PPT-A mRNA Expression Research Articles

Strain differences in the development of cisplatin-induced pica behavior in mice

IntroductionPica behavior, kaolin ingestion, in rats and mice can be used as an assessment of nausea and vomiting; however, we observed that the incidence of pica behavior in ICR strain mice varied markedly. We investigated the susceptibility of four strains of mice (ICR, BALB/c, C57BL/6, and DBA/2) to the development of pica behavior. MethodsMice received cisplatin (7.5 mg/kg, i.p.) with or without a serotonin 5-HT3 receptor antagonist (granisetron: 0.1 mg/kg, i.p.) or tachykinin NK1 receptor antagonist (fosaprepitant: 30 mg/kg, i.p.), and then their daily kaolin intake was measured for 2 days. We examined the expression of preprotachykinin (PPT)-A mRNA in the medulla of cisplatin-treated mice 8 and 32 h after drug administration. ResultsAll mice except for ICR strain significantly increased kaolin intake after cisplatin administration. Among the tested strains, DBA/2 mice compared to BALB/c and C57BL/6 mice notably showed pica behavior on both days (P < 0.0001). The expression of PPT-A mRNA was significantly increased 8 h after cisplatin administration in all strains, but the increase remained on the second day only in DBA/2 mice (P < 0.05). Granisetron significantly inhibited pica behavior in DBA/2 mice on the first day (P < 0.0001), but not the second day; however, fosaprepitant completely inhibited the pica behavior on both days (P < 0.001). DiscussionThese results indicate that cisplatin-induced pica behavior in mice is likely to be influenced by the genotype, and that DBA/2 mice are useful to analyze the emetogenic or anti-emetic potential of drugs in preclinical studies.

Up-Regulation of the Biosynthesis and Release of Substance P through Wnt/β-Catenin Signaling Pathway in Rat Dorsal Root Ganglion Cells.

To examine regulatory effects of β-catenin on the biosynthesis and release of substance P, a rat chronic constriction injury (CCI) model and a rat dorsal root ganglion (DRG) cell culture model were used in the present study. The CCI treatment significantly induced the overall expression of β-catenin (158 ± 6% of sham) in the ipsilateral L5 DRGs in comparison with the sham group (109 ± 4% of sham). The CCI-induced aberrant expression of β-catenin was significantly attenuated by oral administration of diclofenac (119 ± 6% of the sham value; 10 mg/kg). Importantly, aberrant nuclear accumulation of β-catenin in cultured DRG cells resulted in up-regulation of the PPT-A mRNA expression and the substance P release. The up-regulation of both the PPT-A mRNA expression and the substance P release by either a GSK-3β inhibitor TWS119 (10 μM) or a Wnt signaling agonist Wnt-3a (100 ng/ml) were significantly abolished by an inhibitor of cyclooxygenase-2 (COX-2; NS-398, 1 μM). Collectively, these data suggest that nociceptive input-activated β-catenin signaling plays an important role in regulating the biosynthesis and release of substance P, which may contribute to the inflammation responses related to chronic pain.

Involvement of substance P in the development of cisplatin‐induced acute and delayed pica in rats

Although substance P (SP) and neurokinin NK1 receptors have been reported to be involved in cisplatin-induced acute and delayed emesis, their precise roles remain unclear. Pica, the consumption of non-nutrient materials such as kaolin in rats, can be used as a model of nausea in humans. We investigated the time-dependent changes in cisplatin-induced pica and the involvement of SP and NK1 receptors in this behaviour. Rats were administered cisplatin with or without a daily injection of a 5-HT3 receptor antagonist (granisetron) or an NK1 receptor antagonist (aprepitant), and kaolin intake was then monitored for 5 days. The effects of granisetron on the cisplatin-induced expression of preprotachykinin-A (PPT-A) mRNA, which encodes mainly for SP, and on SP release in the medulla, measured by in vivo brain microdialysis, were also investigated. Cisplatin induced pica within 8 h of its administration that continued for 5 days. Granisetron inhibited the acute phase (day 1), but not the delayed phase (days 2-5), of pica, whereas aprepitant abolished both phases. Within 24 h of the injection of cisplatin, PPT-A mRNA expression and SP release in the medulla were significantly increased; these findings lasted during the observation period and were inhibited by granisetron for up to 24 h. The profiles of cisplatin-induced pica in rats are similar to clinical findings for cisplatin-induced emesis in humans, and we showed that SP production in the medulla and activation of NK1 receptors are involved in this cisplatin-induced pica.

