Dt Sz Mutant Research Articles

P 2 Effects of deep brain stimulation (DBS) in the entopeduncular nucleus (EPN) in dystonic dtsz hamsters

Introduction: DBS of the globus pallidus internus (GPi; entopeduncular nucleus, EPN, in rodents) has become important for the treatment of generalized dystonia. There is evidence that striatal dysfunctions cause a disturbed thalamocortical inhibition via altered neuronal activity in the GPi/EPN. However, the pathophysiology as well as the mechanisms of DBS are largely unknown, which hampers the detection of drug targets. In our project of the CRC “Electrically active implants” we aim to elucidate mechanisms of DBS in animal models of dystonia. We recently found that short-term (3 h) EPN-DBS with 130 Hz (50 µA, 60 µs) improves paroxysmal dystonia in the dt sz mutant hamster 1 and reduces spontaneous excitatory cortico-striatal activity in brain slices of this model 2 , indicating fast effects in synaptic plasticity. In the present study, we therefore examined whether short-term DBS leads to changes of c-Fos, an immunohistochemical marker of neuronal activity, in EPN-related network regions. Materials & Methods: For immunohistochemistry we used brains of stimulated dt s z hamsters, in which DBS had improved dystonia, as well as sham-stimulated and naïve animals and performed cell counting and fluorescence intensity measurements of c-Fos within the basal ganglia network. In addition, we performed double labeling of c-Fos with GAD67 and with parvalbumin (PV), respectively, in brain slices of the same animals. Results: After DBS vs. sham, c-Fos + around the electrode was increased. Unexpectedly, c-Fos + cells were decreased in deep cerebellar nuclei (DCN) after DBS, but no changes became evident within the whole EPN, habenula, ventromedial thalamus, cortex and striatum. Cell counting of c-Fos activated GAD67 + as well as activated PV + cells showed no differences between the groups in motor cortex and striatum. Conclusion: With regard to recent electrophysiological data, we expected c-Fos changes especially in cortical GABAergic neurons after DBS in dt sz hamsters. The lack of changes within the basal ganglia network could be related to the short duration of stimulation or the time interval between stimulation and c-Fos staining. Ongoing long-term EPN stimulations, now feasible by a new fully implantable stimulator 3 , probably lead to more pronounced effects within the network and will be useful to verify the DCN effects. Funded by the DFG within the Collaborative Research Centre 1270 ELAINE.

P 16 Effects of pallidal deep brain stimulation on striatal medium spiny neurons in the animal model of the dystonic dtsz mutant hamster

Introduction: Electric stimulation of the deep brain nuclei, such as globus pallidus internus (GPi), indicated the most relevant therapeutic option for patients with severe dystonias. However, the mechanisms of the deep brain stimulation (DBS) of the GPi are far from understood. Dystonia, a hyperkinetic movement disorder, is thought to result from an imbalance in the direct and indirect pathway arises from disturbance in the striatum. In the dt sz mutant hamster, a model of inherited generalized, paroxysmal dystonia, it has been already shown that the number of parvalbumin-positive gabaergic interneurons declined, which leads to uncontrolled projections via the medium spiny neurons (MSN) (see also Paap, et al. (2020), DOI: 10.1016/j.nbd.2020.105163). We hypothesized that DBS via backfiring, or indirectly via thalamic and cortical coupling, modifies striatal network function. Materials & Methods: The dt sz mutant hamsters were bilaterally implanted with stimulation electrodes targeting the entopeduncular nucleus (EPN; equivalent of the human GPi). DBS (130 Hz, rectangular pulse of 50 µA and 60 µs) and sham-DBS was performed in vivo in unanaesthetized animals for three hours. Acute brain slices of stim- and sham-groups were immediately prepared after the DBS has been terminated. Additionally, acute brain slices were obtained from untreated dt sz mutant hamsters (native group) to assess the effects of electrode implantation. With whole-cell patch clamp recordings, we investigated the spontaneous cortico-striatal synaptic activity and the characteristics of the D1- (direct pathway) and D2- (indirect pathway) MSN, which are capable of being differentiated by adding the D2-agonist sumanirole. Additionally, molecular analysis was performed for studying the DBS effect on ion channel gene expression. Results: In view of spontaneous release activity from cortical projections, our study indicated a strong dampening effect on the frequency of spontaneous miniature excitatory postsynaptic currents (mEPSC) of the stim-group in contrast to sham-group. The specific cellular parameters of D1- and D2-MSN, which include resting membrane potential, input resistance, membrane capacity, rheobase, and firing properties of action potentials, did not differ between native-, sham-, and stim- group. Conclusions: To conclude, while short-term EPN-DBS obviously dampens spontaneous presynaptic glutamate release at cortico-striatal synapses, there is no alteration on MSN properties. Future studies will consider the effects of long-term stimulation on synaptic plasticity by a novel implantable stimulation system which enables DBS over weeks (see also Plocksties et al. (2021). DOI: 10.1088/1741-2552/ac23e1). This study is supported by the German Research Foundation (DFG) within the Collaborative Research Centre (SFB 1270/1 ELAINE 299150580)

