Distribution of Cleaved SNAP-25 in the Rat Brain, following Unilateral Injection of Botulinum Neurotoxin-A into the Striatum.

In Parkinson's disease, hypercholinism in the striatum occurs, with the consequence of disturbed motor functions. Direct application of Botulinum neurotoxin-A in the striatum of hemi-Parkinsonian rats might be a promising anticholinergic therapeutic option. Here, we aimed to determine the spread of intrastriatally injected BoNT-A in the brain as well as the duration of its action based on the distribution of cleaved SNAP-25. Rats were injected with 1 ng of BoNT-A into the right striatum and the brains were examined at different times up to one year after treatment. In brain sections immunohistochemically stained for BoNT-A, cleaved SNAP-25 area-specific densitometric analyses were performed. Increased immunoreactivity for cleaved SNAP-25 was found in brain regions other than the unilaterally injected striatum. Most cleaved SNAP-25-ir was found in widespread areas ipsilateral to the BoNT-A injection, in some regions, however, immunoreactivity was also measured in the contralateral hemisphere. There was a linear relationship between the distance of a special area from the injected striatum and the time until its maximum averaged immunoreactivity was reached. Moreover, we observed a positive relationship for the area-specific distance from the injected striatum and its maximum immunoreactivity as well as for the connection density with the striatum and its maximum immunoreactivity. The results speak for a bidirectional axonal transport of BoNT-A after its application into the striatum to its widespread connected parts of the brain. Even one year after BoNT-A injection, cleaved SNAP-25 could still be detected.

Food Restriction in Mice Induces Food-Anticipatory Activity and Circadian-Rhythm-Related Activity Changes.

Anorexia nervosa (AN) is characterized by emaciation, hyperactivity, and amenorrhea. To what extent AN-related symptoms are due to food restriction or neuronal dysfunction is currently unknown. Thus, we investigated the relevance of food restriction on AN-related symptoms. Disrupted circadian rhythms are hypothesized to contribute to the pathophysiology of AN. Starvation was induced by restricting food access in early adolescent or adolescent mice to 40% of their baseline food intake until a 20% weight reduction was reached (acute starvation). To mimic chronic starvation, the reduced weight was maintained for a further 2 weeks. Locomotor activity was analyzed using running wheel sensors. The circadian-rhythm-related activity was measured using the tracking system Goblotrop. Amenorrhea was determined by histological examination of vaginal smears. All cohorts showed an increase in locomotor activity up to 4 h before food presentation (food-anticipatory activity, FAA). While amenorrhea was present in all groups except in early adolescent acutely starved mice, hyperactivity was exclusively found in chronically starved groups. Adolescent chronically starved mice showed a decrease in circadian-rhythm-related activity at night. Chronic starvation most closely mimics AN-related behavioral changes. It appears that the FAA is a direct consequence of starvation. The circadian activity changes might underlie the pathophysiology of AN.

Transmembrane protein 119 is neither a specific nor a reliable marker for microglia.

Microglia are the resident innate immune cells of the central nervous system (CNS) parenchyma. To determine the impact of microglia on disease development and progression in neurodegenerative and neuroinflammatory diseases, it is essential to distinguish microglia from peripheral macrophages/monocytes, which are eventually equally recruited. It has been suggested that transmembrane protein 119 (TMEM119) serves as a reliable microglia marker that discriminates resident microglia from blood-derived macrophages in the human and murine brain. Here, we investigated the validity of TMEM119 as a microglia marker in four in vivo models (cuprizone intoxication, experimental autoimmune encephalomyelitis (EAE), permanent filament middle cerebral artery occlusion (fMCAo), and intracerebral 6-hydroxydopamine (6-OHDA) injections) as well as post mortem multiple sclerosis (MS) brain tissues. In all applied animal models and post mortem MS tissues, we found increased densities of ionized calcium-binding adapter molecule 1+ (IBA1+ ) cells, paralleled by a significant decrease in TMEM119 expression. In addition, other cell types in peripheral tissues (i.e., follicular dendritic cells and brown adipose tissue) were also found to express TMEM119. In summary, this study demonstrates that TMEM119 is not exclusively expressed by microglia nor does it label all microglia, especially under cellular stress conditions. Since novel transgenic lines have been developed to label microglia using the TMEM119 promotor, downregulation of TMEM119 expression might interfere with the results and should, thus, be considered when working with these transgenic mouse models.

