Stimulated serotonin release from hyperinnervated terminals subsequent to neonatal dopamine depletion regulates striatal tachykinin, but not enkephalin gene expression

Dopamine (DA) depletion in neonatal rodents results in depressed tachykinin and elevated enkephalin gene expression in the adult striatum (STR). Concurrently, serotonin (5-HT) fibers sprout to hyperinnervate the DA-depleted anterior striatum (A-STR). The present study was designed to determine if increased 5-HT release from sprouted terminals influences dysregulated preprotachykinin (PPT) and preproenkephalin (PPE) mRNA expression in the DA-depleted STR. Three-day-old Sprague-Dawley rat pups received bilateral intracerebroventricular injections of vehicle or the DA neurotoxin 6-hydroxydopamine (6-OHDA, 100 microg). Two months later, rats received a single intraperitoneal injection of vehicle or the acute 5-HT releasing agent p-chloroamphetamine (PCA; 10 mg/kg). Rats were killed 4 h later and striata processed for monoamine content by HPLC-ED and mRNA expression by in situ hybridization within specific subregions of the A-STR and posterior striatum (P-STR). 6-OHDA treatment severely (>98%) reduced striatal DA levels, while 5-HT content in the A-STR was significantly elevated (doubled), indicative of 5-HT hyperinnervation. Following 6-OHDA, PPT mRNA levels were depressed 60-66% across three subregions of the A-STR and 52-59% across two subregions of the P-STR, while PPE mRNA expression was elevated in both the A-STR (50-62%) and P-STR (55-82%). PCA normalized PPT mRNA levels in all regions of the DA-depleted A-STR and P-STR, yet did not alter PPE levels in either dorsal central or medial regions from 6-OHDA alone, but reduced PPE to control levels in the dorsal lateral A-STR. These data indicate that increased 5-HT neurotransmission, following neonatal 6-OHDA treatment, primarily influences PPT-containing neurons of the direct striatal output pathway.     

NMDA receptor antagonism modifies the synergistic regulation of striatal tachykinin gene expression induced by dopamine D(1) and serotonin(2) receptor stimulation following neonatal dopamine depletion

Co-application of SKF-38393 (dopamine D(1) agonist; 1 mg/kg) and DOI (serotonin(2) agonist; 1 mg/kg) induced a synergistic increase in striatal preprotachykinin (PPT) mRNA levels in adult rats 60 days after neonatal intracerebroventricular injection of 6-hydroxydopamine. This magnitude of response was not observed in intact (vehicle-injected) rats and was restricted to the dorsomedial (DM, 333+/-25% of lesion) subregion of the anterior striatum, with smaller increases observed in the dorsolateral striatum (DL, 206+/-26% of lesion). A single i.p. injection of MK-801 (NMDA antagonist; 0.1 mg/kg) administered prior to dopamine D(1) (D(1)) and serotonin(2) (5-HT(2)) receptor co-stimulation suppressed the synergistic regulation of PPT mRNA expression in the DM striatum, but also produced a large increase in PPT message levels within the DL striatum (321+/-17% of lesion). These data suggest that the synergistic regulation of PPT mRNA within the DM striatum induced by D(1)/5-HT(2) receptor co-stimulation in the dopamine lesioned rat is dependent on NMDA receptor activity. However, MK-801 may simultaneously potentiate striatal PPT mRNA expression by a separate mechanism due to the changed environment of the dopamine-depleted basal ganglia.     

Adenosine A2a receptor antagonists attenuate striatal adaptations following dopamine depletion

The motor symptoms of Parkinson's disease (PD) are widely thought to arise from an imbalance in the activity of the two major striatal efferent pathways following the loss of dopamine (DA) signaling. In striatopallidal, indirect pathway spiny projection neurons (iSPNs), intrinsic excitability rises following the loss of inhibitory D2 receptor signaling. Because these receptors are normally counterbalanced by adenosine A2a adenosine receptors, antagonists of these receptors are being examined as an adjunct to conventional pharmacological therapies. However, little is known about the effects of sustained A2a receptor antagonism on striatal adaptations in PD models. To address this issue, the A2a receptor antagonist SCH58261 was systemically administered to DA-depleted mice. After 5 days of treatment, the effects of SCH58261 on iSPNs were examined in brain slices using electrophysiological and optical approaches. SCH58261 treatment did not prevent spine loss in iSPNs following depletion, but did significantly attenuate alterations in synaptic currents, spine morphology and dendritic excitability. In part, these effects were attributable to the ability of SCH58261 to blunt the effects of DA depletion on cholinergic interneurons, another striatal cell type that co-expresses A2a and D₂ receptors. Collectively, these results suggest that A2a receptor antagonism improves striatal function in PD models by attenuating iSPN adaptations to DA depletion.     