Deep brain stimulation by optimized stimulators in a phenotypic model of dystonia: Effects of different frequencies

Deep brain stimulation (DBS) of the globus pallidus internus (GPi, entopeduncular nucleus, EPN, in rodents) has become important for the treatment of generalized dystonia, a severe and often intractable movement disorder. It is unclear if lower frequencies of GPi-DBS or stimulations of the subthalamic nucleus (STN) are of advantage. In the present study, the main objective was to examined the effects of bilateral EPN-DBS at different frequencies (130 Hz, 40 Hz, 15 Hz) on the severity of dystonia in the dtsz mutant hamster. In addition, STN stimulations were done at a frequency, proven to be effective by the present EPN-DBS in dystonic hamsters. In order to obtain precise bilateral electrical stimuli with magnitude of 50 μA, a pulse width of 60 μs and defined frequencies, it was necessary to develop a new optimized stimulator prior to the experiments. Since the individual highest severity of dystonic episodes is known to be reached within three hours after induction in dtsz hamsters, the duration of DBS was 180 min. During DBS with 130 Hz the severity of dystonia was significantly lower within the third hour than without DBS in the same animals (p < 0.05). DBS with 40 Hz tended to exert antidystonic effects after three hours, while 15 Hz stimulations of the EPN and 130 Hz stimulations of the STN failed to show any effects on the severity. DBS of the EPN at 130 Hz was most effective against generalized dystonia in the dtsz mutant. The response to EPN-DBS confirms that the dtsz mutant is suitable to further investigate the effects of long-term DBS on severity of dystonia and neuronal network activities, important to give insights into the mechanisms of DBS.

Expression of metabotropic glutamate 5 receptors in the striatum and cortex and effects of modulators on the severity of dystonia in the phenotypic dtsz model

The metabotropic glutamate 5 (mGlu5) receptor has been suggested as therapeutic target for L-Dopa-induced dyskinesia which is often associated with dystonic symptoms. Therefore, we investigated the acute effects of the non-competitive mGlu5 receptor antagonist fenobam as well as the positive modulator 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) on the severity of inherited dystonia in the mutant dtsz hamster, a phenotypic model with age-dependent episodes of dystonia. Fenobam did not exert significant antidystonic effects (20-50 mg/kg intraperinoneal, i.p.). CDPPB (10, 20 mg/kg i.p.) which was expected to worsen dystonia also failed to show any effects on the severity of dystonia. Interestingly, CDPPB caused axial dyskinesia in addition to the dystonic symptoms in mutant hamsters. This adverse effect could not be observed in non-dystonic control hamsters, indicating possible changes in the expression of mGlu5 receptors in dystonic hamsters. The mGlu5 receptor mRNA did not differ between the dtsz mutant and control hamsters, while immunohistochemical studies indicated that the mGlu5 receptor expression was about 35% higher in striatum and cortex of mutant hamsters at the age of high dystonia severity scores, notably not after spontaneous remission of dystonia, compared to age-matched controls. This difference in mGlu5 receptor protein may be due to altered protein conformation instead of protein level, as western blots revealed similar amounts of monomeric and dimeric protein in mutant hamsters versus control. Thus, the present data do not provide clear evidence for an important role of the mGlu5 receptor in the pathophysiology and as a therapeutic target for types of inherited dystonia.