Olfactory Bulb D2/D3 Receptor Availability after Intrastriatal Botulinum Neurotoxin-A Injection in a Unilateral 6-OHDA Rat Model of Parkinson's Disease.

Olfactory deficits occur as early non-motor symptoms of idiopathic Parkinson's disease (PD) in humans. The first central relay of the olfactory pathway, the olfactory bulb (OB), depends, among other things, on an intact, functional crosstalk between dopaminergic interneurons and dopamine receptors (D2/D3R). In rats, hemiparkinsonism (hemi-PD) can be induced by unilateral injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle (MFB), disrupting dopaminergic neurons of the substantia nigra pars compacta (SNpc). In a previous study, we showed that subsequent injection of botulinum neurotoxin-A (BoNT-A) into the striatum can reverse most of the pathological motor symptoms and normalize the D2/D3R availability. To determine whether this rat model is suitable to explain olfactory deficits that occur in humans with PD, we examined the availability of D2/D3R by longitudinal [18F]fallypride-PET/CT, the density of tyrosine hydroxylase immunoreactivity in the OB, olfactory performance by an orienting odor identification test adapted for rats, and a connectome analysis. PET/CT and immunohistochemical data remained largely unchanged after 6-OHDA lesion in experimental animals, suggesting that outcomes of the 6-OHDA hemi-PD rat model do not completely explain olfactory deficits in humans. However, after subsequent ipsilateral BoNT-A injection into the striatum, a significant 8.5% increase of the D2/D3R availability in the ipsilateral OB and concomitant improvement of olfactory performance were detectable. Based on tract-tracing meta-analysis, we speculate that this may be due to indirect connections between the striatum and the OB.

Antidepressant-Like Properties of Intrastriatal Botulinum Neurotoxin-A Injection in a Unilateral 6-OHDA Rat Model of Parkinson's Disease.

Parkinson's patients often suffer from depression and anxiety, for which there are no optimal treatments. Hemiparkinsonian (hemi-PD) rats were used to test whether intrastriatal Botulinum neurotoxin-A (BoNT-A) application could also have antidepressant-like properties in addition to the known improvement of motor performance. To quantify depression- and anxiety-like behavior, the forced swim test, tail suspension test, open field test, and elevated plus maze test were applied to hemi-PD rats injected with BoNT-A or vehicle. Furthermore, we correlated the results in the forced swim test, open field test, and elevated plus maze test with the rotational behavior induced by apomorphine and amphetamine. Hemi-PD rats did not show significant anxiety-like behavior as compared with Sham 6-OHDA- + Sham BoNT-A-injected as well as with non-injected rats. However, hemi-PD rats demonstrated increased depression-like behaviors compared with Sham- or non-injected rats; this was seen by increased struggling frequency and increased immobility frequency. Hemi-PD rats intrastriatally injected with BoNT-A exhibited reduced depression-like behavior compared with the respective vehicle-receiving hemi-PD animals. The significant effects of intrastriatally applied BoNT-A seen in the forced swim test are reminiscent of those found after various antidepressant drug therapies. Our data correspond with the efficacy of BoNT-A treatment of glabellar frown lines in treating patients with major depression and suggest that also intrastriatal injected BoNT-A may have some antidepressant-like effect on hemi-PD.

Repeated intrastriatal application of botulinum neurotoxin-A did not influence choline acetyltransferase-immunoreactive interneurons in hemiparkinsonian rat brain - A histological, stereological and correlational analysis.

