Increased adult neurogenesis in mice with a permanent overexpression of the postsynaptic 5-HT1A receptor.

Depression is among the leading causes of disability and disease burden. Recent studies point to an involvement of altered serotonin1A receptor (5-HT1AR) -mediated adult neurogenesis in depression. However, the exact underlying mechanisms remain unclear, mainly due to the complexity of the serotonergic system with its various receptors and their locations. Mice with permanent overexpression of postsynaptic 5-HT1ARs (OE mice) represent a unique tool for investigating the involvement of postsynaptic 5-HT1ARs in this context. Correct 5-HT1AR coupling and functioning has been demonstrated earlier, indicating that more postsynaptic 5-HT1ARs can be activated in these mice. Initially we examined morphometric parameters of the dentate gyrus (DG) and the prefrontal cortex as they are involved in adult hippocampal neurogenesis and/or depression. The volume of the DG in OE mice was increased in comparison to wildtype controls. We therefore investigated parameters of adult neurogenesis by the bromodeoxyuridine method. Proliferation and survival of newborn cells in the DG of OE mice were significantly increased. Significant increases in survived neurons were only detected in the female but not in the male subgroup. Additional staining for early precursor cells (Sox2) and progenitor cells of the neuronal lineage (doublecortin) showed an increase in type-1/2a as well as in type-2b/3 cells in OE mice. Our study suggests a leading role of the postsynaptic 5-HT1AR in adult hippocampal neurogenesis and might open an important link to depression.

Lower KV7.5 Potassium Channel Subunit Expression in an Animal Model of Paroxysmal Dystonia.

Dystonia is a hyperkinetic disabling movement disorder. In the dt(sz) hamster, a model of paroxysmal dystonia, pronounced antidystonic effects of the KV7.2-5 potassium channel opener retigabine and aggravation of dystonia by a selective KV7.2-5 blocker indicated a pathophysiological role of an abnormal expression of KV7 channels. We therefore investigated the expression of KV7 subunits in brains of dystonic hamsters. While KV7.2 and KV7.3 subunits were unaltered, lower KV7.5 mRNA levels became evident in motor areas and in limbic structures of dystonic hamsters. The KV7.2/3 subunit-preferring channel opener N-(6-chloropyridin-3-yl)-3,4- difluorobenzamide (ICA 27243; 10-30 mg/kg i.p.) failed to reduce the severity of dystonia in mutant hamsters, suggesting that the previously observed antidystonic action of retigabine is mediated by the activation of KV7.5 channels. The experiments indicate a functional relevance for KV7.5 channels in paroxysmal dystonia. We suggest that compounds highly selective for subtypes of KV7 channels, i.e. for KV7.5, may provide new therapeutic approaches.

Role of striatal NMDA receptor subunits in a model of paroxysmal dystonia.

Dystonia is a movement disorder in which abnormal plasticity in the basal ganglia has been hypothesized to play a critical role. In a model of paroxysmal dystonia, the dt(sz) mutant hamster, previous studies indicated striatal dysfunctions, including an increased long-term potentiation (LTP). Beneficial effects were exerted by subunit-unspecific antagonists at NMDA receptors, which blocked LTP. NR2B subtype selective antagonists aggravated dystonia after systemic treatment in dt(sz) hamsters, suggesting that beneficial effects involved the NR2A receptor subtype. In the present study, NVP-AAM077, an antagonist with preferential activity on NR2A-containing NMDA receptors, exerted significant antidystonic effects in mutant hamsters after systemic administration (20 and 30mg/kg i.p.) and delayed the onset of a dystonic episode after intrastriatal injections (0.12 and 0.24µg). As shown by present electrophysiological examinations in corticostriatal slices of dt(sz) hamsters and non-dystonic control hamsters, NVP-AAM077 (50nM) completely blocked LTP in dt(sz) slices, but did not exert significant effects on LTP in non-dystonic controls. In contrast, the NR2B antagonist Ro 25-6981 (1-10µmol) reduced LTP to a lower extent in dt(sz) mutant hamsters than in control animals. By using quantitative RT-PCR, the NR2A/NR2B ratio was found to be increased in the striatum, but not in the cortex of mutant hamsters in comparison to non-dystonic controls. These data indicate that NR2A-mediated activation may be involved in the pathophysiology of paroxysmal dystonia. Since significant antidystonic effects were observed after systemic administration of NVP-AAM077 already at well tolerated doses, antagonists with preferential activity on NR2A-containing NMDA receptors could be interesting candidates for the treatment of dystonia.

The K(V)7.2/3 preferring channel opener ICA 27243 attenuates L-DOPA-induced dyskinesia in hemiparkinsonian rats.