Nitric Oxide-NGF Mediated PPTA/SP, ADNP, and VIP Expression in the Peripheral Nervous System

Nerve growth factor (NGF)-deprivation or axotomy of dorsal root ganglion (DRG) neurons causes stress, which they cope by triggering various mechanisms. Among several molecular changes, in the present study, we demonstrate preprotachykinin-A-substance P (PPTA-SP) and activity-dependent neuroprotective protein-vasoactive intestinal peptide (ADNP-VIP) expression pattern using DRG neurons-Schwann cells coculture and axotomy model. In the presence of NGF, DRG cultures showed high levels of PPTA and ADNP mRNA expression, which were significantly suppressed in the absence of NGF and/or nitric oxide synthase (NOS) inhibition by NG-nitro-L-arginine methyl ester (L-NAME), suggesting that both NGF and nitric oxide (NO) can regulate PPTA and ADNP expression. However, treating coculture with NO donor, diethylenetriamine nitric oxide (DETA-NO) did not increase PPTA and ADNP expression in the presence or absence of NGF, although there was a marginal increase in ADNP expression in the absence of NGF. NGF-deprivation increases endogenous NO; thus, DETA-NO had no further effect on PPTA and ADNP expression. Alternatively, NGF produced from NO-stimulated Schwann cells influence gene expression. In addition, interestingly, DETA-NO treatment of Schwann cells alone suppresses both PPTA and ADNP, suggesting differential response of DRG neurons-Schwann cells coculture to DETA-NO. SP and ADNP immunostaining of axotomized DRGs revealed significant reduction in SP and ADNP compared to intact DRG, which was partially recovered in neuronal NOS blocker, 7-nitroindazole (7-NI)-treated DRGs, particularly intense ADNP staining in satellite glia. As ADNP is VIP-responsive gene, we further explored VIP expression in DRGs. Axotomy increased VIP in DRG neurons, but 7-NI treatment caused intense VIP staining in satellite glia. These observations suggest a complex interaction of NO-NGF with PPTA/SP and ADNP-VIP in neuron-glial communication when neurons are stressed.

Increase in substance P precursor mRNA in noninflamed small-bowel sections in patients with Crohn’s disease

Background Neuropeptides, such as substance P (SP), are mediators of neurogenic inflammation and play an important role in inflammatory disorders. To further investigate the role of the SP pathway in inflammatory bowel disease (IBD), we analyzed the following in normal intestinal tissue specimens and in tissue specimens from patients with Crohn’s disease (CD) and ulcerative colitis (UC): neurokinin receptor-1 (NK-1R); its isoforms (NK-1R-L and NK-1R-S); its ligand SP, encoded by preprotachykinin-A (PPT-A); and the SP-degradation enzyme, neutral endopeptidase (NEP). Methods Real-time quantitative reverse transcription–polymerase chain reaction was used to simultaneously determine the expression of NK-1R-L, NK-1R-S, and PPT-A. Protein levels of NK-1R and NEP were determined by immunoblot analysis. Results In noninflamed small-bowel tissue samples of CD patients, PPT-A mRNA expression was significantly increased, whereas there was no difference between inflamed or noninflamed UC and normal intestinal tissue samples. Examining subgroups of diverse intestinal segments from CD and UC samples with various levels of inflammation revealed no differences in NK-1R-L and NK-1R-S mRNA expression, whereas there was a tendency toward overall lower NK-1R-S mRNA copy numbers. Immunoblot analysis showed upregulation of NK-1R protein levels in cases of IBD, with more pronounced enhancement in cases of CD than in UC. For NEP, there were no differences in protein levels in normal, CD, and UC intestinal tissues. Comments These observations suggest a contribution of SP and its receptor, NK-1R, in the local inflammatory reaction in IBD and particularly in ileal CD. Moreover, significant upregulation of PPT-A mRNA in the noninflamed ileum of these patients suggests an influence of inflamed intestines on their healthy counterparts.

