Preprotachykinin mRNA Expression Research Articles

Characterization of striatum-mediated behavior and neurochemistry in the DJ-1 knock-out rat model of Parkinson's disease

The recently developed DJ-1 knockout (KO) rat models the DJ-1 (or PARK7) loss-of-function mutation responsible for one form of early-onset familial Parkinson's disease (PD). Prior studies demonstrate that DJ-1 KO rats present progressive dopamine (DA) cell body degeneration in the substantia nigra pars compacta between 4 and 8 months of age. Furthermore, as some motor deficits emerge before the significant loss of DA cells, this mutation may yield a period of DA neuron dysfunction preceding cell death that may also contribute to cognitive impairments in early PD. However, cognitive functions subserved by corticostriatal circuitry, as well as additional alterations to the neurochemistry of monoamine systems, are largely uncharacterized in the DJ-1 KO rat. We therefore assessed a variety of striatally-mediated behavioral tasks, as well as the integrity of dopamine and serotonin systems, in male DJ-1 KO rats and wild-type (WT) controls at 4, 6, and 8 months of age. We demonstrate that DJ-1 KO rats exhibited motor impairments, but have intact goal-directed control over behavior in an appetitive instrumental learning task. Further, preprotachykinin mRNA expression, a post-synaptic indicator of DA signaling, was significantly decreased in 4-month DJ-1 KO rats, while DA transporter binding in the dorsal striatum did not differ between genotypes at any of the ages examined. Striatal tyrosine hydroxylase levels were significantly increased in 8-month DJ-1 KO rats and tended to be higher than WT at 4 and 6 months. Lastly, serotonin transporter binding was increased in the medial and orbitofrontal cortices of 4-month old DJ-1 KO rats. These results suggest that the nigrostriatal dopaminergic and prefrontal serotoninergic systems are altered early in the progression of DJ-1 KO pathology, despite no overt loss of the DA innervation of the striatum, and thus may be associated with early alterations in the functions of corticostriatal systems.

Phasic‐like stimulation of the medial forebrain bundle augments striatal gene expression despite methamphetamine‐induced partial dopamine denervation

Methamphetamine-induced partial dopamine depletions are associated with impaired basal ganglia function, including decreased preprotachykinin mRNA expression and impaired transcriptional activation of activity-regulated, cytoskeleton-associated (Arc) gene in striatum. Recent work implicates deficits in phasic dopamine signaling as a potential mechanism linking methamphetamine-induced dopamine loss to impaired basal ganglia function. This study thus sought to establish a causal link between phasic dopamine transmission and altered basal ganglia function by determining whether the deficits in striatal neuron gene expression could be restored by increasing phasic dopamine release. Three weeks after pretreatment with saline or a neurotoxic regimen of methamphetamine, rats underwent phasic- or tonic-like stimulation of ascending dopamine neurons. Striatal gene expression was examined using in situ hybridization histochemistry. Phasic-like, but not tonic-like, stimulation induced immediate-early genes Arc and zif268 in both groups, despite the partial striatal dopamine denervation in methamphetamine-pretreated rats, with the Arc expression occurring in presumed striatonigral efferent neurons. Phasic-like stimulation also restored preprotachykinin mRNA expression. These results suggest that disruption of phasic dopamine signaling likely underlies methamphetamine-induced impairments in basal ganglia function, and that restoring phasic dopamine signaling may be a viable approach to manage long-term consequences of methamphetamine-induced dopamine loss on basal ganglia functions.

Implication of substance P neuronal system in the amygdala as a possible mechanism for hypergravity-induced motion sickness