Striatal tachykinin and enkephalin mRNAs are normalized by serotonin2 and NMDA manipulation following dopamine depletion

We have examined the effects of serotonin2 (5-HT2) stimulation and NMDA antagonism on preprotachykinin (PPT) and preproenkephalin (PPE) gene regulation in the dopamine (DA) depleted striatum. Following DA lesions, PPT mRNA expression was reduced (dorsomedial (DM) 44 +/- 9%, dorsolateral (DL) 40 +/- 4%), whereas PPE message levels were elevated (DM 207+/-28%, DL 198+/-25%). Within this state of dysregulated gene activity, DOI (5-HT2 agonist) increased PPT message levels (174 +/- 5%, DM; 153 +/- 13%, DL) without affecting PPE gene expression. In addition, MK-801 (NMDA antagonist) decreased PPE message levels (DM 59 +/- 10%, DL 52 +/- 7%) without significantly altering PPT mRNA expression. Combined application of DOI and MK-801 resulted in normalization of both PPTand PPE message. Statistical analysis revealed no drug interactions in this paradigm suggesting independent mechanisms for 5-HT2 and NMDA receptors in controlling neuropeptide production following DA depletion.     

The adaptation of enkephalin, tachykinin and monoamine neurons of the basal ganglia following neonatal dopaminergic denervation is dependent on the extent of dopamine depletion.

This study examined whether dopamine (DA) is necessary for the normal development of striatal enkephalin and striatonigral tachykinin peptide systems. The neurotoxin, 6-hydroxydopamine (6-OHDA) was used to induce DA deficiency on the third day of the postnatal period in Sprague-Dawley rat pups. The animals were sacrificed at 60 days of age. The levels of Met5-enkephalin (ME) and substance P (SP) were determined by radioimmunoassay and preproenkephalin (PPE) and preprotachykinin (PPT) mRNA abundance in the striatum were assessed by hybridization analysis. The concentrations of DA, 5-hydroxytryptamine (5-HT) and their acid metabolites were determined by high-pressure liquid chromatography with electrochemical detection. The lesioned animals were grouped on the basis of the degree of loss of DA, and changes in ME, SP and 5-HT systems were correlated with respect to the degree of DA loss. The nature and extent of the changes in these systems were dependent on the degree of DA depletion. A loss of more than 90% DA was necessary to result in increased levels of ME and its PPE mRNA and reduced levels of SP and its PPT mRNAs; however, increased levels of 5-HT could be observed at a lower degree of DA loss. The results indicate that the normal development of enkephalin and tachykinin and 5-HT systems of basal ganglia are dependent on the availability of DA and/or the integrity of the nigrostriatal dopaminergic neurons. The results are relevant to our further understanding of the neurobiology of DA deficiency disorders.     

Serotonin 2A receptor gene polymorphism is not associated with completed suicide.

Several lines of evidence indicate that a serotonergic dysfunction is involved in the biological susceptibility to suicide. Recently, the A-1438G polymorphism of the serotonin 2A (5-HT2A) receptor gene has been suggested to be associated with suicide, but the results are inconsistent. We examined whether the A-1438G polymorphism of the 5-HT2A receptor gene was associated with suicide itself using 151 Japanese completed suicides. No significant difference in genotype distribution or allele frequencies of the polymorphism was found between the completed suicides and the comparison group. We conclude that the A-1438G polymorphism of the 5-HT2A receptor gene is not likely to have a major effect on the biological susceptibility of suicide.     

MK-801 prevents dopamine D1 but not serotonin 2A stimulation of striatal preprotachykinin mRNA expression

We examined dopamine (DA) and serotonin (5-HT) receptor-mediated influences on striatal preprotachykinin (PPT, tachykinin precursor) mRNA regulation in organotypic slice cultures. A 3 h exposure to SKF-38393 (10 microM, DA D1 agonist) or DOI (10 microM, 5-HT2 agonist) increased PPT mRNA levels to 196.4% and 154.0%, respectively. Responses to SKF-38393 were prevented by SCH-23390 (10 microM, D1 antagonist) whereas DOI-stimulated increases were prevented by ketanserin (10 microM, 5-HT2A antagonist). Since striatal tachykinin neurons also possess NMDA receptors that regulate gene expression, stimulation of PPT message levels was examined in the presence of MK-801, a non-competitive NMDA antagonist. Alone, MK-801 (10 nM) did not significantly alter basal PPT message levels. However, MK-801 prevented SKF-38393-stimulated increases in PPT mRNA expression while DOI-induced expression was not affected. These results provide evidence that D1 regulation of striatal tachykinin expression is dependent on NMDA-type glutamate neurotransmission while 5-HT2A regulation appears independent.     

