Alexander Hawlitschka

Intrastriatal botulinum toxin abolishes pathologic rotational behaviour and induces axonal varicosities in the 6-OHDA rat model of Parkinson's disease.

Central pathophysiological pathways of basal ganglia dysfunction imply a disturbed interaction of dopaminergic and cholinergic circuits. In Parkinsons disease (PD) imbalanced cholinergic hyperactivity prevails in the striatum. Interruption of acetylcholine (ACh) release in the striatum by locally injected botulinum neurotoxin A (BoNT-A) has been studied in the rat 6-hydroxydopamine (6-OHDA) model of PD (hemi-PD). The hemi-PD was induced by injection of 6-OHDA into the right medial forebrain bundle. Motor dysfunction provoked by apomorphine-induced contralateral rotation was completely reversed for more than 3 months by ipsilateral intrastriatal application of 1-2 ng BoNT-A. Interestingly, BoNT-A injected alone into the right striatum of naïve rats caused a slight transient ipsilateral apomorphine-induced rotation, which lasted only for about one month. Immunohistochemically, large axonal swellings appeared within the striatum injected with BoNT-A, which we tentatively named BoNT-A-induced varicosities. They contained either choline acetyltransferase or tyrosine hydroxylase. These findings suggest a selective inhibition of evoked release of ACh by locally applied BoNT-A. Intrastriatal application of BoNT-A may antagonize localized relative functional disinhibited hypercholinergic activity in neurodegenerative diseases such as PD avoiding side effects of systemic anti-cholinergic treatment.

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. Parkinsons 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.

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

Parkinsons 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.

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 Parkinsons disease.

Intrastriatal administration of botulinum neurotoxin A normalizes striatal D2R binding and reduces striatal D1R 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 (D1R, D2R, and DAT). The striatal D2R 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 D2R. BoNT‐A treatment reduced the asymmetry in both apomorphine‐induced rotational behavior and D2R expression, with the latter returning to normal values 5 months after intervention. D1R expression was significantly reduced, while DAT concentrations showed no alteration. Independent of the treatment, higher interhemispheric symmetry in raclopride binding to D2R 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.

Effects of systemic PSI administration on catecholaminergic cells in the brain, adrenal medulla and carotid body in Wistar rats

Traditional Parkinsons disease models in rats have several disadvantages. A promising alternative in terms of a more physiological model was proposed by McNaught et al. [McNaught, K.S., Perl, D.P., Brownell, A.L., Olanow, C.W., 2004. Systemic exposure to proteasome inhibitors causes a progressive model of Parkinsons disease. Ann. Neurol. 56, 149-162.] inhibiting the proteasomal protein degradation in vivo where they observed in Sprague-Dawley rats distinct symptoms of Parkinsons disease, a typical slow progredient loss of dopaminergic neurons in the substantia nigra and a lack of dopaminergic afferences in the striatum. We administered to Wistar rats a synthetic proteasome inhibitor (PSI) analogous to the published method. Locomotor changes were analysed by a footprint test. Brain slices containing the substantia nigra and the striatum were stained immunohistochemically against tyrosine hydroxylase, neuronal nuclei antigen, glial fibrillary acidic protein, alpha-synuclein and microglia. Standard histological stainings (haematoxylin eosin or Nissl) were also performed. The proteasome inhibitor effect on the glomerular layer of the olfactory bulb, the adrenal medulla and the carotid body was examined. We observed no PSI-induced motor deficits and loss of tyrosine hydroxylase immunoreactivity in the substantia nigra or the striatum. However, we detected a distinct increase of tyrosine hydroxylase immunoreactivity in the glomerular layer of the olfactory bulb and in the adrenal medulla. Our results fall in line with reports of other research groups which failed to reproduce the original report, but here for the first time McNaughts model could not be reproduced in Wistar rats. The observed effects on the olfactory bulb and peripheral catecholaminergic organs speak for an impermeability of the blood brain barrier for PSI.

Dopamine, Noradrenaline and Serotonin Receptor Densities in the Striatum of Hemiparkinsonian Rats following Botulinum Neurotoxin-A Injection

Parkinsons disease (PD) is characterized by a degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) that causes a dopamine (DA) deficit in the caudate-putamen (CPu) accompanied by compensatory changes in other neurotransmitter systems. These changes result in severe motor and non-motor symptoms. To disclose the role of various receptor binding sites for DA, noradrenaline, and serotonin in the hemiparkinsonian (hemi-PD) rat model induced by unilateral 6-hydroxydopamine (6-OHDA) injection, the densities of D1, D2/D3, α1, α2, and 5HT2A receptors were longitudinally visualized and measured in the CPu of hemi-PD rats by quantitative in vitro receptor autoradiography. We found a moderate increase in D1 receptor density 3 weeks post lesion that decreased during longer survival times, a significant increase of D2/D3 receptor density, and 50% reduction in 5HT2A receptor density. α1 receptor density remained unaltered in hemi-PD and α2 receptors demonstrated a slight right-left difference increasing with post lesion survival. In a second step, the possible role of receptors on the known reduction of apomorphine-induced rotations in hemi-PD rats by intrastriatally injected Botulinum neurotoxin-A (BoNT-A) was analyzed by measuring the receptor densities after BoNT-A injection. The application of this neurotoxin reduced D2/D3 receptor density, whereas the other receptors mainly remained unaltered. Our results provide novel data for an understanding of the postlesional plasticity of dopaminergic, noradrenergic and serotonergic receptors in the hemi-PD rat model. The results further suggest a therapeutic effect of BoNT-A on the impaired motor behavior of hemi-PD rats by reducing the interhemispheric imbalance in D2/D3 receptor density.

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.

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 Parkinsons 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.