Nanoparticle Design and Characterization for Catalytic Applications in Sustainable Chemistry

GABAergic disinhibition has been suggested to play a critical role in the pathophysiology of several basal ganglia disorders, including dystonia, a common movement disorder. Previous studies have shown a deficit of striatal GABAergic interneurons (IN) in the dtsz mutant hamster, one of the few phenotypic animal models of dystonia. However, mechanisms underlying this deficit are largely unknown. In the present study, we investigated the migration and maturation of striatal IN during postnatal development (18 days of age) and at age of highest severity of dystonia (33 days of age) in this hamster model. In line with previous findings, the density of GAD67-positive IN and the level of parvalbumin mRNA, a marker for fast spiking GABAergic IN, were lower in the dtsz mutant than in control hamsters. However, an unaltered density of Nkx2.1 labeled cells and Nkx2.1 mRNA level suggested that the migration of GABAergic IN into the striatum was not retarded. Therefore, different factors that indicate maturation of GABAergic IN were determined. While mRNA of the KCC2 cation/chloride transporters and the cytosolic carboanhydrase VII, used as markers for the so called GABA switch, as well as BDNF were unaltered, we found a reduced number of IN expressing the alpha1 subunit of the GABAA-receptor (37.5%) in dtsz hamsters at an age of 33 days, but not after spontaneous remission of dystonia at an age of 90 days. Since IN shift expression from alpha2 to alpha1 subunits during postnatal maturation, this result together with a decreased parvalbumin mRNA expression suggest a delayed maturation of striatal GABAergic IN in this animal model, which might underlie abnormal neuronal activity and striatal plasticity.

P 76 Maturation deficits in striatal interneurons in a phenotypic model of dystonia

GABAergic disinhibition has been suggested to play a critical role in the pathophysiology of several basal ganglia disorders, including dystonia, a common incurable movement disorder characterized by involuntary muscle contractions resulting in abnormal movements and/or postures. Previous studies have shown a deficit of striatal GABAergic interneurons (IN) in the dtsz mutant hamster, one of the few phenotypic animal models of dystonia. The resulting lack of inhibition of striatal projection neurons is likely involved in development of dystonic episodes which is corroborated by antidystonic effects of GABA potentiating drugs in the animal model and in dystonia patients. However, mechanisms underlying this deficit in striatal IN are largely unknown. In the present study, we investigated the migration and maturation of striatal IN during postnatal development (18 days of age) and at age of highest severity of dystonia (33 days of age) in the dtsz hamster by immunohistochemistry and real time PCR. In line with previous findings, the density of GAD67 positive IN and the level of parvalbumin mRNA, a marker for fast spiking GABAergic IN, were lower in mutant hamsters than in controls. However, an unaltered density of Nkx2.1 labeled cells and Nkx2.1 mRNA level indicated that the migration of GABAergic IN into the striatum was not retarded. KCC2 cation/chloride transporter and cytosolic carboanhydrase VII mRNA expression, used as markers for maturation of striatal GABAergic signaling, as well as expression of BDNF were unaltered. However, we found a reduced number of IN expressing the alpha1 subunit of the GABAA-receptor (37.5%) in dtsz hamsters at an age of 33 days, but not after spontaneous remission of dystonia at an age of 90 days. Since IN shift expression from alpha2 to alpha1 subunits during postnatal maturation, this result together with a decreased parvalbumin mRNA expression suggest a delayed maturation of striatal GABAergic IN in this animal model, which might underlie abnormal neuronal activity and striatal plasticity. The potential link between alterations in GABAA receptor subunits and GABAergic disinhibition in dystonia deserves further attention in research on the pathophysiology and therapeutic targets.