In Parkinson's disease, dopamine depletion leads to hyperactivity of cholinergic interneurons in the caudate-putamen (CPu). Botulinum neurotoxin-A (BoNT-A) inhibits the release of acetylcholine in the peripheral nervous system and is also thought to act as a local anticholinergic drug when injected intrastriatally. In hemiparkinsonian (hemi-PD) rats, a unilateral intrastriatal injection of 1 ng BoNT-A significantly diminished apomorphine-induced rotation behavior for at least 3 months, the effect fading thereafter. A second intrastriatal BoNT-A application, 6 months after the first one, led to a stronger and longer-lasting, beneficial behavioral reaction. As a single BoNT-A injection was not cytotoxic in the rat striatum and resembled BoNT-A treatment in clinical practice, here, we investigated the structural outcome of repeated intrastriatal BoNT-A injections with respect to striatal volume, the number of choline acetyltransferase-immunoreactive (ChAT-ir) interneurons and of the length of their dendritic arbors, and the numeric density of ChAT-ir BoNT-A-induced varicosities (BiVs). Repeated unilateral intrastriatal BoNT-A application decreased the volume of the injected CPu, but did not significantly change the number of striatal ChAT-ir interneurons. Also, the total dendrite length of ChAT-ir interneurons after repeated BoNT-A application resembled the values in double vehicle-injected hemi-PD rats. In repeatedly BoNT-A-injected hemi-PD rats, the numeric density of ChAT-ir BiVs in the CPu was increased compared with rats only intrastriatally injected once with BoNT-A. Even repeated BoNT-A injections in rat striata did not cause substantial morphological changes in ChAT-ir neuron, except for the increased numeric density of ChAT-ir BiVs.

Botulinum Neurotoxin-A Injected Intrastriatally into Hemiparkinsonian Rats Improves the Initiation Time for Left and Right Forelimbs in Both Forehand and Backhand Directions.

Forelimb stepping is a widely used test for the assessment of forelimb akinesia in hemiparkinsonian (hemi-PD) rats. The initiation time (IT) is considered the most sensitive parameter in the stepping test procedure. Here we propose a novel, reliable, and simple method for the measurement of IT of both forelimbs in both forehand and backhand directions in rats. Evaluating the same videos taken for quantifying adjusting steps, IT measurements were done without additional experiments. This is in contrast to the classical approach introduced by Olsson et al. (1995), in which separate experiments are necessary. We successfully applied our approach to hemi-PD rats intrastriatally treated with botulinum neurotoxin-A (BoNT-A). In naïve rats, an IT of about 0.62 s was found, and in right-sided hemi-PD rats the IT of the left forepaw increased to about 3.62 s. These hemi-PD rats showed, however, reduced ITs of the impaired left forepaws 1 month and the second time 7 months after induction of hemi-PD via the injection of 1 ng BoNT-A into the ipsilateral striatum, depending on post BoNT-A survival time. The method described offers the possibility of a precise and animal-friendly evaluation of IT in rats, including the beneficial effect of BoNT-A treatment in hemi-PD rats.

Repeated Intrastriatal Botulinum Neurotoxin-A Injection in Hemiparkinsonian Rats Increased the Beneficial Effect on Rotational Behavior.

Injection of botulinum neurotoxin-A (BoNT-A) into the striatum of hemiparkinsonian (hemi-PD) rats reduced apomorphine-induced rotation behavior significantly, for at least 3 months. Thereafter, rotation behavior increased again. We injected hemi-PD rats with 1 ng BoNT-A twice, the second injection following 6 months after the first one and tested the rats for apomorphine-induced rotations and spontaneous motor behaviors, i.e., corridor task and stepping test. To test the hypothesis that BoNT-A reduced striatal hypercholinism in hemi-PD rats, the acetylcholinesterase inhibitor donepezil was injected prior to separate apomorphine-induced rotation tests. In hemi-PD rats, the first BoNT-A injection led to a clear reduction of the apomorphine-induced rotations, and the second BoNT-A injection to a more massive and prolonged reaction. In hemi-PD rats whose apomorphine-induced rotation behavior was strongly reduced by an intrastriatal BoNT-A, subsequent donepezil injections led to significant increases of the rotation rate. Concerning corridor task and stepping test, neither first nor second BoNT-A injections changed hemi-PD rats' behavior significantly. The data give evidence for the possibility of repeated intrastriatal administrations of BoNT-A, for treatment of motor symptoms in experimental hemi-PD over a longer time.

Acetylcholine Neurotransmitter Receptor Densities in the Striatum of Hemiparkinsonian Rats Following Botulinum Neurotoxin-A Injection.