Depletion of striatal β-phenylethylamine following dopamine but not 5-HT denervation

The effects of lesions of the substantia nigra (electrolytic 2 mA 10 sec, or 6-OHDA 2 or 8 micrograms) and of the midbrain raphé nuclei (electrolytic 2 X 1.0 mA 10 sec) at 7 days postlesion on striatal levels of beta-phenylethylamine, DA, DOPAC, HVA, 5-HT and 5-HIAA and on hypothalamic levels of beta-phenylethylamine, DA, NA, 5-HT and 5-HIAA were investigated. In the presence of deprenyl (2 mg kg-1 2 hr SC), both electrolytic and 6-OHDA-induced dopamine-depleting lesions of the nigra but not 5-HT-depleting lesions of the raphé nuclei resulted in a marked decrease in the accumulation of beta-phenylethylamine. The marked reduction in accumulation of striatal beta-phenylethylamine in response to lesions of the substantia nigra indicates that the intraneuronal compartment is a major site of striatal beta-phenylethylamine synthesis. An equivalent decrease (approximately 40%) in the accumulation of 5-HT was observed following electrolytic lesions of the substantia nigra or raphé nuclei after administration of L-5-HTP (200 mg kg-1 hr IP). As L-5-HTP at the dose employed in this study is taken up non-selectively by both DA- and 5-HT-containing neurones the loss of L-AAD following nigral and raphé lesions was apparently equivalent. These results indicate that depletion of beta-phenylethylamine may not be simply attributable to a general loss of L-AAD following lesions of monoamine-containing neurones and suggest either co-localisation of beta-phenylethylamine and DA or the existence of distinct beta-phenylethylamine-containing neurones.     

Differential regulation of the dopamine D1, D2 and D3 receptor gene expression and changes in the phenotype of the striatal neurons in mice lacking the dopamine transporter

Mice with a genetic disruption of the dopamine transporter (DAT-/-) exhibit locomotor hyperactivity and profound alterations in the homeostasis of the nigrostriatal system, e.g. a dramatic increase in the extracellular dopamine level. Here, we investigated the adaptive changes in dopamine D1, D2 and D3 receptor gene expression in the caudate putamen and nucleus accumbens of DAT-/- mice. We used quantitative in situ hybridization and found that the constitutive hyperdopaminergia results in opposite regulations in the gene expression for the dopamine receptors. In DAT-/- mice, we observed increased mRNA levels encoding the D3 receptor (caudate putamen, +60-85%; nucleus accumbens, +40-107%), and decreased mRNA levels for both D1 (caudate putamen, -34%; nucleus accumbens, -45%) and D2 receptors (caudate putamen, -36%; nucleus accumbens, -33%). Furthermore, we assessed the phenotypical organization of the striatal efferent neurons by using double in situ hybridization. Our results show that in DAT+/+ mice, D1 and D2 receptor mRNAs are segregated in two different main populations corresponding to substance P and preproenkephalin A mRNA-containing neurons, respectively. The phenotype of D1 or D2 mRNA-containing neurons was unchanged in both the caudate putamen and nucleus accumbens of DAT-/- mice. Interestingly, we found an increased density of preproenkephalin A-negative neurons that express the D3 receptor mRNA in the nucleus accumbens (core, +35%; shell, +46%) of DAT-/- mice. Our data further support the critical role for the D3 receptor in the regulation of D1-D2 interactions, an action being restricted to neurons coexpressing D1 and D3 receptors in the nucleus accumbens.     

Expression of D1 but not D2 dopamine receptors in striatal neurons producing neurokinin B in rats