Altered postnatal maturation of striatal GABAergic interneurons in a phenotypic animal model of dystonia

GABAergic disinhibition has been suggested to play a critical role in the pathophysiology of several basal ganglia disorders, including dystonia, a common movement disorder. Previous studies have shown a deficit of striatal GABAergic interneurons (IN) in the dtsz mutant hamster, one of the few phenotypic animal models of dystonia. However, mechanisms underlying this deficit are largely unknown. In the present study, we investigated the migration and maturation of striatal IN during postnatal development (18days of age) and at age of highest severity of dystonia (33days of age) in this hamster model. In line with previous findings, the density of GAD67-positive IN and the level of parvalbumin mRNA, a marker for fast spiking GABAergic IN, were lower in the dtsz mutant than in control hamsters. However, an unaltered density of Nkx2.1 labeled cells and Nkx2.1 mRNA level suggested that the migration of GABAergic IN into the striatum was not retarded. Therefore, different factors that indicate maturation of GABAergic IN were determined. While mRNA of the KCC2 cation/chloride transporters and the cytosolic carboanhydrase VII, used as markers for the so called GABA switch, as well as BDNF were unaltered, we found a reduced number of IN expressing the alpha1 subunit of the GABAA-receptor (37.5%) in dtsz hamsters at an age of 33days, but not after spontaneous remission of dystonia at an age of 90days. Since IN shift expression from alpha2 to alpha1 subunits during postnatal maturation, this result together with a decreased parvalbumin mRNA expression suggest a delayed maturation of striatal GABAergic IN in this animal model, which might underlie abnormal neuronal activity and striatal plasticity.

The α2β3γ2 GABAA receptor preferring agonist NS11394 aggravates dystonia in the phenotypic dtsz model

Dystonia is a movement disorder, characterized by involuntary muscle contractions resulting in abnormal movements and/or postures. Antidystonic effects of benzodiazepines in patients with different types of dystonia could be replicated in the dtsz mutant hamster, a phenotypic model of paroxysmal dystonia. Compounds with preferred binding at specific subunits of the gamma aminobutyric acid type A (GABAA) receptor may provide a more beneficial spectrum of effects in comparison with benzodiazepines. We therefore examined the effects of the α1β3γ2 GABAA receptor preferring compound zolpidem (2.0–10.0mg/kg i.p.) and of the α2β3γ2 GABAA receptor preferring compound NS11394 (3.0–30mg/kg i.p.) on the severity of dystonia in the dtsz mutant in comparison with the benzodiazepine clonazepam (0.5–1.0mg/kg i.p.). As expected, clonazepam exerted pronounced antidystonic effects. While zolpidem showed moderate beneficial effects, NS11394 significantly increased the severity of dystonia. The present results indicate for the first time that positive GABAA receptor modulators show contrary effects on dystonia dependent on their preference for alpha-subunits. The potential link between alterations in GABAA receptor subunits and GABAergic disinhibition in dystonia deserves further attention in research on the pathophysiology and therapeutic targets.

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 dtsz 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 dtsz 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 dtsz hamsters and non-dystonic control hamsters, NVP-AAM077 (50nM) completely blocked LTP in dtsz 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 dtsz 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.