Spontaneous involuntary dystonic and choreatic movements induced by L-DOPA (L-DOPA-induced dyskinesias (LID)) represent a severe complication of long-time pharmacotherapy in Parkinson's disease that deserves novel therapeutics. Previous studies demonstrated antidyskinetics effect of the KV7.2-7.5 channel opener retigabine after acute and chronic treatment in a rat model of LID. We hypothesized that this effect was mainly mediated by KV7.2/3 channels located on striatal projection neurons, as an increased activity of these neurons seems to be involved in the pathophysiology of LID. We therefore examined the acute effects of the KV7.2/3 preferring channel opener ICA 27243 (N-(6-chloro-pyridin-3-yl)-3,4-difluoro-benzamide, 5-15 mg/kg i.p.) on LID in this animal model. Ten and 15 mg ICA 27243 significantly reduced abnormal involuntary movements (AIM) while no negative impact on the antiparkinsonian effect of L-DOPA was observed. However, at the end of the testing session (180 min) AIM scores increased after application of both doses. Further studies have to clarify if this can be avoided by a different application regime. Nevertheless, the present results suggest that selective openers of KV7.2/3 channels might be interesting candidates for the treatment of LID as antidyskinetic effects occurred at well-tolerated doses and did not interfere with the antiparkinsonian effect of L-DOPA.

Changes in dynorphin immunoreactivity but unaltered density of enkephalin immunoreactive neurons in basal ganglia nuclei of genetically dystonic hamsters.

Dystonia is regarded as a basal ganglia disorder. In the dt(sz) hamster, a genetic animal model of paroxysmal dystonia, previous studies demonstrated a reduced density of striatal GABAergic interneurons which inhibit striatal GABAergic projection neurons. Although the disinhibition of striatal GABAergic projection neurons was evidenced in the dt(sz) hamster, alterations in their density have not been elucidated so far. Therefore, in the present study, the density of striatal methionin-(met-) enkephalin (ENK) immunoreactive GABAergic neurons, which project to the globus pallidus (indirect pathway), was determined in dt(sz) and control hamsters to clarify a possible role of an altered ratio between striatal interneurons and projection neurons. Furthermore, the immunoreactivity of dynorphin A (DYN), which is expressed in entopeduncular fibers of striatal neurons of the direct pathway, was verified by gray level measurements to illuminate the functional relevance of an enhanced striato-entopeduncular neuronal activity previously found in dt(sz) hamsters. While the density of striatal ENK immunoreactive (ENK(+) ) neurons did not significantly differ between mutant and control hamsters, there was a significantly enhanced ratio between the DYN immunoreactive area and the whole area of the EPN in dt(sz) hamsters compared to controls. These results support the hypothesis that a disbalance between a reduced density of striatal interneurons and an unchanged density of striatal projection neurons causes imbalances in the basal ganglia network. The consequentially enhanced striato-entopeduncular inhibition leads to an already evidenced reduced activity and an altered firing pattern of entopeduncular neurons in the dt(sz) hamster.

Effects of pharmacological entopeduncular manipulations on idiopathic dystonia in the dt(sz) mutant hamster.

The pathophysiology of idiopathic dystonias is still unknown, but it is regarded as a basal ganglia disorder. Previous experiments in the dt(sz) hamster, a model of primary paroxysmal dystonia, demonstrated reduced discharge rates and an abnormal pattern within the entopeduncular nucleus (EPN), a basal ganglia output structure. To clarify if this is based on abnormal gamma-aminobutyric acid(GABA)ergic or glutamatergic input, microinjections into the EPN were done in mutant hamsters in the present study. The GABA(A) receptor antagonists pentylenetetrazole and bicuculline exerted moderate antidystonic effects, while previous systemic administrations worsened dystonia in the dt(sz) mutant. GABA-potentiating drugs, i.e., the GABA(A) receptor agonist muscimol and the GABA transporter inhibitor 1,2,5,6-tetrahydro-1-[2-[[(diphenylmethylene)amino]oxy]ethyl]-3-pyridinecarboxy-lic acid (NNC-711), which are known to improve dystonia after systemic treatment in mutant hamsters, did not exert significant effects after EPN injections, but NNC-711 tended to increase the severity at the highest dose (2.5 ng bilateral). The NMDA receptor antagonist D(-)-2-amino-5-phosphopentanoic acid (AP-5) retarded the onset of a dystonic attack. However, this effect was not dose dependent and the AMPA receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzol(f)quinoxaline (NBQX) alone or in combination with AP-5 and NNC-711, also failed to show any effects on dystonia. The present data do not provide clear evidence for an enhanced striatal GABAergic input or a reduced glutamatergic activation of the EPN via the subthalamic nucleus, i.e., more pronounced antidystonic effects of GABA(A) receptor antagonists and stronger prodystonic effects of GABAmimetics and glutamate receptor antagonists were expected. Nevertheless, previously found changes in entopeduncular activity probably play a critical pathophysiological role in dystonic hamsters.