Stress induces substance P in vagal sensory neurons innervating the mouse airways

Tachykinins-like substance P (SP) have been shown to play an important role in initiating and perpetuating airway inflammation. Furthermore, they are supposed to be released into tissues in response to stress. The aim of this study was to investigate the effects of stress alone or in combination with allergic airway inflammation on SP expression in sensory neurons innervating the mouse airways. Balb/c mice were systemically sensitized to ovalbumin (OVA), followed by allergen aerosol exposure, and compared with non-sensitized controls. Additionally, OVA-sensitized and -challenged and non-sensitized mice were exposed to sound stress. SP expression in airway-specific and overall vagal sensory neurons of the jugular and nodose ganglion complex was analysed using retrograde neuronal tracing in combination with immunohistochemistry. Preprotachykinin A (PPT-A) mRNA, the precursor for SP, was quantified in lung tissue by real-time PCR. Bronchoalveolar lavage (BAL) fluid was obtained, and cell numbers and differentiation were determined. Stress and/or allergic airway inflammation significantly increased SP expression in retrograde-labelled vagal sensory neurons from the mouse lower airways compared with controls [stress: 15.7+/-0.8% (% of retrograde-labelled neurons, mean+/-SEM); allergen: 17.9+/-0.4%; allergen/stress: 13.1+/-0.7% vs. controls: 6.3+/-0.3%]. Similarly, SP expression increased in overall vagal sensory neurons identified by the neuronal marker protein gene product (PGP) 9.5 [stress: 9.3+/-0.6% (% of PGP 9.5-positive neurons, means+/-SEM); allergen: 12.5+/-0.4%; allergen/stress: 10.2+/-0.4% vs. controls: 5.1+/-0.3%]. Furthermore, stress significantly increased PPT-A mRNA expression in lung tissue from OVA-sensitized and -challenged animals, and immune cells were identified as an additional source of SP in the lung by immunohistochemistry. Associated with enhanced neuronal SP expression, a significantly higher number of leucocytes were found in the BAL following allergen exposure. Further, stress significantly increased allergen-induced airway inflammation identified by increased leucocyte numbers in BAL fluids. The central event of sound stress leads to the stimulation of SP expression in airway-specific neurons. However, in sensitized stressed mice an additional local source of SP (probably inflammatory cells) might enhance allergic airway inflammation.

The effect of CP96,345 on the expression of tachykinins and neurokinin receptors in acute pancreatitis

Acute pancreatitis (AP) is a life-threatening condition that involves an acute inflammatory process in the pancreas. The involvement of tachykinins and neurokinin receptors in acute pancreatitis has been described only recently, despite their long-established role in inflammatory conditions. Among these, substance P (SP) is believed to play a central role in exacerbating the inflammatory process by acting through neurokinin-1 receptor (NK1R). Treatment with the NK1R antagonist, CP96,345, results in protection against caerulein-induced acute pancreatitis in mice. However, the mechanism by which NK1R and SP worsen the condition is still unclear. In the present study, we have investigated the effect of NK1R blockage on the expression of preprotachykinin genes and neurokinin receptors in acute pancreatitis. In the pancreas, CP96,345 treatment resulted in suppression of the elevation of SP concentration, preprotachykinin-A gene (PPT-A) mRNA expression, and NK1R mRNA and protein expression. In the lungs, the antagonist was found to suppress the increase in SP concentration, PPT-A mRNA expression and preprotachykinin-C gene (PPT-C) mRNA expression. However, the antagonist treatment further promoted the accumulation of pulmonary NK1R mRNA and protein expression. Neurokinin-2 receptor (NK2R) mRNA expression was not detected in normal pancreas. However, up-regulated expression of the mRNA for this receptor was observed during acute pancreatitis and treatment with CP96,345 further increased this expression. Pulmonary NK2R mRNA expression was found to be reduced during acute pancreatitis and CP96,345 treatment normalized this reduction. Neurokinin-3 receptor (NK3R) mRNA expression was absent in both pancreas and lung. These data have provided valuable information regarding the regulation of tachykinins and neurokinin receptors during acute pancreatitis.