We previously reported that motion sickness was prevented in rats with amygdala lesion and that provocative motion stimuli increased the number of Fos-positive neurons in the amygdala, suggesting that the amygdala is one of the neural substrates involved in the development of motion sickness. NK-1 receptors in the brain stem and amygdala are thought to play an important role in emesis and affective disorders, respectively. In the present study, to elucidate a role of substance P neuronal system and NK-1 receptors in the brain stem and amygdala in the development of motion sickness, we measured changes in gene expression of NK-1 receptors and preprotachykinin, a precursor of substance P, using quantitative real-time PCR methods in solitary tract nucleus and amygdala in rats after provocative motion stimuli induced by 2G hypergravity load. Effects of systemic administration of CP-99,994, an antagonist for NK-1 receptors, on hypergravity-induced motion sickness were also examined using pica behavior, eating non-nutritive substances such as kaolin, as an index of motion sickness in rats. Hypergravity-induced motion sickness was inhibited by CP-99,994 with a dose-dependent and enantioselective manner. Preprotachykinin mRNA expression was increased in basolateral nucleus of amygdala and solitary tract nucleus after hypergravity load for 3h, whereas NK-1 receptor mRNA expression was not changed by hypergravity in amygdala and solitary tract nucleus. Present results suggest that 2G hypergravity load activated the substance P neuronal system in amygdala as well as in the brain stem and this activation would be related to the development of motion sickness.

Nimesulide inhibits protein kinase C epsilon and substance P in sensory neurons – comparison with paracetamol

In this paper we describe new actions of nimesulide and paracetamol in cultured peripheral neurons isolated from rat dorsal root ganglia (DRG). Both drugs were able to decrease in a dose-dependent fashion the number of cultured DRG neurons showing translocation of protein kinase C epsilon (PKCɛ) caused by exposure to 1 μM bradykinin or 100 nM thrombin. In addition, the level of substance P (SP) released by DRG neurons and the level of preprotachykinin mRNA expression were measured in basal conditions and after 70 minutes or 36 hours of stimulation with nerve growth factor (NGF) or with an inflammatory soup containing bradykinin, thrombin, endothelin-1, and KCl. Nimesulide (10 μM) significantly decreased the mRNA levels of the SP precursor preprotachykinin in basal and in stimulated conditions, and decreased the amount of SP released in the medium during stimulation of neurons with NGF or with the inflammatory soup. The effects of paracetamol (10 μM) on such response was lower. Nimesulide completely inhibited the release of prostaglandin E2 (PGE2) from DRG neurons, either basal or induced by NGF and by inflammatory soup, while paracetamol decreased PGE2 release only partially. Our data demonstrate, for the first time, a direct effect of two drugs largely used as analgesics on DRG neurons. The present results suggest that PKCɛ might be a target for the effect of nimesulide and paracetamol, while inhibition of SP synthesis and release is clearly more relevant for nimesulide than for paracetamol mechanism of action.

Regulatory effects of insulin-like growth factor-1 on the expression of sensory neuropeptide mRNAs in cultured dorsal root ganglion neurons with excitotoxicity induced by glutamate

To determine the effects of insulin-like growth factor-1 (IGF-1) on the expression of preprotachykinin (PPT) mRNA encoding substance P (SP) and calcitonin gene-related peptide (CGRP) mRNA in cultured dorsal root ganglion (DRG) neurons with excitotoxicity induced by glutamate (Glu). DRGs were dissected from embryonic day 15 Wistar rats. DRG neurons were dissociated and cultured for 48 h and then exposed to Glu (0.2 mmol/L) or Glu (0.2 mmol/L) plus IGF-1 (5 nmol/L, 10 nmol/L and 20 nmol/L) for 12 h. The DRG neurons in control group were exposed to only growth media throughout the experiment. After that, the living DRG neurons were observed under inverted phase contrast microscope and microphotographs were taken. The expression levels of PPT and CGRP mRNAs were detected by reverse transcription-polymerase chain reaction (RT-PCR). IGF-1 could inhibit Glu-induced shortening of neurite. Besides, IGF-1 could significantly increase the levels of PPT mRNA and CGRP mRNA in primary cultured DRG neurons with Glu-induced excitotoxicity, in a dose-dependent manner. IGF-1 may exert neuroprotective effects on DRG neurons against Glu-induced excitotoxicity, probably through regulating the expression levels of PPT and CGRP mRNAs.