Cholinergic neurotransmission has a pivotal function in the caudate-putamen, and is highly associated with the pathophysiology of Parkinson's disease. Here, we investigated long-term changes in the densities of the muscarinic receptor subtypes M1, M2, M3 (mAchRs) and the nicotinic receptor subtype α4ß2 (nAchRs) in the striatum of the 6-OHDA-induced hemiparkinsonian (hemi-PD) rat model using quantitative in vitro receptor autoradiography. Hemi-PD rats exhibited an ipsilateral decrease in striatal mAchR densities between 6 and 16%. Moreover, a massive and constant decrease in striatal nAchR density by 57% was found. A second goal of the study was to disclose receptor-related mechanisms for the positive motor effect of intrastriatally injected Botulinum neurotoxin-A (BoNT-A) in hemi-PD rats in the apomorphine rotation test. Therefore, the effect of intrastriatally injected BoNT-A in control and hemi-PD rats on mAchR and nAchR densities was analyzed and compared to control animals or vehicle-injected hemi-PD rats. BoNT-A administration slightly reduced interhemispheric differences of mAchR and nAchR densities in hemi-PD rats. Importantly, the BoNT-A effect on striatal nAchRs significantly correlated with behavioral testing after apomorphine application. This study gives novel insights of 6-OHDA-induced effects on striatal mAchR and nAchR densities, and partly explains the therapeutic effect of BoNT-A in hemi-PD rats on a cellular level.

Unilateral Botulinum Neurotoxin-A Injection into the Striatum of C57BL/6 Mice Leads to a Different Motor Behavior Compared with Rats.

Different morphological changes in the caudate-putamen (CPu) of naïve rats and mice were observed after intrastriatal botulinum neurotoxin-A (BoNT-A) injection. For this purpose we here studied various motor behaviors in mice (n = 46) longitudinally up to 9 months after intrastriatal BoNT-A administration as previously reported for rats, and compared both outcomes. Apomorphine- and amphetamine-induced rotational behavior, spontaneous motor behavior, as well as lateralized neglect were studied in mice after the injection of single doses of BoNT-A into the right CPu, comparing them with sham-injected animals. Unilateral intrastriatal injection of BoNT-A in mice induced significantly increased contralateral apomorphine-induced rotations for 1 to 3 months, as well as significantly increased contralateral amphetamine-induced rotations 1 to 9 months after injection. In rats (n = 28), unilateral BoNT-A injection also induced significantly increased contralateral apomorphine-induced rotations 3 months after injection, but did not provoke amphetamine-induced rotations at all. Lateralized sensorimotor integration, forelimb preference, and forelimb stepping were significantly impaired on the left side. The differences in motor behaviors between rats and mice may be caused by different BoNT-A effects on cholinergic and catecholaminergic fibers in rat and mouse striata, interspecies differences in striatal receptor densities, and different connectomes of the basal ganglia.

Experimental Intrastriatal Applications of Botulinum Neurotoxin-A: A Review.

Parkinson’s disease (PD) is one of the most frequent neurodegenerative disorders. Its main pathophysiological characteristic is the loss of dopaminergic neurons in the substantia nigra pars compacta followed by a lack of striatal dopaminergic input and a consequent disinhibition of tonically active cholinergic interneurons. The resulting striatal hypercholinism causes major motor symptoms in PD. Anticholinergic pharmacotherapies have antiparkinsonian effects on motor symptoms, but, due to systemic actions, also numerous severe side effects occur on a regular basis. To circumvent these side effects, a local anticholinergic therapy acting exclusively in the striatum would be reasonable. Botulinum neurotoxin-A (BoNT-A) is synthesized by Clostridium botulinum and blocks the release of acetylcholine from the presynaptic bouton. For several decades, BoNT-A has been used successfully for medical and cosmetic purposes to induce controlled paralyses of single muscles. Our group and others investigated the experimental treatment of striatal hypercholinism by the direct injection of BoNT-A into the striatum of rats and mice as well as of hemiparkinsonian animal models. This review gives an overview of the most important results of the experimental intrastriatal BoNT-A application, with a focus on hemiparkinsonian rats.

[18F]fallypride-PET/CT Analysis of the Dopamine D2/D3 Receptor in the Hemiparkinsonian Rat Brain Following Intrastriatal Botulinum Neurotoxin A Injection.

