamine (6-OHDA) lesion, were higher in dyskinetic than non-dyskinetic animals, and positively correlated with the rats' dyskinesia scores. By contrast, striatal preprotachykinin mRNA expression and D2 receptor-radioligand binding were not significantly associated with dyskinesia. Among all these markers, PDyn mRNA levels showed the most pronounced treatment-dependence (three times higher in the L-DOPA-treated group than in saline-injected lesion-only controls), and the strongest correlation with the rats' dyskinesia scores (r2 = 0.82). However, a multiple regression equation including the three factors, GAD67 mRNA levels in the GP, GAD67 mRNA in the lateral CPu, and striatal PDyn mRNA, gave a better fit for dyskinesia scores than PDyn mRNA alone (r2 = 0.92). The results show that L-DOPA-induced dyskinesia is associated with overexpression of PDyn and GAD67 mRNA in the striatal projection neurons, and GAD67 mRNA levels in the globus pallidus. Due to its treatment-dependent expression, and strong correlation with the associated dyskinetic symptoms, striatal PDyn mRNA, in particular, may play a role in the mechanisms of behavioural sensitization brought about by the drug.

L-DOPA-induced dyskinesia in the rat is associated with striatal overexpression of prodynorphin- and glutamic acid decarboxylase mRNA

Rats sustaining unilateral near-complete 6-hydroxydopamine lesions of the mesostriatal dopamine pathway received daily injections of 3, 4 dihydroxyphenyl-l-alanine (L-DOPA, 8 mg/kg plus 15 mg/kg benserazide) for 3 weeks. During this period, about 50% of the rats gradually developed abnormal involuntary movements, lasting for 2-3 h following each L-DOPA dose. Rats were killed 3 days after the last L-DOPA injection, and sections through the striatum were processed for in situ hybridization histochemistry. Within the L-DOPA-treated group, levels of preproenkephalin (PPE) mRNA, glutamic acid decarboxylase (GAD67) mRNA, and prodynorphin (PDyn) mRNA in the dopamine-denervated caudate-putamen, as well as GAD67 mRNA expression in the globus pallidus ipsilateral to the 6-hydroxydopamine (6-OHDA) lesion, were higher in dyskinetic than non-dyskinetic animals, and positively correlated with the rats' dyskinesia scores. By contrast, striatal preprotachykinin mRNA expression and D2 receptor-radioligand binding were not significantly associated with dyskinesia. Among all these markers, PDyn mRNA levels showed the most pronounced treatment-dependence (three times higher in the L-DOPA-treated group than in saline-injected lesion-only controls), and the strongest correlation with the rats' dyskinesia scores (r2 = 0.82). However, a multiple regression equation including the three factors, GAD67 mRNA levels in the GP, GAD67 mRNA in the lateral CPu, and striatal PDyn mRNA, gave a better fit for dyskinesia scores than PDyn mRNA alone (r2 = 0.92). The results show that L-DOPA-induced dyskinesia is associated with overexpression of PDyn and GAD67 mRNA in the striatal projection neurons, and GAD67 mRNA levels in the globus pallidus. Due to its treatment-dependent expression, and strong correlation with the associated dyskinetic symptoms, striatal PDyn mRNA, in particular, may play a role in the mechanisms of behavioural sensitization brought about by the drug.

Increase of preprotachykinin mRNA and substance P immunoreactivity in spared dorsal root ganglion neurons following partial sciatic nerve injury.

Complete sciatic nerve injury reduces substance P (SP) expression in primary sensory neurons of the L4 and L5 dorsal root ganglia (DRG), due to loss of target-derived nerve growth factor (NGF). Partial nerve injury spares a proportion of DRG neurons, whose axons lie in the partially degenerating nerve, and are exposed to elevated NGF levels from Schwann and other endoneurial cells involved in Wallerian degeneration. To test the hypothesis that SP is elevated in spared DRG neurons following partial nerve injury, we compared the effects of complete sciatic nerve transection (CSNT) with those of two types of partial injury, partial sciatic nerve transection (PSNT) and chronic constriction injury (CCI). As expected, a CSNT profoundly decreased SP expression at 4 and 14 days postinjury, but after PSNT and CCI the levels of preprotachykinin (PPT) mRNA, assessed by in situ hybridization, and the SP immunoreactivity (SP-IR) of the L4 and L5 DRGs did not decrease, nor did dorsal horn SP-IR decrease. Using retrograde labelling with fluorogold to identify spared DRG neurons, we found that the proportion of these neurons expressing SP-IR 14 days after injury was much higher than in neurons of normal DRGs. Further, the highest levels of SP-IR in individual neurons were detected in ipsilateral L4 and L5 DRG neurons after PSNT and CCI. We conclude that partial sciatic nerve injury elevates SP levels in spared DRG neurons. This phenomenon might be involved in the development of neuropathic pain, which commonly follows partial nerve injury.