Effect of Estrogen on Bladder Nociception in Rats

We assessed the effect of ovariectomy and estrogen replacement on nociceptive responses to bladder distention in a rat model. Female Sprague-Dawley rats (Harlan) underwent ovariectomy or sham surgery. Visceromotor responses (abdominal contractions) to bladder distention were determined 3 to 4 weeks later under isoflurane anesthesia. In rat subsets estrogen was chronically replaced with a subcutaneous estrogen pellet vs a placebo pellet or acutely replaced by subcutaneous injection 24 hours before testing. Effects of estrogen withdrawal were examined in another group of rats by implanting a pellet and explanting the pellet 24 hours before testing. Uterine weight was measured to assess the estrogen dose. Visceromotor responses to bladder distention were significantly less vigorous in ovariectomized rats vs controls. Acute estrogen replacement increased visceromotor responses in these rats but chronic estrogen replacement did not. Sudden chronic estrogen withdrawal resulted in increased visceromotor responses. Uterine weight was consistent with the physiological estrogen dose. Estrogen alone was not sufficient to produce increased nociceptive responses but an acute decrease in estrogen resulted in increased visceromotor responses. These data suggest that the pronociceptive effects of estrogen may be due to a mismatch between peripheral vs central and/or genomic vs nongenomic effects of the hormone, which occur during rapidly decreasing estrogen levels.

Dysfunction of the cortico‐basal ganglia‐cortical loop in a rat model of early parkinsonism is reversed by metabotropic glutamate receptor 5 antagonism

This study examined the cellular correlates of the akinetic deficits produced in Wistar rats by discrete bilateral 6-hydroxydopamine (6-OHDA) striatal infusions in the dorsolateral striatum, mimicking the preferential denervation of the motor striatal territory in early symptomatic stage of Parkinson's disease (PD). Intraneuronal gene expression of cytochrome oxidase subunit I (COI), a metabolic index of neuronal activity, was increased in the subthalamic nucleus, substantia nigra pars reticulata and decreased in frontal cortical areas, but paradoxically unchanged in the striatum, globus pallidus, entopeduncular nucleus and ventrolateral thalamic nucleus. Neither preproenkephalin A nor preprotachykinin mRNA expression, markers of striatal projection neurons, were modified in the denervated striatal area despite 90% loss of dopamine (DA) terminals. Preproenkephalin A mRNA expression was however, decreased in the nondepleted striatal region, suggesting compensatory increase of dopamine tone from those spared areas. A chronic treatment with the metabotropic glutamate receptor 5 (mGluR5) antagonist 2-methyl-6-(phenylethylnyl)-pyridine (MPEP), which alleviated the akinetic disorders produced by the lesion, reversed the lesion-induced variations of COI gene expression, moderately increased this marker in the structures unaffected by the lesion and did not modify the striatal neuropeptides gene expression. These data suggest that the expression of akinetic deficits in early parkinsonism is associated with focused metabolic changes in the cortico-basal ganglia-cortical loop downstream of the striatum and pallidal complex.

Failure of MK-801 to suppress D1 receptor-mediated induction of locomotor activity and striatal preprotachykinin mRNA expression in the dopamine-depleted rat

N-methyl- d-aspartate receptor antagonism exerts suppressive influences over dopamine D1 receptor-mediated striatal gene expression and locomotor behavior in the intact rat. The present study examined the effects of the N-methyl- d-aspartate receptor antagonist MK-801 on locomotor activity and striatal preprotachykinin mRNA expression stimulated by the D1 agonist (±)6-chloro-7, 8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1 H-3-benzazepine hydrobromide in rats with bilateral dopamine lesions. Two months after neonatal dopamine lesions with 6-hydroxydopamine, rats were challenged with (±)6-chloro-7, 8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1 H-3-benzazepine hydrobromide (1.0mg/kg) 15 min after administration of the N-methyl- d-aspartate receptor antagonist MK-801 (0.1mg/kg). In the intact rat, MK-801 prevented the induction of striatal preprotachykinin mRNA by D1 agonism. Similarly, direct infusion of (±)6-chloro-7, 8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1 H-3-benzazepine hydrobromide (3.0μg) into the intact striatum produced an increase in locomotor activity that was suppressed by MK-801 (1.0μg) co-infusion. In the dopamine-depleted rat, MK-801 (0.1mg/kg) administered prior to (±)6-chloro-7, 8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1 H-3-benzazepine hydrobromide (1.0mg/kg) increased, rather than suppressed, striatal preprotachykinin mRNA levels. Intrastriatal infusion of MK-801 (1.0μg) failed to inhibit D1-mediated induction of motor activity in dopamine-depleted animals. Together, these data provide further support that N-methyl- d-aspartate receptor antagonists lose their ability to block D1-mediated behavioral activation following dopamine depletion. The activation, rather than suppression, of tachykinin neurons of the direct striatonigral pathway may play a facilitatory role in this mechanism.

