Integration of atomic force microscopy and a microfluidic liquid cell for aqueous imaging and force spectroscopy.

We have designed and built a microfluidic liquid cell capable of high-resolution atomic force microscope (AFM) imaging and force spectroscopy. The liquid cell was assembled from three molded poly(dimethylsiloxane) (PDMS) pieces and integrated with commercially purchased probes. The AFM probe was embedded within the assembly such that the cantilever and tip protrude into the microfluidic channel. This channel is defined by the PDMS assembly on the top, a PDMS gasket on all four sides, and the sample substrate on the bottom, forming a liquid-tight seal. Our design features a low volume fluidic channel on the order of 50 nl, which is a reduction of over 3-5 orders of magnitude compared to several commercial liquid cells. This device facilitates testing at high shear rates and laminar flow conditions coupled with full AFM functionality in microfluidic aqueous environments, including execution of both force displacement curves and high resolution imaging.

Inducible nitric oxide synthase expression elicited in the mouse brain by inflammatory mediators circulating in the cerebrospinal fluid.

Expression of inducible nitric oxide synthase (iNOS) protein was studied in the brain after intracerebroventricular injections of interferon (IFN)-gamma, and IFN-gamma combined with lipopolysaccharide (LPS) or tumor necrosis factor (TNF)-alpha, compared to ovalbumin as control. Wild-type mice and mice with targeted deletion of the IFN-gamma receptor gene were used. Findings based on iNOS immunoreactivity were evaluated at 1, 2, 4 and 7 days post-injection, using also quantitative image analysis and double labeling with glial cell markers. IFN-gamma administration induced iNOS immmunostaining in activated microglia and macrophages in the parenchyma surrounding the ventricular system, several cortical fields and fiber tracts. IFN-gamma-elicited iNOS immunoreactivity was down-regulated after 1 day. The number of iNOS-immunopositive cells was significantly enhanced by co-administration of LPS or TNF-alpha; IFN-gamma+TNF-alpha injections also resulted in longer persistence of iNOS immunoreactivity. No immunopositive cells were seen in the brain of IFN-gamma receptor knockout mice after IFN-gamma administration; very few immunostained macrophages were detected in these cases, mostly around the injection needle track, after co-administration of LPS or TNF-alpha. Western blot analysis confirmed a marked iNOS induction in the brain of wild-type mice 24 h after IFN-gamma+LPS injections. The findings show that inflammatory mediators circulating in the cerebrospinal fluid induce in vivo iNOS in the brain with topographical selectivity and temporal regulation. The data also demonstrate that the signaling cascade activated by IFN-gamma binding to its receptor is critical for iNOS induction, and the synergistic action of LPS and TNF-alpha as iNOS inducers in brain cells is largely mediated by the receptor-regulated action of IFN-gamma.

Microglia activation in a model of sleep disorder: an immunohistochemical study in the rat brain during Trypanosoma brucei infection.

Microglial cells play a key role in the events triggered by infection, injury or degeneration in the central nervous system not only as scavenger cells but also as immune effector elements. We analyzed the features and distribution of cells of the microglia/macrophage lineage with OX-42 and ED-1 immunohistochemistry in the brain of experimental rats infected with the extracellular parasite Trypanosoma brucei. Such experimental infection provides a rat model of sleeping sickness or African trypanosomiasis, and is hallmarked in its advanced stages by severe alterations of the animals' sleep structure. In infected rats a remarkable activation of microglia, revealed by OX-42 immunoreactivity, became evident in the 3rd week post-infection in periventricular and subpial brain regions, with a prevalence in the hypothalamus. These features were concomitant with the onset of sleep anomalies, monitored with electroencephalographic recordings. Microglia activation increased in the following weeks, paralleling the progressive alterations of sleep parameters, and was most marked in the terminal stages of the infection, corresponding to the 6th-7th weeks. In addition, ED-1-immunoreactive macrophages and ramified microglia, confined to hypothalamic periventricular and basal regions, were evident after 4 weeks of disease. Degeneration of neuronal perikarya was not detected histologically in the infected brains at any time point. These data provide evidence for a reaction of microglia and macrophages in the brain of trypanosome-infected rats, and point out a selective distribution of these activated cells. The findings are discussed in relation to the animals' sleep disorder during trypanosome infection.

Altered neuronal activity rhythm and glutamate receptor expression in the suprachiasmatic nuclei of Trypanosoma brucei-infected rats.