Thalamo-striatal deafferentation affects preproenkephalin but not preprotachykinin gene expression in the rat striatum

This study examined the effects of thalamo-striatal deafferentation on preprotachykinin and preproenkephalin mRNA expression in the rat neostriatum, using quantitative in situ hybridization histochemistry. Unilateral ibotenate-induced intralaminar thalamic lesion produced a significant decrease in preproenkephalin mRNA levels (−27%) restricted to the ipsilateral striatum at 5 days post-lesion. At 12 days post-lesion, significant decreases in striatal preproenkephalin mRNA expression were found on both brain sides. This post-lesional response was more pronounced in the ipsilateral (−32%) than contralateral (−18%) striatum. All these changes were homogeneously distributed between the dorsolateral and ventromedial parts of the striatum. In parallel, no significant change in preprotachykinin mRNA expression was found at either 5 or 12 days after thalamic lesion, when considering the striatum as a whole. However, at 5 days post-lesion, the regional analysis revealed a slight decrease (−17%) in preprotachykinin mRNA expression, confined to the dorsolateral part of the ipsilateral striatum. These results show that thalamic lesion preferentially affects preproenkephalin vs. preprotachykinin gene expression in the striatum, suggesting, at the first site, a predominant influence of thalamo-striatal inputs on the enkephalin-containing striato-pallidal pathway. However, given that the thalamo-striatal projection is strictly ipsilateral, the bilateralization of the down-regulation of preproenkephalin mRNA expression at 12 days post-lesion suggests an involvement of interhemispheric adaptive mechanisms via cortical networks.

The effect of aging on the response of striatal preproenkephalin and preprotachykinin mRNA contents to chronic haloperidol treatment in rats: measurement by solution-hybridization RNase protection assay

The preproenkephalin (PPeK) and preprotachykinin (PPT) mRNA contents in 3-, 10- and 23-month-old rats in the striatum were measured by solution hybridization-RNase protection assay after 3 weeks of haloperidol injection. Haloperidol increased striatal PPek mRNA. There was no age-related difference in the response of striatal PPeK mRNA to chronic haloperidol treatment. The PPT mRNA decreased by 21% after the haloperidol treatment in young rats only. Meanwhile, age decreased the PPT mRNA by 27 and 24% in 10- and 23-month-old rats, respectively. It is concluded that there is a difference in the effects of aging on the response of PPek and PPT mRNA contents to haloperidol and that the loss of PPT mRNA response in 10- and 23-month-old rats might be due to the change of dopamine system of the striatum in these rats.

Effects of risperidone and haloperidol on tachykinin and opioid precursor peptide mRNA levels in the caudate-putamen and nucleus accumbens of the rat.

We investigated whether the two output pathways of the striatum are differently affected by the novel atypical drug risperidone and the conventional typical antipsychotic drug haloperidol. To this end, changes in mRNA levels of preproenkephalin-A, preproenkephalin-B, and preprotachykinin were determined in the rat striatum following chronic drug treatment for 14 days, using quantitative in situ hybridization. Furthermore, we studied the contribution of the dopamine D2 and serotonin 5-HT2A antagonist components of risperidone in establishing its effects on neuropeptide mRNA levels in the striatum. The results showed that both risperidone and haloperidol had major effects on the preproenkephalin-A mRNA and thus on the indirect striatal output route, whereas they had minor effects on preproenkephalin-B and preprotachykinin mRNA, contained by the direct output route. When both drugs were administered in the same dose, preproenkephalin-A mRNA was much more elevated by haloperidol than by risperidone. However, when doses of risperidone and haloperidol were modified to attain comparable dopamine D2 receptor occupancy, the drugs had comparable effects on preproenkephalin-A mRNA levels. It was further found that 5-HT2A/C receptor blockade with ritanserin had only modest effects on preproenkephalin-B and preprotachykinin mRNA levels and did not affect preproenkephalin-A mRNA levels. We conclude that risperidone and haloperidol, administered in the same dose, differently affect the striatal output routes. Furthermore, the results suggest that the effects of risperidone on neuropeptide mRNA levels are fully accounted for by its D2 antagonism and that no indication exists for a role of 5-HT2A receptor blockade in this action.