Acute pancreatitis results in referred mechanical hypersensitivity and neuropeptide up-regulation that can be suppressed by the protein kinase inhibitor k252a

Although pain is a cardinal feature of pancreatitis, its pathogenesis is poorly understood and treatment remains difficult. Nociceptive sensitization in several somatic pain models has been associated with activation of protein kinases including trkA, protein kinase C, and protein kinase A. We therefore tested the hypothesis that systemic treatment with a kinase inhibitor, k252a, known to inhibit all of these kinases would alleviate pain in an animal model of pancreatitis. Von Frey filament testing of somatic referral regions was evaluated as a method to measure referred pain in a rat model of acute necrotizing pancreatitis induced by L-arginine. Rats with pancreatitis showed increased sensitivity to abdominal stimulation with Von Frey filament. This referred mechanical sensitivity was associated with an 8-fold increase in levels of phosphorylated trkA in the pancreas and with significant up-regulation of both calcitonin gene-related peptide and preprotachykinin mRNA expression in thoracic dorsal root ganglia and with increased calcitonin gene-related peptide and substance P immunoreactivity in spinal cord segment T10. Treatment with the kinase inhibitor k252a suppressed the phosphorylation of trkA in the pancreas as well as reversed both the behavioral changes and the increase in neuropeptide expression associated with pancreatitis.

Increase of preprotachykinin mRNA in the uninjured mandibular neurons after rat infraorbital nerve transection

An increasing number of studies are suggesting that the adjacent uninjured primary afferents contribute to the mechanisms of neuropathic pain following peripheral nerve injury. In this study, we report that transection of the infraorbital nerve, a major branch of the maxillary nerve, causes exaggerated face grooming to normally innocuous mechanical stimuli in the skin territory of the uninjured mandibular nerve, and increases the expression of preprotachykinin mRNA in the primary afferent neurons in the mandibular zone in the trigeminal ganglia. Considering the various functions of substance P in the sensory transmission process, the increase in preprotachykinin mRNA in the uninjured primary afferent neurons may be one of the mechanisms of pain-related behavior in this neuropathic pain model.

Compensatory regulation of striatal neuropeptide gene expression occurs before changes in metabolic activity of basal ganglia nuclei

Compensatory mechanisms delay the appearance of parkinsonian symptoms. However, both the order of appearance and potential interactions of compensatory mechanisms acting within the nigrostriatal pathway as well as inside and outside the basal ganglia are not clear. We hypothesize that, after the striatal dopaminergic homeostasis breakdown, a modification in the expression of several striatal markers (neuropeptide precursors and dopamine receptors) may occur before a change in the activity of both globus pallidus (GP) and substantia nigra pars reticulata (SNr) in response to a partial nigrostriatal lesion. The present data show, in MPTP-treated mice, that preproenkephalin-A and preprotachykinin mRNA expression and D 3 receptor binding are modified without changes in cytochrome oxidase metabolic activity in both GP and SNr, respectively. These changes in neuropeptide expression would compensate for the dopamine depletion-induced changes in inhibitory GABAergic input from the striatum to GP and SNr. It also indicates that nondopaminergic compensatory mechanisms inherent to the basal ganglia are activated before those residing outside the basal ganglia.

Long-term post-synaptic consequences of methamphetamine on preprotachykinin mRNA expression.

Exposure to repeated high doses of methamphetamine produces long-term toxicity to central monoamine systems and alters striatonigral pathway function 3 weeks after exposure. To determine whether these changes in the striatonigral pathway persist for longer we examined neuropeptide mRNA expression in the striatum and cytochrome oxidase activity in the output nuclei of the basal ganglia after treatment with multiple high doses of methamphetamine. Rats exposed to multiple high doses of methamphetamine had significant depletion in dopamine and serotonin content, decreases in tyrosine hydroxylase immunoreactivity, and decreases in preprotachykinin mRNA expression, 6 and 12 weeks after methamphetamine treatment. Preprotachykinin mRNA expression was significantly reduced by approximately 20% in the middle striatum and approximately 32% in the caudal striatum, 6 weeks after treatment. Twelve weeks after treatment, preprotachykinin mRNA expression continued to be significantly reduced by approximately 20% in the middle striatum and approximately 14% in the caudal striatum. Cytochrome oxidase histochemical staining in the entopeduncular nucleus and substantia nigra pars reticulata was not significantly different from that in controls at either time point. These data suggest that neurotoxic regimens of methamphetamine induce changes in striatonigral neurons that persist for up to 3 months, although there is some recovery.