Persistent changes of corticostriatal plasticity in dtsz mutant hamsters after age-dependent remission of dystonia

Abnormal plasticity in the cortico-basal ganglia–thalamocortical loop has been suggested to represent a key factor in the pathophysiology of dystonia. In a model of primary paroxysmal dystonia, the dtsz mutant hamster, previous experiments have shown a strongly increased long-term potentiation (LTP) in comparison to non-dystonic control hamsters. These basal changes, i.e. in the absence of dystonia, were found in young animals at an age of 5weeks, when the age-dependent dystonia in dtsz mutant reaches highest severity. In the present study we examined in corticostriatal slices (1) whether the increases in synaptic plasticity can be modulated by stressful stimuli which induce dystonic episodes in young mutant hamsters, and (2) whether increases of LTP persist after spontaneous remission of dystonia in animals older than 10weeks. The present data show that in slices of young mutant hamsters the extent of LTP was not influenced by the presence of dystonia: In comparison to age-matched control hamsters, LTP was increased in mutant hamsters independent of preceding stressful stimulation. After remission of dystonia, i.e., in older dtsz mutant hamsters >10weeks, only LTP could be elicited, while in preparations from age-matched control hamsters, either LTP or long-term depression developed, depending on previous behavioral challenge. We conclude that in mature brain, corticostriatal connections have the potential for changes in metaplasticity, while in dtsz mutant hamsters this metaplasticity is persistently infringed even though stress-inducible dystonic symptoms are lost.

P.1.d.008 The involvement of an intact prefrontal cortex–hippocampal circuit in the forced swim test in male and female rats

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 dtsz 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 dtsz 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 30 mg/kg i.p.) and delayed the onset of a dystonic episode after intrastriatal injections (0.12 and 0.24 μg). As shown by present electrophysiological examinations in corticostriatal slices of dtsz hamsters and non-dystonic control hamsters, NVP-AAM077 (50 nM) completely blocked LTP in dtsz slices, but did not exert significant effects on LTP in non-dystonic controls. In contrast, the NR2B antagonist Ro 25-6981 (1–10 μmol) reduced LTP to a lower extent in dtsz 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.

Autoradiographic and pharmacological studies on the role of dopamine D3 receptors in genetically dystonic ( dtsz) 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. [ 125I]7-[[(E)-3-iodoprop-2-enyl]-propylamino]-5,6,7,8-tetrahydronaphthalen-2-ol ([ 125I]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 40 mg/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 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.

Extracellular amino acid levels in the striatum of the dtsz mutant, a model of paroxysmal dystonia

The pathophysiology of idiopathic dystonia is still unknown, but it is regarded as a basal ganglia disorder. Previous studies indicated an involvement of a striatal GABAergic disinhibition and a cortico-striatal glutamatergic overactivity in the manifestation of stress-inducible dystonic episodes in the dt sz hamster, a model of idiopathic paroxysmal dystonia. These investigations were carried out postmortem or in anesthetized animals. In the present study, in vivo microdialysis in conscious, freely-moving dt sz and non-dystonic control hamsters was used to examine the levels of GABA, aspartate, glutamate, glutamine, glycine and taurine in each animal during following conditions: (1) at baseline in the absence of dystonia, (2) during an episode of paroxysmal dystonia precipitated by stressful stimuli, (3) during a recovery period and (4) at baseline after complete recovery. In comparison to non-dystonic controls, which were treated in the same manner as the dystonic animals, no differences could be detected under basal conditions. The induction of a dystonic episode in mutant hamsters led to higher contents of glycine in these animals in comparison to stressed but non-dystonic controls. Significant changes of glycine levels within the animal groups were not detected. The levels of the excitatory amino acids glutamate, glutamine and aspartate as well as the levels of the inhibitory amino acids GABA and taurine did not differ between the animal groups or between the periods of measurement. The higher levels of glycine might contribute to the manifestation of paroxysmal dystonia in dt sz hamsters, although unaltered glutamate, glutamine and aspartate levels do not support the hypothesis of a critical involvement of a cortico-striatal overactivity. It seems that a deficiency of GABAergic interneurons, found by previous immunohistochemical examinations, does not lead to reduced extracellular GABA levels in the striatum.