Neostriatal projection neurons are known to be largely divided into two groups, striatoentopeduncular/striatonigral and striatopallidal neurons, which mainly express D1 and D2 dopamine receptors, respectively. Recently, a small population of neostriatal neurons have been reported to produce neurokinin B (NKB), and send their axons mainly to the basal forebrain regions. To reveal which type of dopamine receptors were expressed by these NKB-producing neurons, we examined rat striatal neurons by combining immunofluorescence labeling for preprotachykinin B (PPTB), the precursor of NKB, and fluorescence in situ hybridization labeling for dopamine receptors. Fluorescent signals for D1 receptor mRNA were detected in 85-89% of PPTB-immunopositive neurons in the neostriatum, accumbens nucleus and lateral stripe of the striatum, whereas almost no signal for D2 receptor was observed in PPTB-positive striatal neurons. To further reveal intracellular signaling downstream of D1 receptor in PPTB-producing neurons, we used a double immunofluorescence labeling method to study the localization of some substrates for protein kinase A (PKA), which was known to be activated by D1 receptor. Although only 3-7% of PPTB-immunopositive striatal neurons displayed immunoreactivity for dopamine- and cAMP-regulated phosphoprotein of 32 kDa, a well-known PKA substrate expressed in the two major groups of neostriatal projection neurons, 60-64% of PPTB-positive striatal neurons exhibited immunoreactivity for striatal-enriched tyrosine phosphatase. These results suggest that NKB-producing neostriatal neurons are similar to striatoentopeduncular/striatonigral neurons in the usage of dopamine receptor subtypes, but different from the two major groups of neostriatal projection neurons in terms of the downstream signaling of dopamine receptors.     

Reserpine treatment stimulates enkephalin and D2 dopamine receptor gene expression in the rat striatum.

We investigated the effect of catecholamine depletion on gene expression for preproenkephalin A (PPA) and D2 dopamine receptor (D2R) in the rat nigrostriatal complex, using quantitative Northern blot analysis. The D2R probe indifferently recognizes the two mRNA isoforms generated by alternative splicing from the same gene. Short-term and chronic reserpine treatment increase the level of PPA and D2R mRNA in the striatum in a complex manner. For short-term treatment, we injected 10 mg/kg of reserpine the first day, 5 mg/kg 24 h later and sacrificed the rats at various times after the last injection. This treatment resulted in an increase of the level of PPA mRNA by 50% and D2R mRNA up to 150%. For chronic treatment, we injected 0.5 mg/kg of reserpine for 21 days, sacrificed the rats one day after the last injection and observed an increase in PPA and D2R mRNA levels by 100%. Statistical analysis revealed that the PPA mRNA level after chronic treatment was significantly higher from the one obtained after short-term treatment while no such difference was observed for the D2R mRNA. In contrast, reserpine treatment does not modify the level of D2R mRNA in the substantia nigra suggesting that catecholamine depletion has postsynaptic but not presynaptic consequences in the rat nigrostriatal complex. These results demonstrate that reserpine acts at the gene or the mRNA level to induce dopamine supersensitivity in striatal dopaminoceptive neurons.     

Sprouting of striatal serotonin nerve terminals following selective lesions of nigro-striatal dopamine neurons in neonatal rat

The effects of neonatal intracisternal 6-hydroxydopamine (6-OHDA; 50 micrograms) treatment on striatal serotonin (5-HT) nerve terminals in rat have been characterized using histo- and neurochemical methods. The 6-OHDA lesion caused a 60% reduction of striatal dopamine (DA) concentration when analyzed in the adult stage, while 5-HT levels were increased by about 40% and 3H-5-HT uptake in vitro was increased by about 60%. Using computerized image analysis, a marked increase in 5-HT-like immunoreactive terminal density was found in both rostral (+200%) and caudal (+50%) striatum. Pretreatment with the DA uptake blocker amfolenic acid completely counteracted the 6-OHDA-induced alterations in both DA and 5-HT neurons in the striatum, while pretreatment with the noradrenaline uptake blocker desipramine had no significant effects. Regional analysis of 5-HT levels in the CNS after neonatal 6-OHDA treatment or the combined desipramine + 6-OHDA treatment showed no significant effect in any of the brain areas analyzed, apart from the observed 5-HT increase in striatum. It was furthermore observed that the striatal 5-hydroxyindoleacetic acid (5-HIAA)/5-HT ratio was decreased, while the 3,4-dihydroxyphenylacetic acid (DOPAC)/DA ratio was increased following the 6-OHDA lesion, indicating compensatory mechanisms in turnover of transmitters. These alterations were completely reversed after pretreatment with amfolenic acid. The present results support the view that the 5-HT hyperinnervation following neonatal 6-OHDA treatment is a collateral sprouting response induced by lesioning of the striatal DA neurons.     

Changes in D2 but not D1 receptor binding in the striatum following a selective lesion of striatopallidal neurons

We have used the immunotoxin OX7/saporin, a suicide transport agent, to selectively lesion striatopallidal neurons. Following injection of OX7/saporin into the globus pallidus, in situ hybridization for preproenkephalin mRNA was examined in the striatum to confirm successful retrograde neurotoxicity. Comparison of D1 and D2 receptor binding in the striatum demonstrated that D2 but not D1 receptor binding sites are localized to striatopallidal neurons.     