The parasites Trypanosoma brucei cause African trypanosomiasis (sleeping sickness), a severe neuropsychiatric disease with marked disturbances of sleep-wake alternation. The sites of brain lesions are not well characterized. The present experimental investigation is focused on the hypothalamic suprachiasmatic nuclei, which play a role of a biological clock entraining endogenous rhythms in the mammalian brain. The electrophysiological properties of these neurons were analyzed in slice preparations from trypanosome-infected rats. The neuronal spontaneous activity, which shows a circadian oscillation, was markedly altered in the infected animals, displaying a reduced firing rate and phase advance of its circadian peak. The direct retinal fibers, which play a pivotal role in entrainment of the circadian pacemaker, displayed a normal density and distribution in the suprachiasmatic nuclei of infected animals after intraocular tracer injections in vivo. At the postsynaptic level, immunohistochemistry and Western blotting revealed in the suprachiasmatic nuclei of infected rats a selective decrease of the expression of glutamate AMPA GluR2/3 and NMDAR1 receptor subunits that gate retinal afferents. These data disclose an impairment of the neuronal functions in the biological clock in African trypanosomiasis, and may serve to unravel functional and molecular mechanisms behind endogenous rhythm disturbances.

Distribution and temporal regulation of the immune response in the rat brain to intracerebroventricular injection of interferon-gamma.

The response to intracerebroventricular administration of interferon (IFN)-gamma was examined in the adult Wistar rat brain: major histocompatibility complex (MHC) antigens class I and II, CD8 and CD4 antigens, and the macrophage/microglia antigen OX42 were analyzed in respect to saline-injected cases over 1 week. The glial cell type expressing MHC antigens was characterized with double labeling. IFN-gamma was thus found to induce MHC class I and II expression in microglia, identified by tomato lectin histochemistry, and not in GFAP-immunostained astrocytes. MHC antigen-expressing microglia was detected in the periventricular parenchyma, several fields of the cerebral cortex, cerebellum, major fiber tracts, and brainstem superficial parenchyma. Different gradients of density and staining intensity of the MHC-immunopositive elements were observed in these regions, in which MHC class I antigens persisted up to 1 week, when MHC class II induction had declined. Quantitative analysis pointed out the proliferation of OX42-immunoreactive cells in periventricular and basal brain regions. CD8+ T cells were observed in periventricular regions, basal forebrain, and fiber tracts 3 days after IFN-gamma injection and their density markedly increased by 7 days. CD4+ T cells were also seen and they were fewer than CD8+ ones. However, numerous CD4+ microglial cells were observed in periventricular and subpial regions, especially 1 week after IFN-gamma injection. Our data indicate that this proinflammatory cytokine mediates in vivo microglia activation and T cell infiltration in the brain and that the cells involved in this immune response display a regional selectivity and a different temporal regulation of antigen expression.

NADPH-diaphorase in antral gastritis of childhood.

BACKGROUND: Nitric oxide has an important role in the pathophysiology of the gastric mucosa. However, to date, it is not clear if nitric oxide plays a cytoprotective or cytotoxic role in the pathogenesis of mucosal lesion. METHODS: We have used the NADPH-diaphorase histochemistry that selectively stains cells containing nitric oxide synthase, the enzyme that catalyzes the production of nitric oxide on the antral gastric mucosa of children with antral gastritis. RESULTS: In the lamina propria of the mucosa, the presence of the enzymatic activity was found in perivascular round cells and nerve fibers. In the epithelium, focal positivity to NADPH-diaphorase was found in superficial cells, mainly located in the extrusive zones. The epithelial cells in the pits and glands were negative, in the mucous layer, Helicobacter pylori were also stained by NADPH-diaphorase. A single H. pylori-infected child who was also examined after eradication of the H. pylori showed during the control examination absence of microorganisms and reduction of the NADPH-diaphorase-positive cells in the mucosa. CONCLUSIONS: Our results demonstrate that, in gastric mucosa, endogenous and exogenous structures express a NADPH-diaphorase activity.

Age-dependent induction of nitric oxide synthase activity in facial motoneurons after axotomy.

The facial nerve was transected in rats at different postnatal ages, from birth to early adulthood. NADPH-diaphorase histochemistry was performed to analyze the induction of nitric oxide synthase, the synthetic enzyme of the free radical nitric oxide, in injured facial motoneurons. In addition, in situ nick-end labeling of DNA fragmentation (TUNEL technique) was performed after axotomy at birth, to verify the occurrence of apoptosis in the damaged facial motoneurons. A striking age-dependency was found in the induction of nitric oxide synthase activity in axotomized facial motoneurons. NADPH-diaphorase positivity was not detectable in these neurons 1 and 2 days after axotomy at birth, when apoptotic changes were evident and marked. In addition, NADPH-diaphorase staining was hardly detectable in the facial nucleus 4 days after axotomies at birth, when extensive motoneuron loss was evident. NADPH-diaphorase positivity was instead induced in the facial motoneurons axotomized from the end of the first postnatal week to adulthood, when the nerve cell loss was less severe than in newborns. However, the time course of the enzyme activity induction varied considerably in relation to the animals' age. These findings are discussed in relation to the role of nitric oxide in motoneuron death or protective response to injury and of oxidative stress in neurodegeneration.