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 μg/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.

Decreased density of amygdaloid parvalbumin-positive interneurons and behavioral changes in dystonic hamsters (Mesocricetus auratus).

The dtsz hamster represents a model of primary paroxysmal nonkinesiogenic dyskinesia in which dystonic episodes can be induced by stress and anxious stimuli. This disease is regarded as a basal ganglia disorder. In fact, a deficit of striatal interneurons could play a key role in the pathophysiology in dystonic hamsters. Because the involvement of limbic structures cannot be excluded so far, the density of parvalbumin-immunoreactive (PV+) interneurons was determined in the basolateral amygdala in the present study. Compared with nondystonic hamsters, the density of PV+ interneurons was moderately decreased in the dtsz mutant. The functional consequence of this finding was examined by behavioral analyses. Examinations in the elevated plus maze and in a modified open field failed to disclose an enhanced anxiety-related behavior in dtsz hamsters (Mesocricetus auratus). A lower acoustic startle response and a stronger habituation in mutant hamsters than in controls correlated with a decreased body weight. Interestingly, prepulse inhibition was absent in mutant hamsters. The latter finding suggests a disturbed sensorimotor gating that can be related to alterations in both the basal ganglia nuclei and in limbic structures.

Age-related changes in parvalbumin-positive interneurons in the striatum, but not in the sensorimotor cortex in dystonic brains of the dtsz mutant hamster

In the dt sz hamster, a model of paroxysmal dystonia, an age-dependent increase in the activity of striatal projection neurons has been hypothesized to be based on a deficit of striatal parvalbumin-immunoreactive (PV +) interneurons at an age of most marked expression of dystonia (30–40 days of life). In the present study, the spontaneous age-dependent remission of paroxysmal dystonia in older dt sz hamsters (age > 90 days) was found to coincide with a normalization of the density of striatal PV + interneurons. Furthermore, the arborization of these interneurons was lower in 31 day old dt sz hamsters, but was even higher in dt sz mutant at an age of > 90 days than in control animals. Double-labeling with bromodeoxyuridine failed to show a retarded proliferation, while the number of interneurons with strong expression of PV mRNA was lower in young mutant hamsters. As shown by unaltered density of PV + interneurons in sensorimotor cortex of 31 day old dt sz hamsters, PV containing interneurons are not reduced throughout the whole brain at the sensitive age. The present data suggest that a retarded postnatal maturation of striatal PV + interneurons plays a critical role in paroxysmal dystonia.

Effects of intrastriatal injections of glutamate receptor antagonists on the severity of paroxysmal dystonia in the dtsz mutant

Imbalances of the glutamatergic system are implicated in the pathophysiology of various basal ganglia disorders, but few is known about their role in dystonia, a common neurological syndrome in which involuntary muscle co-contractions lead to twisting movements and abnormal postures. Previous systemic administrations of glutamate receptor antagonists in dt sz hamsters, an animal model of primary paroxysmal dystonia, exerted antidystonic effects and electrophysiological experiments pointed to an enhanced corticostriatal glutamatergic activity. In order to examine the pathophysiological relevance of these findings, we performed striatal microinjections of the α-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropanoic acid (AMPA) receptor antagonist 2,3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX) and the N-methyl- d-aspartate (NMDA) receptor antagonists d(−)-2-amino-5-phosphopentanoic acid (AP-5), ( R)-(+)-3-amino-1-hydroxypyrrolidin-2-one (HA-966) and dizocilpine (MK-801). The striatal application of NBQX reduced the severity and increased the latency to onset of dystonia significantly only at a dosage of 0.08 μg per hemisphere, lower (0.03 μg) and higher dosages (0.16 μg and 0.32 μg) failed to exert comparable effects on the severity. None of the striatal injected NMDA receptor antagonists influenced the severity of the dystonic attacks in the mutant hamster. The combined application of NBQX (0.08 μg) with AP-5 (1.0 μg) failed to exert synergistic antidystonic effects, but the beneficial effect on the severity of dystonia of the single application of NBQX was reproduced. Therefore, corticostriatal glutamatergic overactivity mediated by AMPA receptors, but not by NMDA receptors, is possibly important for the manifestation of dystonic attacks in the dt sz hamster mutant.

