Svenja E. Sander

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 dtsz 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 dtsz hamsters cannot be excluded. Therefore, we determined the density of calretinin-immunoreactive (CR+) interneurons in the dtsz 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 dtsz 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 (dtsz hamsters: -38%, controls: -54%). These results demonstrate that alterations of striatal interneuron density in dtsz 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 dtsz mutant at an age of maximum expression of dystonia.

Age-related changes in striatal nitric oxide synthase-immunoreactive interneurones in the dystonic dt sz mutant hamster

The dt sz mutant hamster represents a model of paroxysmal dyskinesia in which dystonic episodes can be age‐dependently induced by stress. GABAergic interneurones which co‐express calcium binding proteins were found to be reduced in the striatum of the dtsz mutant. Other types of striatal interneurones have so far not been examined. In the present study, we therefore determined the density of nitric oxide synthase (NOS)‐immunoreactive interneurones in the striatum of the dtsz mutant in comparison with nondystonic control hamsters. At the age of most marked expression of dystonia (30–40 days of life), the density of NOS‐positive interneurones was decreased in the striatum of dtsz hamsters (−21%) in comparison with age‐matched nondystonic control hamsters. Spontaneous remission of dystonia (age >90 days) coincided with a normalization of the density of NOS‐reactive interneurones within the whole striatum of dtsz hamsters, but there remained a reduced density in distinct subregions. Together with previous findings the present data indicate that the development of striatal interneurones is retarded in mutant hamsters. The age‐related deficit of NOS‐reactive interneurones may at least in part contribute to an abnormal activity of striatal GABAergic projection neurones and thereby to the age–dependent dystonic syndrome in the dtsz mutant.

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

Brivaracetam and seletracetam, two new SV2A ligands, improve paroxysmal dystonia in the dtsz 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.

Towards optimized anesthesia protocols for stereotactic surgery in rats: Analgesic, stress and general health effects of injectable anesthetics. A comparison of a recommended complete reversal anesthesia with traditional chloral hydrate monoanesthesia

Although injectable anesthetics are still widely used in laboratory rodents, scientific data concerning pain and distress during and after stereotactic surgery are rare. However, optimal anesthesia protocols have a high impact on the quality of the derived data. We therefore investigated the suitability of recommended injectable anesthesia with a traditionally used monoanesthesia for stereotactic surgery in view of optimization and refinement in rats. The influence of the recommended complete reversal anesthesia (MMF; 0.15mg/kg medetomidine, 2mg/kg midazolam, 0.005mg/kg fentanyl; i.m.) with or without reversal and of chloral hydrate (430mg/kg, 3.6%, i.p.) on various physiological, biochemical and behavioral parameters (before, during, after surgery) was analyzed. Isoflurane was also included in stress parameter analysis. In all groups, depth of anesthesia was sufficient for stereotactic surgery with no animal losses. MMF caused transient exophthalmos, myositis at the injection site and increased early postoperative pain scores. Reversal induced agitation, restlessness and hypothermia. Even the low concentrated chloral hydrate led to peritonitis and multifocal liver necrosis, corresponding to increased stress hormone levels and loss in body weight. Increased stress response was also exerted by isoflurane anesthesia. Pronounced systemic toxicity of chloral hydrate strongly questions its further use in rodent anesthesia. In view of undesired effects of MMF and isoflurane, thorough consideration of anesthesia protocols for particular research projects is indispensable. Reversal should be restricted to emergency situations. Our data support further refinement of the current protocols and the importance of sham operated controls.

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.

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 dtsz 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 dtsz 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 dtsz 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 dtsz 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 dtsz 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 dtsz hamster. Synapse 2011.

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.

Pharmacological and autoradiographic studies on the pathophysiological role of GABAB receptors in the dystonic hamster: pronounced antidystonic effects of baclofen after striatal injections

The GABA(B) receptor (GABA(B)R) agonist baclofen is known to have a beneficial potency in patients who suffer from dystonia, a neurological syndrome characterized by involuntary co-contractions of opposing muscles. The underlying mechanisms of this movement disorder are still unclear. Previous studies in the dt(sz) hamster, an animal model of primary paroxysmal dystonia, revealed alterations of the GABAergic system, including a reduction of striatal GABAergic interneurons and an altered GABA(A) receptor (GABA(A)R) binding in several brain regions. In order to clarify the pathophysiological role of central GABA(B)Rs in the hamster mutant, we performed pharmacological and receptor autoradiographic studies. Systemic administration of the GABA(B)R agonist (R)-baclofen (1.5, 2.5 and 3.5 mg/kg i.p.) produced pronounced antidystonic effects in the dt(sz) hamster. Striatal microinjections of baclofen (0.125, 0.25 and 0.5 microg/0.5 microl) also strongly reduced the severity of dystonia. Single striatal administration of the selective GABA(B)R antagonist CGP 35348 [(3-Aminopropyl)(diethoxymethyl)phosphinic acid, 5 and 10 microg/0.5 microl] did not influence the severity of dystonia, but antagonized the antidystonic effect of baclofen. For receptor autoradiographic studies, [H3]-CGP 54626 ([S-(R*,R*)]-[3-[[1-(3,4-Dichlorophenyl)ethyl]amino]-2-hydroxypropyl](cyclohexylmethyl)phosphinic acid) binding was determined in dt(sz) hamsters in comparison to non-dystonic control hamsters. [H3]-CGP 54626 binding was not altered in motor areas but in some limbic structures of dt(sz) hamsters. In view of the absence of striatal changes in GABA(B) binding, the strong antidystonic effect of baclofen after its striatal microinjection is probably related to a suppression of a pathophysiologically increased synaptic activity.

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.