Down‐regulation of amygdala preprotachykinin A mRNA but not 3H‐SP receptor binding sites in subjects affected by mood disorders and schizophrenia

Increasing evidence suggests that substance P (SP) neurokinin-1 (NK1) receptors are involved in stress and emotional responses, representing a potential target for the treatment of anxiety and depression in humans. Given the important role of the amygdaloid complex in the regulation of emotional behavior, we examined the mRNA levels of preprotachykinin A [PPT-A, a precursor of both SP and neurokinin A (NKA)] and 3H-SP binding sites in the amygdala of patients affected by bipolar disorder, major depression or schizophrenia as compared with matched control individuals. By means of in situ hybridization, a significant reduction of PPT-A mRNA expression levels was detected in the three diagnostic groups, mainly in the basal, lateral and accessory basal amygdaloid nuclei, but not in the temporal cortical area proximal to the amygdala. Receptor autoradiography performed on adjacent sections showed no change in 3H-SP binding sites as compared with controls. No significant correlation was found between levels of PPT-A mRNA expression or binding sites and subject age, gender, hemisphere side, cause of death or history of substance misuse (marijuana, alcohol, cocaine/amphetamine). An inverse relationship between PPT-A mRNA expression levels and lifetime antipsychotic treatment (Fluphenazine) in the schizophrenic and bipolar disorder groups was found. Post-mortem delay was also negatively correlated with NK1 binding sites. The results confirm an involvement of the tachykinins in psychiatric disorders, suggesting there is a generalized impairment of the SP system in the amygdala in mood disorders and schizophrenia rather than this being a disease-related phenomenon.

The expression of PPTA and c-fos mRNA in dog caudal spinal trigeminal nucleus induced by traumatic occlusion

PPTA and c-fos mRNA expression were detected in dog caudalis subnucleus of trigeminal spinal tract nucleus (VC) induced by trauma occlusion in order to investigate orofacial pain mechanism. The occlusal surface of the first and second maxillary right molars in 15 dogs were unilaterally raised 1.5 mm with casting Ni-Cr inlay which were fixed in Class I hole. On days 3, 7, 14, 30 and 60 after teeth operation, the VC of right and left sides were removed. PPTA and c-fos mRNAs were detected in experimental and control groups with reverse transcription-polymerase chain reaction (RT-PCR). (1) The basal levels of PPTA and c-fos mRNAs were extremely low and poorly detectable in VC in control animals. (2) The expression of PPTA mRNA in VC of traumatic side was up regulated from 3 days after inlay was fixed in molar and reached peak level during 14 to 30 days and then down-regulated gradually and no significant difference was noted between 60 days group and control group. (3) c-fos mRNA expression was more intense during 3 to 7 days compared with the control group but undetectable in the other experimental period. (4) Both PPTA and c-fos mRNAs expression in VC of trauma occlusal side were more intense than that in the contralateral side. The present results show that both PPTA and c-fos mRNA expression are elevated in dog's VC induced by traumatic occlusion. The primary afferent terminal of orofacial area is sensitized, which suggest one kind of mechanism of orofacial pain in the condition of traumatic occlusion.