Simultaneous quantitation of substance P-encoding preprotachykinin alternatively spliced mRNAs and substance P receptor NK-1 mRNA by an RNase protection assay

Tachykinins form a family of peptides with neurotransmitter/neuromodulator function. Four tachykinins, substance P, neurokinin A, neuropeptide γ and neuropeptide K, are encoded by the same PreProTachykinin (PPT) gene. Alternatively spliced mRNAs encode different combinations of these peptides (Brown, E.R., Harlan, R.E., Krause, J.E., Gonadal steroid regulation of substance P (SP) and SP-encoding mRNA in the rat anterior pituitary and hypothalamus, Endocrinology, 126 (1990) 330–340 [1]; Krause, J.E., Chirgwin, J.M., Carter, M.S., Xu, Z.S., Hershey, A.D., Three rat preprotachykinin mRNAs encode the neuropeptides substance P and neurokinin A, Proc. Natl. Acad. Sci. USA, 84 (1987) 881–885 [10]). The proportion of PPT mRNAs varies from tissue to tissue (Carter, M.S., Krause, J.E., Structure, expression, and some regulatory mechanisms of the rat preprotachykinin gene encoding substance P, neurokinin A, neuropeptide K, and neuropeptide γ, J. Neurosci., 10 (1990) 2203–2214 [2]), and within the rat hypothalamus according to the estrous cycle-related hormonal status (Gautreau, A., Duval, P., Kerdelhué, B., Variations in substance P-encoding preprotachykinin and substance P receptor NK-1 mRNA transcripts in the rat hypothalamus throughout the estrous cycle: a correlation between amounts of β-preprotachykinin and NK-1 mRNA, Mol. Brain Res., (1997) in press [7]). Tachykinin receptors as well as tachykinins are regulated at the mRNA level. A fully quantitative method is needed to deal with the complex physiological regulation of the tachykinin system. Here, we describe an RNase protection assay that allows the simultaneous quantitation of alternatively spliced PPT mRNAs, Substance P receptor NK-1 mRNA, and glyceraldehyde-3-phosphodehydrogenase (GAPDH) mRNA as an internal control, in the rat hypothalamus. The advantages of this method are its high sensitivity (0.1 pg) and a wide range of linearity (more than 3 orders of magnitude). Moreover, this protocol provides guidelines to set up a quantitative multiprobe RNase protection assay for other genes.

Effect of capsaicin on substance P and nerve growth factor in adjuvant arthritic rats

We have investigated the effect of capsaicin pretreatment (50 mg kg −1 s.c.) on substance P, preprotachykinin (PPT) mRNA, and nerve growth factor (NGF), plus its high-affinity receptor, trkA, in adult rats with adjuvant arthritis. Twenty one days after induction of adjuvant arthritis, sciatic nerve levels of substance P were significantly increased whilst there was a small but non-significant increase in γ-PPT mRNA and substance P in L 4/L 5 dorsal root ganglia (DRG). NGF levels in sciatic nerve and foot skin as well as DRG trkA mRNA were unaltered after 21 days arthritis suggesting that NGF may not play a role in chronic inflammation. Capsaicin treatment of naive rats significantly reduced substance P in all tissues and NGF levels in the sciatic nerve. In contrast, γ-PPT mRNA and trkA mRNA expression in DRG were significantly increased after capsaicin treatment. The nervous and skin tissues used in this study were harvested from the same rats in which we had previously shown that capsaicin pretreatment significantly attenuated the severity of arthritis (Cruwys, S.C., Garrett, N.E. and Kidd, B.L., Sensory denervation with capsaicin attenuates inflammation and nociception in arthritic rats, Neurosci. Lett., 193 (1995) 205–207). Arthritis in capsaicin-treated rats had no effect on substance P or NGF levels in any tissue when compared with capsaicin-treated control rats, suggesting that pharmacological impairment of the sensory nervous system can reduce the severity of inflammatory joint disease.

Effects of deoxycorticosterone acetate and diazepam on neuropeptidergic neurons in rat striatum

The neurosteroid tetrahydrodeoxycorticosterone (THDOC) interacts with gamma-aminobutyric acid (GABA)/ benzodiazepine (BZ) receptors. To test the hypothesis that THDOC works partially through mechanisms associated with GABAA/BZ receptor function, deoxycorticosterone acetate (DOCA) and the benzodiazepine, diazepam (DZ), were administered short- (1 day) and long-term (11 days). Levels of mRNA for dynorphin, preprotachykinin and preproenkephalin in the striatum of adult male Sprague-Dawley rats were measured by in situ hybridization. Acute DOCA and DZ treatment produced parallel neuropeptide mRNA profiles, whereas chronic DOCA and DZ treatment yielded different patterns of neuropeptide gene expression. Chronic DZ treatment resulted in no significant increase in salt intake whereas chronic DOCA activated salt appetite. We suggest that acute DZ and DOCA interact with GABAA/BZ receptors; however, the results of chronic treatment suggest that DZ and DOCA operate through dissimilar mechanisms.