Intrastriatal injection of botulinum neurotoxin A (BoNT-A) results in improved motor behavior of hemiparkinsonian (hemi-PD) rats, an animal model for Parkinson's disease. The caudate-putamen (CPu), as the main input nucleus of the basal ganglia loop, is fundamentally involved in motor function and directly interacts with the dopaminergic system. To determine receptor-mediated explanations for the BoNT-A effect, we analyzed the dopamine D2/D3 receptor (D2/D3R) in the CPu of 6-hydroxydopamine (6-OHDA)-induced hemi-PD rats by [18F]fallypride-PET/CT scans one, three, and six months post-BoNT-A or -sham-BoNT-A injection. Male Wistar rats were assigned to three different groups: controls, sham-injected hemi-PD rats, and BoNT-A-injected hemi-PD rats. Disease-specific motor impairment was verified by apomorphine and amphetamine rotation testing. Animal-specific magnetic resonance imaging was performed for co-registration and anatomical reference. PET quantification was achieved using PMOD software with the simplified reference tissue model 2. Hemi-PD rats exhibited a constant increase of 23% in D2/D3R availability in the CPu, which was almost normalized by intrastriatal application of BoNT-A. Importantly, the BoNT-A effect on striatal D2/D3R significantly correlated with behavioral results in the apomorphine rotation test. Our results suggest a therapeutic effect of BoNT-A on the impaired motor behavior of hemi-PD rats by reducing interhemispheric changes of striatal D2/D3R.

Intrastriatal administration of botulinum neurotoxin A normalizes striatal D2 R binding and reduces striatal D1 R binding in male hemiparkinsonian rats.

Cerebral administration of botulinum neurotoxin A (BoNT-A) has been shown to improve disease-specific motor behavior in a rat model of Parkinson disease (PD). Since the dopaminergic system of the basal ganglia fundamentally contributes to motor function, we investigated the impact of BoNT-A on striatal dopamine receptor expression using in vitro and in vivo imaging techniques (positron emission tomography and quantitative autoradiography, respectively). Seventeen male Wistar rats were unilaterally lesioned with 6-hydroxydopamine (6-OHDA) and assigned to two treatment groups 7 weeks later: 10 rats were treated ipsilaterally with an intrastriatal injection of 1 ng BoNT-A, while the others received vehicle (n = 7). All animals were tested for asymmetric motor behavior (apomorphine-induced rotations and forelimb usage) and for striatal expression of dopamine receptors and transporters (D1 R, D2 R, and DAT). The striatal D2 R availability was also quantified longitudinally (1.5, 3, and 5 months after intervention) in 5 animals per treatment group. The 6-OHDA lesion alone induced a unilateral PD-like phenotype and a 13% increase of striatal D2 R. BoNT-A treatment reduced the asymmetry in both apomorphine-induced rotational behavior and D2 R expression, with the latter returning to normal values 5 months after intervention. D1 R expression was significantly reduced, while DAT concentrations showed no alteration. Independent of the treatment, higher interhemispheric symmetry in raclopride binding to D2 R was generally associated with reduced forelimb akinesia. Our findings indicate that striatal BoNT-A treatment diminishes motor impairment and induces changes in D1 and D2 binding site density in the 6-OHDA rat model of PD.

Intrastriatally injected botulinum neurotoxin-A differently effects cholinergic and dopaminergic fibers in C57BL/6 mice.

Unilateral intrastriatal BoNT-A injection abolished apomorphine-induced rotational behavior in a rat model of hemiparkinsonism (hemi-PD) up to 6months. It was hypothesized that the beneficial effect of botulinum neurotoxin-A (BoNT-A) grounded on the reduction of the Parkinson's diseases (PD) associated striatal hypercholinism. Intrastriatal injection of BoNT-A was not cytotoxic in rat brain, but neuronal fiber swellings in the BoNT-A infiltrated striata appeared and named BoNT-A-induced varicosities (BiVs). In the rat BiVs were immunoreactive (ir) either for choline acetyltransferase (ChAT) or tyrosine hydroxylase (TH). In the present study the structural effect of unilateral intrastriatal BoNT-A injection in the naïve mouse brain was analyzed to extend possible therapeutic BoNT-A applications to genetical Parkinsonian strains. We investigated the effect of a single dose of 25pg BoNT-A injected into the right caudate-putamen (CPu) for up to 9months, and of increasing doses up to 200pg on striatal volume, number of ChAT-ir interneurons, and numeric density and volume of the ChAT-ir BiVs in comparison to the uninjected hemisphere. Intrastriatal BoNT-A injection did not alter the number of ChAT-ir interneurons irrespective of survival time and dosage tested. However, the numeric density of the ChAT-ir BiVs at a dose of 25pg increased from 1 to 3months after BoNT-A, followed by a time dependent decrease. In parallel, with increasing BoNT-A survival time, the mean BiV volume increased as the number of small BiVs decreased. Interestingly, in contrast to rats we did not find TH-ir BiVs in BoNT-A injected mouse striatum.