Substance P Induced Preprotachykinin-A mRNA, Neutral Endopeptidase mRNA and Substance P in Cultured Normal Fibroblasts

In certain skin diseases, stress can modulate the induction and/or progression of cutaneous manifestations. However, little is known about the circuit in neuroendocrine and in the immune systems of the skin. To address this question, we have analyzed the regulatory mechanisms of autocrine induction of substance P (SP) by cultured normal human fibroblasts that compose the major population of the skin and might augment stress-induced skin inflammatory responses. In nonstimulated conditions, normal fibroblasts express a moderate amount of preprotachykinin-A (PPT-A), a precursor of SP mRNA, and exogenous SP significantly upregulated PPT-A mRNA expression. Maximum response of SP peptide and SP mRNA in fibroblasts was observed 1–3 h after stimulation with SP. In contrast, the expression of neutral endopeptidase (NEP), a cell surface peptide with hydrolyzing activity of SP, was increased in fibroblasts stimulated with SP after 24 h. The administration of NEP inhibitor (phosphoramidon) to the fibroblasts induced higher SP production. In addition, the neurokinin (NK) receptor antagonists (spantide, FK224 and FK888) and protein synthesis inhibitor (cycloheximide) inhibited SP production by 30–40% of control response. In immunostaining study, specific cytoplasmic staining of SP was observed in fibroblasts stimulated with SP. Finally, we confirmed that the nucleotide sequence of the PPT-A expressed in fibroblasts perfectly corresponded to the gene bank human PPT-A cDNA. This is the first report that SP mRNA, NEP mRNA and SP peptide can be induced by normal human skin fibroblasts in response to exogenous SP, and that fibroblast-derived SP might play an important role in the induction and acceleration of certain cutaneous diseases.

Regulation of striatal neuropeptide mRNAs: effects of the 5-HT(2) antagonist SR46349B in adult rats with a neonatal 6-hydroxydopamine lesion.

The intrastriatal injection of 6-hydroxydopamine (6-OHDA) in newborn rats produces a marked striatal dopamine (DA) depletion, accompanied by a serotonin (5-HT) hyperinnervation and an up-regulation of 5-HT receptors. The aim of the present study was to investigate whether the increase in 5-HT(2) receptors could compensate for some of the DA lesion-induced effects, such as the increase in striatal preproenkephalin (PPE) and the decrease in preprotachykinin A (PPT-A) mRNA levels. Three months after the DA lesion, the effect of the selective 5-HT(2) antagonist SR46349B was investigated by a subacute treatment (10 mg/kg, IP, twice per day for 3.5 days). In sham-operated rats, the blockade of 5-HT(2) receptors decreased PPE mRNA levels in the striatum and, by contrast, had no effect on PPT-A mRNA levels. In rats with a unilateral neonatal DA lesion, SR46349B had no more effect on PPE mRNA levels in the intact striatum and was unable to modify the lesion induced-increase in PPE mRNA. The decrease in PPT-A mRNA levels induced by the neonatal DA lesion was not changed after SR46349B treatment in the posterior part of the lesioned striatum. Our results suggest that SR46349B indirectly decreases PPE mRNA levels in striatopallidal neurons in intact animals through a desinhibition of DA neuron activity. This is further evidenced by the lack of PPE mRNA changes in the DA lesioned striatum despite the up-regulation of 5-HT(2) receptor transmission induced in this model. Finally, the absence of any effect of 5-HT(2) antagonist on the expression of PPT-A mRNA in intact animals is discussed. The precise role of 5-HT(2) receptor on PPT-A mRNA biosynthesis after a neonatal lesion should be clarified by further experiments using 5-HT(2) agonists.

In situ hybridization analysis of preprotachykinin-A mRNA levels in young and old rats.

Preprotachykinin-A (PPT-A) mRNA levels in discrete rat brain regions were examined. Analysis of silver grains revealed a 19.2% and 31.5% statistically significant decrease in PPT-A mRNA in the dorsal and ventral caudate putamen (d-CPu and v-CPu), respectively, a 30% lower expression of PPT-A mRNA in the bed nucleus of the stria terminalis (BNST), a 33.7% decrease in PPT-A mRNA in the habenula (Hb), and a 30% decrease of PPT-A mRNA levels in the posterodorsal part of the medial amygdala (MePD). Results show that aging of the CNS is associated with widespread changes in tachykinin gene expression, suggesting that alterations in the tachykinergic system may have implications in the physiopathology of the elderly.