Upregulation of neurokinin-1 receptor expression in rat spinal cord by an N-terminal metabolite of substance P.

Chronic inflammatory conditions are associated with an upregulation of both substance P (SP) and neurokinin-1 (NK-1) receptors in the dorsal spinal cord. These receptors have been implicated in hyperalgesia as well as stress-induced analgesia. On the basis of the release of SP during chronic pain, and its rapid metabolism, we tested the hypothesis that SP metabolites regulate the synthesis of either SP or NK-1 receptors in the spinal cord. We measured expression of preprotachykinin mRNA and NK-1 receptor mRNA following intrathecally administered substance P(1-7) (SP1-7), the major metabolite of SP in rat, and following capsaicin, a compound known to induce release of endogenous SP. SP(1-7) and capsaicin each increased NK-1 receptor mRNA in the spinal cord (6 h) followed by an increase in NK-1 receptor-immunoreactivity (24 h and 1 week). D-SP(1-7), a D-isomer and antagonist of SP(1-7), did not mimic the effect of SP(1-7), indicating stereoselectivity. Instead, D-SP(1-7) prevented the upregulation of NK-1 receptor immunoreactivity that was induced by capsaicin injected intrathecally, suggesting that the effect of capsaicin is also mediated by SP N-terminal metabolites. In contrast, the decrease in SP synthesis produced by capsaicin was not dependent on SP metabolites as SP(1-7) failed to decrease either preprotachykinin mRNA content in dorsal root ganglia (6 h) or SP immunoreactivity in the lumbar spinal cord (24 h and 1 week). In addition, the effects of capsaicin on SP synthesis were not prevented by D-SP(1-7). Thus, SP metabolites, at times and doses that are antinociceptive, appear to enhance SP-mediated signal transduction by upregulating NK-1 receptor expression without affecting SP synthesis.

Stress response, tachykinin, and cutaneous inflammation.

In the last decade, several new aspects of glucocorticoid (GC)-actions on immune cells have been recognized. This recognition has been largely obtained through clinical observations of stress-induced exacerbations of certain dermatologic diseases. To clarify whether GC modulates cutaneous inflammatory reactions besides its known anti-inflammatory effect, first we examined the effect of long-term application of topical GC on several kinds of inflammatory responses induced in the murine model and demonstrated that these regimens significantly augmented the classical contact sensitivity reaction, the croton oil-induced irritant reaction, and the IgE-mediated biphasic cutaneous reaction. In addition, large dose topical steroid and its withdrawal enhanced scratching behavior in hapten-challenged mice. This augmented scratching behavior correlated with the induction of preprotachykinin mRNA expression in the challenged skin. In an in vitro experiment, a low-dose, stress-induced level of glucocorticoid significantly upregulated hapten-induced proinflammatory cytokine (IL1alpha) production by murine keratinocyte cell line Pam 212 and induced substance P peptide production from cultured human keratinocytes. Our results suggest that unsuitable use of GC in addition to stress-induced GC may modulate immune function in the skin through aberrant production of tachykinin, such as substance P or other epidermal cell derived cytokines.

Neonatal dopamine depletion reveals a synergistic mechanism of mRNA regulation that is mediated by dopamine D1 and serotonin 2 receptors and is targeted to tachykinin neurons of the dorsomedial striatum