Botulinum Neurotoxin A Injected Ipsilaterally or Contralaterally into the Striatum in the Rat 6-OHDA Model of Unilateral Parkinson's Disease Differently Affects Behavior.

Parkinson's disease (PD) is one of the most frequent neurodegenerative disorders. The loss of dopaminergic neurons in the substantia nigra leads to a disinhibition of cholinergic interneurons in the striatum. Pharmacotherapeutical strategies of PD-related hypercholinism have numerous adverse side effects. We previously showed that ipsilateral intrastriatal injections of 1 ng in unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats inhibit apomorphine-induced rotation behavior significantly up to 6 months. In this study, we extended the behavioral testing of ipsilateral botulinum neurotoxin A (BoNT-A)-injection and additionally investigated the impact of intrastriatal BoNT-A-injections contralateral to the 6-OHDA-lesioned hemisphere on the basal ganglia circuity and motor functions. We hypothesized that the interhemispheric differences of acetylcholine (ACh) concentration seen in unilateral hemi-PD should be differentially and temporally influenced by the ipsilateral or contralateral injection of BoNT-A. Hemi-PD rats were injected with 1 ng BoNT-A or vehicle substance into either the ipsilateral or contralateral striatum 6 weeks after 6-OHDA-lesion and various behaviors were tested. In hemi-PD rats intrastriatal ipsilateral BoNT-A-injections significantly reduced apomorphine-induced rotations and increased amphetamine-induced rotations, but showed no significant improvement of forelimb usage and akinesia, lateralized sensorimotor integration and also no effect on spontaneous locomotor activity. However, intrastriatal BoNT-A-injections contralateral to the lesion led to a significant increase of the apomorphine-induced turning rate only 2 weeks after the treatment. The apomorphine-induced rotation rate decreases thereafter to a value below the initial rotation rate. Amphetamine-induced rotations were not significantly changed after BoNT-A-application in comparison to sham-treated animals. Forelimb usage was temporally improved by contralateral BoNT-A-injection at 2 weeks after BoNT-A. Akinesia and lateralized sensorimotor integration were also improved, but contralateral BoNT-A-injection had no significant effect on spontaneous locomotor activity. These long-ranging and different effects suggest that intrastriatally applied BoNT-A acts not only as an inhibitor of ACh release but also has long-lasting impact on transmitter expression and thereby on the basal ganglia circuitry. Evaluation of changes of transmitter receptors is subject of ongoing studies of our group.

Intrastriatal injection of botulinum neurotoxin-A is not cytotoxic in rat brain - A histological and stereological analysis.

Parkinson's disease (PD) is caused by progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta, resulting in a deficiency of dopamine in the striatum and an increased release of acetylcholine by tonically active interneurons. Botulinum neurotoxin-A (BoNT-A) is well known for blocking transmitter release by cholinergic presynaptic terminals. Treating striatal hypercholinism by local application of BoNT-A could be a possible new local therapy option of PD. In previous studies of our group, we analyzed the effect of BoNT-A injection into the CPu of 6-OHDA lesioned hemiparkinsonian rats. Our studies showed that BoNT-A application in hemiparkinson rat model is capable of abolishing apomorphine induced rotations for approximately 3 months. Regularly occurring axonal swellings in the BoNT-A infiltrated striata were also discovered, which we named BoNT-A induced varicosities (BiVs). Résumé: Here we investigated the long-term effect of the injection of 1ng BoNT-A into the right CPu of naive Wistar rats on the number of ChAT-ir interneurons as well as on the numeric density and the volumetric size of the BiVs in the CPu. Significant differences in the number of ChAT-ir neurons between the right BoNT-A treated CPu and the left untreated CPu were not detected up to 12 month post BoNT-A injection. The numeric density of BiVs in the treated CPu reached a maximum 3 months after BoNT-A treatment and decreased afterwards, whereas the volume of single BiVs increased steadily throughout the whole time course of the experiment.

Reduced cerebellar neurodegeneration after combined therapy with cyclodextrin/allopregnanolone and miglustat in NPC1: a mouse model of Niemann-Pick type C1 disease.