Birth-related expression of c- fos, c- jun and substance P mRNAs in the rat brainstem and pia mater: possible relationship to changes in central chemosensitivity

In situ hybridization was used to characterize respiration-related areas of the brainstem activated around the time of birth as well as their postnatal sensitivity to CO 2. Levels of mRNA corresponding to the immediate early genes (IEG), c- fos and c- jun, and of substance P precursor, ppt-A, were determined in rat fetuses (E21) and neonatal pups (1 h, 1 day and 6 days after normal birth) and after exposure to hypercapnia (12% CO 2 for 1 h). Transient increases in c- fos mRNA were observed in the central chemoreceptor area of the ventral medullary surface (VMS), in the lateral reticular nucleus (LRN), in the nucleus of the solitary tract (NTS), and in the nucleus raphé pallidus (RPA) 1 h after birth. Increased expression of c- fos mRNA in the VMS could also be evoked by hypercapnia and this response was particularly pronounced 1 day after birth. On the other hand, c- jun mRNA could be detected already at E21 in the hypoglossal nucleus (XII) and LRN and these levels were not significantly altered at 1 h after birth. There was, however, an increase in the expression of c- jun mRNA in the pia mater surrounding the brainstem after birth. At 1 day after birth, c- jun mRNA levels had decreased in the LRN and pia mater, and later on (6 days after birth) in XII. Furthermore, the ppt-A mRNA level in NTS increased immediately after birth and remained high 1 and 6 days later. These results suggest that (a) the central chemoreceptor area of the VMS, as well as the NTS, LRN, RPA and pia mater are activated following birth; (b) the VMS, but not the other structures examined, can be activated immediately after birth by hypercapnia; and (c) increased expression of ppt-A mRNA may be related to the transition of respiratory control at birth.

Intrastriatal grafting of a GDNF-producing cell line protects striatonigral neurons from quinolinic acid excitotoxicity in vivo.

Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor with a therapeutic potential in neurodegenerative disorders. GDNF is expressed in the adult striatum, but its signalling tyrosine kinase receptor, c-ret, has not been detected in this structure by in situ hybridization. In the present work, we first examined c-ret and GDNF receptor alpha 1 (GFR-alpha 1) expression using an RNAse protection assay, and found that both receptors are expressed in the adult rat striatum. We then examined whether GDNF was able to regulate the phenotype and/or prevent the degeneration of striatal projection neurons in a well-characterized model of excitotoxic damage. A fibroblast cell line, engineered to overexpress GDNF, was grafted in adult rats striatum 24 h before quinolinic acid (QUIN) injection. QUIN injection alone or in combination with the control cell line induced a loss of glutamic acid decarboxylase 67 (GAD)-, preprotachykinin A (PPTA)-, prodynorphin (DYN)- and preproenkephalin (PPE)-positive neurons. GDNF selectively prevented: (i) the loss of a subpopulation of striatonigral neurons expressing GAD and PPTA; (ii) the atrophy of PPTA-positive neurons; and (iii) the decrease in GAD, PPTA and DYN mRNA expression, after QUIN injection. Moreover, in unlesioned animals, GDNF increased the size of PPTA-positive neurons and up-regulated their mRNA levels. In contrast, GDNF showed no effect in intact or lesioned striatopallidal PPE-positive neurons. Thus, our findings show that GDNF selectively regulates the phenotype and protects striatonigral neurons from QUIN-induced excitotoxicity, suggesting that GDNF may be used for the treatment of striatonigral degenerative disorders, e.g. Huntington's disease and multiple system atrophy.

Regulation of preprotachykinin-A mRNA in genetic hypertensive and normotensive rats