Antidystonic effects of Kv7 (KCNQ) channel openers in the dtsz mutant, an animal model of primary paroxysmal dystonia

Mutations in neuronal Kv7 (KCNQ) potassium channels can cause episodic neurological disorders. Paroxysmal dyskinesias with dystonia are a group of movement disorders which are regarded as ion channelopathies, but the role of Kv7 channels in the pathogenesis and as targets for the treatment have so far not been examined. In the present study, we therefore examined the effects of the activators of neuronal Kv7.2/7.3 channels retigabine (5, 7.5, 10 mg kg(-1) i.p. and 10, 20 mg kg(-1) p.o.) and flupirtine (10, 20 mg kg(-1) i.p.) and of the channel blocker 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone (XE-991, 3 and 6 mg kg(-1) i.p.) in the dt sz mutant hamster, a model of paroxysmal dyskinesia in which dystonic episodes occur in response to stress. Retigabine (10 mg kg(-1) i.p., 20 mg kg(-1) p.o.) and flupirtine (20 mg kg(-1) i.p.) significantly improved dystonia, while XE-991 caused a significant aggravation in the dt sz mutant. The antidystonic effect of retigabine (10 mg kg(-1) i.p.) was counteracted by XE-991 (3 mg kg(-1) i.p.). These data indicate that dysfunctions of neuronal Kv7 channels deserve attention in dyskinesias. Since retigabine and flupirtine are well tolerated in humans, the present finding of pronounced antidystonic efficacy in the dt sz mutant suggests that neuronal Kv7 channel activators are interesting candidates for the treatment of dystonia-associated dyskinesias and probably of other types of dystonias. The established analgesic effects of Kv7 channel openers might contribute to improvement of these disorders which are often accompanied by painful muscle spasms.

Acetylcholine receptor binding and cholinergic interneuron density are unaltered in a genetic animal model of primary paroxysmal dystonia

The underlying pathophysiological mechanisms of hereditary types of paroxysmal dyskinesias are still unknown, but basal ganglia dysfunctions seem to play a critical role. In fact, numerous pharmacological, neurochemical, immunohistochemical and electrophysiological investigations in the dt sz hamsters, a unique rodent model of age-dependent primary paroxysmal dystonia, revealed alterations within the basal ganglia, particularly of the GABAergic and dopaminergic neurotransmitter systems. A deficit in several types of striatal GABAergic interneurons in dt sz mutant hamsters seems to play a crucial pathophysiological role, but deficits in other types of striatal interneurons cannot be excluded by previous studies. In view of ameliorating effects of anti-cholinergic drugs in dystonic patients, we therefore investigated the density of striatal cholinergic interneurons in the present study. These interneurons were marked specifically by the enzyme choline acetyltransferase and counted by using a stereological counting method in a blinded fashion. Additionally, acetylcholine receptor binding was determined in mutant and nondystonic control hamsters by autoradiographic analyses with the nonselective muscarinic ligand [ 3H]-quinuclidinyl benzilate (QNB) in 11 brain (sub)regions. There were no significant differences in the density of striatal cholinergic interneurons between dt sz mutant hamsters (789 ± 39 interneurons/mm 3) and nondystonic controls (807 ± 36 interneurons/mm 3). [ 3H]QNB binding was also comparable between mutant and control hamsters. These results point to an unaltered striatal cholinergic neurotransmitter system in dt sz hamsters under basal conditions.