Niemann-Pick type C1 (NPC1) disease is a lysosomal storage disease characterized by a deficiency of NPC1 gene function. The malfunction of protein results in a progressive accumulation of lipids in many organs. A combined approach with substrate-reduction therapy (SRT) and byproduct therapy (BPT) has been shown to ameliorate the disease course in a mutant mouse model (NPC1(-/-)). The present study examines the morphological parameters underlying these changes. For the combined SRT/BPT treatment, NPC1(-/-) mutant mice (NPC1(-/-SRT/BPT)) were injected with allopregnanolone/cyclodextrin weekly, starting at postnatal day (P) 7. Starting at P10, a miglustat injection was administered daily until P23. Thereafter, miglustat was added to the powdered chow. For the sham treatment, both mutant NPC1(-/-) (NPC1(-/-sham)) and wild-type (NPC1(+/+sham)) mice received an NaCl injection and were fed powdered chow without miglustat. Analysis was performed on cerebellar slices by histology and immunohistochemistry. The volumes and cell counts of cerebellar structures were quantified. Additionally, ultrastructural analysis was performed with transmission electron microscopy. In agreement with previous studies, the current study demonstrates Purkinje cell degeneration in the mutant mice, which was partially abrogated by SRT/BPT. The volumes of cerebellar white matter and molecular layer were reduced as well. Also, the number of neurons was reduced in granular and molecular layers. However, only the molecular layer benefited from the therapy, as shown by an increase in the volume and the amount of neurons. The volume and number of neurons of the deep cerebellar nuclei were significantly decreased in mutant mice; an appreciable therapeutic benefit could be demonstrated for the nucleus interpositus.

Behavioral and structural effects of unilateral intrastriatal injections of botulinum neurotoxin a in the rat model of Parkinson's disease.

Botulinum neurotoxin (BoNT) inhibits the release of acetylcholine from presynaptic vesicles through its proteinase activity cleaving the SNARE complex. Parkinson's disease (PD) is associated with locally increased cholinergic activity in the striatum. Therefore, the present study investigates the effect of unilateral intrastriatal BoNT-A injection in naïve rats on striatal morphology; i.e., the total number of Nissl-stained neurons and the volume of caudate-putamen (CPu) were estimated. Furthermore, stainings for markers of gliosis (glial fibrillary acidic protein) and microglia (Iba1) were performed. In addition, the potential beneficial effects of a unilateral intrastriatal injection of BoNT-A on motor activity in the rat model of hemi-PD were evaluated. Hemi-PD was induced by unilateral injection of 6-hydroxydopamine (6-OHDA) into the right medial forebrain bundle. Six weeks later, rats received an ipsilateral intrastriatal injection of BoNT-A. Behaviorally, motor performance was tested. The total number of CPu neurons and the striatal volume were not significantly different between the BoNT-A-injected right and the intact left hemispheres of naïve rats. In hemi-PD rats, intrastriatal BoNT-A abolished apomorphine-induced rotations, increased amphetamine-induced rotations, and tended to improve left forelimb usage. Forced motor function in the accelerod test was not significantly changed by BoNT-A, and open field activity was also unaltered compared with sham treatment. Thus, intrastriatal BoNT-A affects spontaneous motor activity of hemi-PD rats to a minor degree compared with drug-induced motor function. In the future, tests assessing the cognitive and emotional performance should be performed to ascertain finally the potential therapeutic usefulness of intrastriatal BoNT-A for PD.

Intracerebrally applied botulinum neurotoxin in experimental neuroscience.

The use of botulinum neurotoxins (BoNTs) for therapeutic purposes in neuromuscular disorders and peripheral hypercholinergic conditions as well as in aesthetic medicine is widespread and common. BoNTs are also able to block the release of a wide range of transmitters from presynaptic boutons. Therefore, application of BoNTs directly in the central nervous system (CNS) is currently under study with respect to basic research and potentially as a new therapeutic strategy of neurological diseases. Investigations concentrate on effects of intracerebral and intraspinal application of BoNTs in rodents on the impact on spinal, nuclear, limbic and cortical neuronal circuits. In animal model first promising BoNT-induced therapeutical benefit has been shown in the treatment of pain, epilepsy, stroke and Parkinson's disease.