It is well-known that central administration of tachykinins (Tks) inhibit salt intake in rats. Recent studies have shown that conditions that arouse salt appetite, such as adrenalectomy and sodium depletion, induce a decrease in preprotachykinin-A (PPT-A) mRNA in discrete regions of the rat brain, suggesting that reduced levels of PPT-A mRNA in the brain may have a permissive role on the expression of salt appetite. It has also been shown that spontaneously hypertensive rats (SHR) show higher avidity for salty solutions than their normotensive control Wistar–Kyoto (WKY) rats. In this regard, the present study tested whether SHR and WKY rats differ in expression of the gene coding for PPT-A, the precursor for Tks peptides. Using semi-quantitative in situ hybridization histochemistry, we examined the level of PPT-A mRNA in discrete rat brain regions of SHR and WKY rats under no treatment, after 1 or 3 days of Na + depletion. Levels of PPT-A mRNA were analysed in the olfactory tubercle (Tu), in the lateral olfactory tubercle (LOT), in the dorsal and ventral caudate putamen (d/v CPu), in the medial preoptic area (mPOA), in the bed nucleus of the stria terminalis (BNST), in the habenula (Hb) and in the postero-dorsal part of the amygdala (MePD). Semi-quantitative analysis of silver grains revealed a 27.5% lower expression of the PPT-A mRNA levels in SHR opposite to WKY rats under no treatment in v-CPu, mPOA, BNST and Hb. 1 day of Na + depletion reduced PPT-A mRNA levels when opposite to Na +-repleted animals in Tu and mPOA in both SHR and WKY rats. On the other hand, when comparing SHR and WKY rats after 1 day of Na + depletion, a 26% lower level of PPT-A mRNA was detected in Tu and d-CPu of SHR opposite to WKYrats whereas a 14% and an 18% lower level was detected in v-CPu and Hb, respectively. A lower expression of PPT-A mRNA in SHR compared to WKY rats was also found in BNST and MePD, although no statistical significance was detected in these two brain areas. In the last experiment, 3 days of Na + depletion reduced PPT-A mRNA levels in mPOA while negligibly increased mRNA levels in d-CPu and v-CPu, in BNST, Hb and MePD, both in SHR and WKY rats. Conversely, when making comparisons between the two strains, a 35% lower level of PPT-A mRNA in SHR with respect to WKY rats was found after 3 days of Na + depletion in d-CPu, v-CPu and mPOA. A lower gene expression, even though not statistically significant, was found in Tu, LOT, MePD. These findings show a consistent difference of PPT-A mRNA levels in discrete regions of the SHR brain opposite to WKY rats and confirm that 1 day of Na + depletion reduces PPT-A mRNA in discrete brain regions. Since SHR are notoriously more salt-avid than WKY rats and Tks are potent inhibitors of sodium intake, the down-regulation of PPT-A mRNA may contribute to the higher natriophilia and, therefore, to the etiology of the hypertensive disease.

Midbrain dopamine neurons regulate preprotachykinin-A mRNA expression in the rat forebrain during development

Intracerebroventricular 6-hydroxydopamine injections were performed at postnatal days 3 and 6 in animals pretreated with the norepinephrine uptakeblocker desimipramine in order to generate a selective lesion of dopamine neurons. In situ hybridization was then used to analyze preprotachykinin-A (PPT-A) mRNA expression in the lesioned as well as in saline-injected control animals. The midbrain dopaminergic lesion caused a 22–25% increase in the level of PPT-A mRNA in cingulate cortex and frontoparietal cortex when analysed at 2 weeks of age, compared to saline-injected control animals. In contrast, the lesion caused no change in PPT-A mRNA expression in the neonatal caudate-putamen. These results indicate that dopamine neurons downregulate the expression of PPT-A mRNA specifically in cingulate cortex and frontoparietal cortex during early postnatal brain development. In the adult rat forebrain, lesioned at P3 and P6, no change in the level of PPT-A mRNA was seen in cingulate cortex and frontoparietal cortex. However, a 29% decrease in PPT-A mRNA was seen in the lateral caudate-putamen with no significant change in neurons of medial caudate-putamen. Thus, dopamine neurons appears to exert a region specific influence on PPT-A mRNA expression during brain development.