Serotonin is not synthesized, but specifically transported, in the neurons of the hypothalamic dorsomedial nucleus

A small group of neurons in the hypothalamic dorsomedial nucleus (DMN) have been reported to contain serotonin after pharmacological treatments enhancing brain serotonin levels. This study aimed at elucidating whether these neurons are able to synthesize serotonin de novo, and whether they possess a specific serotonin transport mechanism. Serotonin content in these neurons was raised by administration of l -tryptophan and pargyline. Double immunostaining for serotonin and tryptophan hydroxylase (TpOH), the serotonin synthesizing enzyme, revealed that none of the serotonin-containing neuronal somata expressed TpOH. Intracerebro- ventricular colchicine treatment did not result in TpOH-IR in these neurons. Fluoxetine, a specific serotonin transport inhibitor, prevented the accumulation of serotonin in these neurons. The present results thus indicate that the serotonin-containing DMN neurons are not able to synthesize serotonin. Instead, they take up exogenous serotonin via a specific serotonin transport mechanism. As serotonin and DMN are associated with various physiological functions, such as regulation of food intake and modulation of fear and anxiety, the mechanisms revealed in the present study may participate in these clinically important brain functions.     

Striatal depth EEG reveals postsynaptic activity of striatal neurons following dopamine receptor stimulation and blockade

This paper demonstrates a technique for measuring depth electroencephalographic (EEG) recordings from the freely moving mouse. This technique minimizes electrical artifact associated with gross movements by amplifying the current of the EEG signal directly at permanently indwelling electrodes. Stable EEG signals, with high signal-to-noise ratios, can be obtained from these animals while their movement inside the testing cage remains relatively unrestricted. We used this technique to examine the effects of dopamine (DA) receptor agonist and antagonist treatments on depth EEG signals generated within the striatum. Baseline measures of spontaneous striatal EEG activity were obtained prior to drug administration and post-drug measures of striatal activity were subsequently obtained. Apomorphine treatment resulted in desynchronization of striatal EEG signals while haloperidol or sulpiride treatment induced slow wave synchronization. Fast Fourier analysis of EEG signals revealed that DA agonist and antagonist treatment altered spontaneous striatal EEG activity in an opposite manner--relative to baseline signals, apomorphine attenuated low frequency components and augmented higher frequency components of the signal while haloperidol augmented low frequency components and attenuated higher frequency components of the signal. Moreover, mice pretreated with unilateral intracerebral injections of sulpiride and subsequently administered systemic apomorphine simultaneously demonstrated EEG synchronization on the side ipsilateral to the injection of sulpiride and EEG desynchronization on the contralateral side. The population of neurons examined in the medial striatum appear to have the properties of being excitatory to DA agonist stimulation and show decreased activity following DA antagonist treatment. These results suggest that striatal EEG activity may be used as measure of postsynaptic activity of dopaminergic neurons.     

The MAPK pathway is required for depolarization-induced "promiscuous" immediate-early gene expression but not for depolarization-restricted immediate-early gene expression in neurons

Depolarization, growth factors, neurotrophins, and other stimuli induce expression of immediate early genes (IEGs) in neurons. We identified a subset of IEGs, IPD-IEGs, which are induced preferentially by depolarization, but not by neurotrophins or growth factors, in PC12 cells. The "promiscuous" IEGs Egr1 and c-fos, induced by growth factors and neurotrophins, in addition to depolarization, require activation of the MAP kinase signaling pathway for induction in response to KCl depolarization in PC12 cells; MEK1/2 inhibitors block KCl-induced Egr1 and c-fos expression. In contrast, MEK1/2 inhibition has no effect on KCl-induced expression of the known IPD-IEGs in PC12 cells. Additional "candidate" IDP-IEGs were identified by a microarray comparison of genes induced by KCl in the presence vs. the absence of an MEK1/2 inhibitor in PC12 cells. Northern blot analyses demonstrated that representative newly identified candidate IPD-IEGs, as with the known IPD-IEGs, are also induced by a MAP kinase- independent pathway in response to depolarization, both in PC12 cells and in rat primary cortical neurons. Nerve growth factor and epidermal growth factor are unable to induce the expression of the Crem/Icer, Nur77, Nor1, Rgs2, Dusp1 (Mkp1), and Dscr1 genes in PC12 cells, validating their identification as IPD-IEGs. Inhibiting calcium/calmodulin-dependent kinase II (CaMKII), calcineurin, or protein kinase A (PKA) activity prevents KCl-induced IPD-IEG mRNA accumulation, suggesting that the IPD-IEG genes are induced by depolarization in neurons via a combination of calcineurin/PKA- and CaMKII-dependent pathways.