It has been hypothesized that dopamine D1 and serotonin 2 receptors become sensitized to agonist-mediated regulation of gene expression following loss of dopaminergic innervation to the striatum. We have previously demonstrated that the combined administration of dopamine D1 and serotonin 2 receptor agonists to dopamine-depleted adult rats induced preprotachykinin mRNA expression within the periventricular rostral striatum to levels which were significantly different than what could be elicited by either agonist alone. In the present study, we have determined that this phenomenon is revealed only after dopamine depletion. In addition, it is targeted primarily to tachykinin producing neurons of the dorsomedial striatum and is dependent on both dopamine D1 and serotonin 2 receptor activation. Preprotachykinin mRNA levels in the intact striatum were unaltered 4 h following an i.p. injection of either SKF-38393 (1 mg/kg, dopamine D1 partial agonist) or (±)-1-(4-Iodo-2,5-dimethoxyphenyl)-2-aminopropane (DOI 1 mg/kg, serotonin 2 agonist). However, the combined application of both agonists increased (+44%) preprotachykinin message levels, but these changes were restricted to the dorsomedial striatum. In adult animals depleted of dopamine as neonates, striatal preprotachykinin mRNA expression was reduced by approximately 50%. From this lowered level of basal expression, DOI or SKF-38393 raised preprotachykinin mRNA levels within the dorsomedial, but not the dorsolateral striatum. Furthermore, co-stimulation of dopamine D1 and serotonin 2 receptors produced a nearly four-fold induction of preprotachykinin message levels in the dorsomedial striatum that was significantly greater than either agonist alone. Application of both agonists also elevated preprotachykinin mRNA expression within the dorsolateral striatum, but to a lesser extent. All increases in preprotachykinin mRNA resulting from co-application of SKF-38393 and DOI were prevented by pretreatment with either SCH-23390 (1 mg/kg, dopamine D1 antagonist) or ritanserin (1 mg/kg, serotonin 2 antagonist). Alternately, preproenkephalin mRNA expression was unaffected by dopamine D1 receptor stimulation, but was slightly elevated by DOI or both agonists together (42–58%) in intact animals. However, neither agonist treatment in this experiment significantly altered preproenkephalin mRNA expression in the dopamine-depleted striatum which was elevated in response to dopamine lesion alone. Dopamine depletion appears to promote a synergistic interaction between dopamine D1 and serotonin 2 receptors that leads to enhanced expression of striatal preprotachykinin mRNA levels. The localization of this phenomenon to tachykinin neurons of the direct striatonigral pathway specifically within the dorsomedial regions of the rostral striatum may be relevant to the problem of dyskinetic behaviors which arise during the pharmacological treatment of movement disorders.

Expression of Striatal Preprotachykinin mRNA in Symptomatic and Asymptomatic 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Exposed Monkeys Is Related to Parkinsonian Motor Signs

Striatal preprotachykinin (PPT) gene expression and [(3)H]mazindol binding were examined in monkeys exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Some animals (n = 5) became moderately to severely parkinsonian after receiving large doses of MPTP over 9-30 d and remained symptomatic for a relatively short time (3 weeks to 3 months; acutely symptomatic group). A second group of animals (n = 5) received low doses of MPTP (1.5-12 months), developed cognitive impairments but displayed no gross motor deficits (asymptomatic group), and were killed 3-12 months after their final dose of MPTP. Other animals became moderately to severely parkinsonian after receiving escalating doses of MPTP (>6 months; n = 4) or high doses of MPTP (<1 month; n = 1) and remained symptomatic for 2.5-5.75 years (chronically symptomatic group). All MPTP-treated animals had extensive losses of [(3)H]mazindol binding in dorsal striatal sensorimotor regions with asymptomatic animals generally having a lesser degree of damage. However, PPT mRNA levels differed sharply among treatment groups. Symptomatic animals (acutely and chronically parkinsonian) had significantly decreased PPT mRNA levels in most striatal regions. In asymptomatic animals, PPT mRNA expression was not significantly different from that measured in control animals, despite decreases in [(3)H]mazindol binding in some striatal regions of similar magnitude to those observed in symptomatic animals. These observations suggest that PPT gene expression may be directly related to expression of parkinsonian motor symptomatology regardless of duration of MPTP exposure, duration of the parkinsonism, or extent of dopamine denervation. These results imply that the direct striatal output circuit may have a greater contribution to expression of parkinsonian symptomatology than proposed previously.