Nasal mucosa sensitization with toluene diisocyanate (TDI) increases preprotachykinin A (PPTA) and preproCGRP mRNAs in guinea pig trigeminal ganglion neurons

Toluene diisocyanate (TDI) induces respiratory allergy in mammals. Using immunohistochemistry and in situ hybridization histochemistry, the present study examined effects of nasal mucosa sensitization by TDI on the immunoreactivity for substance P (SP) and calcitonin gene-related peptide (CGRP) and on the expression of their mRNAs in guinea pig trigeminal ganglion and their terminals. Single intranasal application of TDI (acute experiment) did not induce nasal allergy-like behaviours and failed to cause changes of SP and CGRP immunoreactivity and in the expression of preprotachykinin A (PPTA) mRNA and preproCGRP mRNA coding for SP and CGRP respectively in the trigeminal ganglion neurons. However, repeated application of TDI (chronic experiment) caused a dramatic increase of SP and CGRP immunoreactivity in peripheral neurites of sensory nerves in the nasal mucosa but a slight increase in the spinal trigeminal nucleus, a decrease of the same immunoreactivities in the cell bodies of the trigeminal ganglion neurons, and an increase of the expression of PPTA and preproCGRP mRNA in the same neurons. These findings suggest that chronic exposure of the nasal mucosa to TDI apparently causes enhancement of both the biosynthesis of SP and CGRP and their axonal transport in the trigeminal system.

Amphetamine and haloperidol modulate preprotachykinin A mRNA expression in rat nucleus accumbens and caudate-putamen

In situ hybridization was used to measure the effect of repeated amphetamine (1.5 mg/kg) and haloperidol (0.5 mg/kg) treatment for 7 days on the expression of preprotachykinin A (PPT-A) mRNA in rat nucleus accumbens (Acb) and caudate-putamen (CPu). Amphetamine elevated the level of PPT-A mRNA in Acb, but not in CPu. Haloperidol decreased the levels in Acb shell and CPu, but not in Acb core. Haloperidol injected together with amphetamine, prevented the amphetamine-induced increase in PPT-A mRNA expression in both Acb core and shell.

Preprotachykinin A mRNA expression in the rat brain during development

Expression of preprotachykinin A (PPT-A) mRNA was analyzed by northern blots using mRNA prepared from rat brain at 12 different developmental stages ranging from embryonic day 15 (E15) to adult. A single PPT-A mRNA of 1.3 kb was detected throughout development. PPT-A mRNA was detected as early as E15 and an approximately 3-fold increase occurred at birth. This amount remained until 3 weeks of age when the level increased, reaching a peak at 5 weeks of age. Adult amounts were approximately 3-fold higher than the levels at birth. The distribution of PPT-A mRNA-expressing cells in rat brain was studied by in situ hybridization on sections from embryonic day 20, postnatal days 4 and 7 as well as adult. Cells expressing PPT-A mRNA were detected in the forebrain at all 4 ages analyzed. However, the hybridization pattern and the labeling intensity varied in different brain regions during development. In cingulate cortex, intense labeling was seen in numerous cells at embryonic day 20 and postnatal days 4 and 7, whereas in the adult cingulate cortex only a few scattered labeled cells were observed. In frontoparietal cortex labeled cells were found from postnatal day 4 to adult, with the highest density of labeled cells at P7. Developmental differences in both the distribution of PPT-A mRNA-expressing cells and the level of PPT-A mRNA expression were also found in caudate-putamen, lateral hypothalamus and amygdala. Thus, our results show several changes in PPT-A mRNA expression during ontogeny, indicating a region and time-specific regulation of PPT-A mRNA expression during brain maturation.

A noxious stimulus induces the preprotachykinin-A gene expression in the rat dorsal root ganglion: a quantitative study using in situ hybridization histochemistry

Using in situ hybridization histochemistry, we examined the biosynthesis of the preprotachykinin-A (PPTA) mRNAs, which code for substance P (SP) and also a related peptide, substance K (SK), in the rat dorsal root ganglion (DRG). The animals were injected with a small amount of formalin in the right hindpaw for nociceptive chemical stimulation. Analysis of hybridization signals in the emulsion autoradiography showed that the lumbar-5 DRG neurons expressing PPTA mRNAs were significantly increased in number and signal intensity on the formalin-injected side. Such elevation of the PPTA mRNA expression in the DRG was observed as early as 3 h after formalin injection. These results show that the expression of the PPTA gene is activated by chemogenic noxious stimuli.