Autoradiographic and pharmacological studies on the role of dopamine D3 receptors in genetically dystonic (dt(sz)) hamsters.

Previous examinations demonstrated periodic increases in striatal extracellular dopamine levels during dystonic attacks and changes in dopamine D1 and D2 receptor binding in the dt(sz) mutant hamster, an animal model of paroxysmal non-kinesiogenic dyskinesia in which dystonic episodes can be induced by stress. Since dopamine D3 receptors are involved in the regulation of striatal dopamine release, D3 receptor function was investigated by autoradiographic and pharmacological examinations in mutant hamsters in the present study. [(125)I]7-[[(E)-3-iodoprop-2-enyl]-propylamino]-5,6,7,8-tetrahydronaphthalen-2-ol ([(125)I]7-OH-PIPAT) binding was not significantly altered in the striatum, n. accumbens, ventral pallidum or cerebellum in dt(sz) hamsters in comparison to non-dystonic control hamsters. In line with the unaltered D3 receptor binding, the preferential dopamine D3 versus D2 receptor antagonist U-99194 (5,6-dimethoxy-N,N-dipropyl-2,3-dihydro-1H-inden-2-amine hydrochloride) did not exert significant effects on the severity of dystonia in dt(sz) hamsters at doses of 10 to 40mg/kg which induced hyperlocomotion. These results suggest that periodic elevations of dopamine levels in these animals are not related to D3 receptor dysfunctions.

Brivaracetam and seletracetam, two new SV2A ligands, improve paroxysmal dystonia in the dt sz mutant hamster.

Previous examinations demonstrated antidystonic effects of the synaptic vesicle protein 2A (SV2A) ligand levetiracetam in the dt(sz) mutant hamster, an animal model of paroxysmal non-kinesiogenic dyskinesia in which dystonic episodes can be induced by stress. In the present study, we examined the effects of the two new, high affinity SV2A ligands, brivaracetam and seletracetam, in comparison to levetiracetam on the severity of dystonia in mutant hamsters. Seletracetam (50 and 75 mg/kg i.p.) and brivaracetam (75 mg/kg i.p.) reduced the severity of dystonia to a comparable extent as levetiracetam (50 and 75 mg/kg i.p.). These data confirm the therapeutic potential of these pyrrolidone derivatives for the treatment of paroxysmal dystonia.

Effects of the kynurenine 3-hydroxylase inhibitor Ro 61-8048 after intrastriatal injections on the severity of dystonia in the dt sz mutant.

Striatal dysfunctions seem to play a key role in the pathophysiology of dystonia in the dt(sz) mutant hamster, a model of paroxysmal non-kinesigenic dyskinesia, in which stress precipitates dystonic episodes. Previous examinations have shown changes in kynurenic acid levels and antidystonic effects of the kynurenine 3-hydroxylase inhibitor 3,4-dimethoxy-N-[4-(3-nitrophenyl)thiazol-2-yl]benzenesulfon-amide (Ro 61-8048) after systemic treatment in dt(sz) hamsters. In the present study, intrastriatal injections of Ro 61-8048 (60-80 microg/hemisphere) significantly reduced the severity of dystonia in dt(sz) hamsters, suggesting that kynurenine 3-hydroxylase inhibitors may be interesting candidates for managing dyskinesias which are related to striatal dysfunction.

Age-dependent alterations of striatal calretinin interneuron density in a genetic animal model of primary paroxysmal dystonia.

Various types of hereditary dystonia are regarded as a basal ganglia disorder, but the underlying mechanisms are still unknown. In the dt hamster, a genetic animal model of age-dependent paroxysmal dystonia, recent studies demonstrated a reduced density of striatal parvalbumin-immunoreactive (PV) GABAergic interneurons at an age of maximum severity of dystonia in comparison with age-matched nondystonic controls. So far, alterations of other types of striatal interneurons in dt hamsters cannot be excluded. Therefore, we determined the density of calretinin-immunoreactive (CR) interneurons in the dt mutant at an age of maximum severity and after spontaneous remission of dystonia in comparison with age-matched nondystonic controls using an image analysis system and a stereologic counting method in a blinded fashion. At an age of maximum severity of dystonia, CR interneuron density was significantly lower in dt hamsters in comparison with controls (-20%), whereas no significant differences between the animal groups could be detected after spontaneous remission of dystonia. The comparison of CR interneuron density between young hamsters with those at an age of > 90 days revealed a significant ontogenetic decrease of CR interneurons in both animal groups (dt hamsters: -38%, controls: -54%). These results demonstrate that alterations of striatal interneuron density in dt mutants are not restricted to PV ones. A deficit of CR interneurons that coexpress GABA may contribute to previous findings of disinhibition of striatal projection neurons in the dt mutant at an age of maximum expression of dystonia.