Early Changes in Neuropeptide mRNA Expression in the Striatum Following Reserpine Treatment

Chronic dopamine depletion produces neurochemical changes within the striatum as well as enhanced behavioral and metabolic responses to dopamine agonists. Changes in striatal neuropeptides have been consistently described, including increased expression of preproenkephalin mRNA and decreased expression of preprotachykinin and prodynorphin mRNA. Acute dopamine depletion following treatment with reserpine also produces enhanced behavioral and metabolic responses to agonist treatment which develop rapidly. In the present study, we used in situ hybridization histochemistry to investigate whether acute neurochemical changes occur following reserpine treatment. We evaluated neuropeptide mRNA expression in the striatum and nucleus accumbens at several time points from 6 to 120 h following single doses of reserpine and AMPT. The aim of these studies was to determine if changes in neuropeptide mRNA expression occur following acute dopamine depletion and whether such changes are specific to the striatum. Changes in striatal neuropeptide mRNA expression developed rapidly. Preproenkephalin mRNA expression by striatopallidal neurons was unchanged at 48 h, but increased by 44% at 120 h. Preprotachykinin mRNA expression in striatonigral neurons was increased at 6 h and then fell, with a maximal decrease of 45% at 48 h and partial recovery by 120 h. Prodynorphin mRNA expression was unchanged. Expression of preproenkephalin and preprotachykinin mRNA was also examined in subregions of the striatum and the nucleus accumbens. Expression of preproenkephalin mRNA was uniform in the striatum and higher in the core than the shell of the nucleus accumbens. Preprotachykinin mRNA expression in the striatum was higher in the lateral quadrants and was higher in the shell than in the core of the nucleus accumbens. The changes in neuropeptide mRNA following treatment with reserpine were only found in the striatum. These data provide further evidence for early alterations in neuronal function in the striatum following acute dopamine depletion and suggest that neuropeptide expression by striatonigral neurons may be more rapidly regulated in response to changes in dopamine levels.

Striatal preprotachykinin mRNA levels are regulated by stimulatory agents and dopamine D1 receptor manipulation in rodent organotypic slice cultures

We have utilized an organotypic slice culture system to determine factors which directly influence the expression of striatal preprotachykinin (PPT) mRNA. Striatal slices were generated from 3-day-old male rat pups and cultured on Millicell-CM inserts in serum-containing media. Under these conditions, striatal PPT mRNA levels fell significantly (−55.7±6.2%) in slices cultured for 2 days in vitro (2DIV) as compared to slices placed in culture for 3 h (0DIV). However, striatal PPT mRNA expression did not decline further in 4DIV cultured slices (−59.6±7.1%). When 2DIV slices were exposed to combined high potassium (K +, 10 mM) and forskolin (10 μM) stimulation for 3 h, PPT mRNA levels were increased within areas of the brain normally associated with tachykinin production. Application of the dopamine (DA) D1 receptor agonist SKF-38393 (10 μM) at 2DIV for 3 h also increased (+162.9±28.9%) PPT mRNA expression, but increases were localized within the striatum. SKF-38393-stimulated increases were completely blocked by the D1 antagonist SCH-23390 (10 μM), which alone had no effect on mRNA levels. However, a 3-h incubation with SKF-38393 on 0DIV slice cultures did not affect PPT mRNA expression whereas SCH-23390 decreased PPT message levels (−24.5±5.4%). These findings indicate that tachykinin gene expression is inducible within slice culture preparations and that the maintenance of normal striatal PPT mRNA levels depends on DA D1 receptor tone.

Effects of antipsychotic drugs on cholecystokinin and preprotachykinin (substance P) mRNA expression in the rat hippocampal formation

To assess the involvement of substance P (SP) and cholecystokinin (CCK) in the effects of antipsychotic drugs, preprotachykinin-A (PPT-A) and CCK mRNA expression was studied in the hippocampal formation using in situ hybridisation following 21 daily i.p. injections with the typical antipsychotic drug haloperidol (1 mg/kg) and the atypical drug clozapine (15 mg/kg). PPT-A mRNA levels were increased in the hippocampal CA3 subregion and in the entorhinal cortex after haloperidol, whereas a decrease was observed in the CA1 after clozapine. CCK mRNA levels increased in the CA1, the entorhinal cortex and in hilus, following both haloperidol and clozapine. It is suggested that earlier findings of increased SP levels in the hippocampal formation of schizophrenics may be a consequence of haloperidol treatment and that reduced hippocampal CCK and CCK mRNA levels found earlier in schizophrenics do not result from antipsychotic drug treatment. These results are consonant to the hypothesis that increased cortical CCK transmission may be beneficial in the treatment of psychosis.

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.