Induction of NADPH-diaphorase activity in the forebrain in a model of intracerebral hemorrhage and its inhibition by the traditional Chinese medicine complex Nao Yi An.

Induction of NADPH-diaphorase (NDP) activity in the rat cerebral cortex was studied after autologous blood injection into the internal capsule as experimental model of intracerebral hemorrhage. The potential inhibitory effect on NDP induction by Nao Yi An (NYA), a complex derived from materials of animal and plant origin used in the treatment of intracerebral hemorrhage in traditional Chinese medicine, was also investigated. In animals without therapeutic treatment 2 and 4 days after injection of autologous blood, NDP activity was highly induced in pyramidal neurons in the neocortex, piriform, and entorhinal cortices, in astrocytes and in phagocytes in the hematoma and the area surrounding it, as well as in the subcortical white matter, and in endothelial cells in both the cortex and subcortical white matter bilaterally. Oral administration of NYA failed to inhibit NDP induction in endothelial cells but demonstrated a strong inhibitory effect on NDP activity induced in pyramidal neurons and astrocytes. NDP induction in phagocytes was also inhibited by the administration of NYA. Altogether the present results suggest that intracerebral hemorrhage in the internal capsule may induce nitric oxide synthase activity in different cell populations in the cortex and that administration of NYA can selectively inhibit such induction and, thus, potentially play a neuroprotective role.

Induction of NADPH-diaphorase activity in the rat forebrain after middle cerebral artery occlusion.

Induction of NADPH-diaphorase (NDP) following ischemic infarction was studied by means of histochemistry in the rat cerebral cortex 1,2,7, and 14 days after distal occlusion of the right middle cerebral artery (MCA). The fine structure of cells in the penumbra region of the necrotic center was also investigated. MCA distal occlusion resulted in ischemic lesion of the frontoparietal cortex of variable extent; NDP induction was detected in neurons, astrocytes, macrophages, and endothelial cells, with regional specificity and a temporal gradient. One, two, and seven days after MCA occlusion, weak NDP positivity was consistently induced in some pyramidal neurons in cortical areas neighboring the necrotic area; NDP induction was also seen in pyramidal neurons of the ipsilateral anterior cingulate and infralimbic cortices and in the tenia tecta. In addition, numerous NDP-positive pyramidal neurons were detected in the contralateral frontoparietal cortex after relatively large ischemic lesions. Two weeks after MCA occlusion, NDP induction in neurons was only evident in the deep cortical layers near the lesion. NDP histochemistry combined with glial fibrillary acidic protein immunofluorescence, performed 7 days after MCA occlusion, indicated that the astrocytes at the periphery of the necrotic area were hypertrophic and some of them were also NDP-positive. One and two days after MCA occlusion, numerous macrophages displaying NDP positivity of variable intensity were seen at the periphery of the necrotic area and in the external capsule of the ischemic cerebral hemisphere. Many endothelial cells in the cortex and subcortical white matter were consistently NDP-positive in all rats. Electron microscopic studies indicated that the area adjacent to the necrotic center was composed of fibrous astrocytes, with the morphological characteristics of proliferation, and numerous lysosome-filled macrophages. Altogether the present results suggest that focal cerebral ischemia may induce in different cell types nitric oxide synthase, which is equivalent to NDP in fixed tissue. The induction of nitric oxide synthase may be related to (1) blood-flow regulation at relatively early postischemic stages, which may decline when collateral circulation is established, and/or (2) cytotoxic or neuroprotective mechanisms.

The chemical heterogeneity of cortical interneurons: nitric oxide synthase vs. calbindin and parvalbumin immunoreactivity in the rat.

Neurons that contain nitric oxide synthase (NOS) type I and the calcium binding proteins calbindin D28k or parvalbumin were simultaneously visualized by means of double immunohistofluorescence in the cerebral cortex of Wistar and Sprague-Dawley rats. All the three immunoreactive cell populations were primarily represented by nonpyramidal neurons. NOS-immunoreactive cells were less numerous than the calbindin- or parvalbumin-immunoreactive ones, and were intermingled with the neurons containing these calcium binding proteins. NOS-immunoreactive cells were separate from the parvalbumin-immunoreactive ones, whereas a minor proportion of them was found to be colocalized with calbindin. The cortical neurons in which NOS and calbindin coexisted were more numerous in the Sprague-Dawley than in the Wistar rats, and displayed an anteroposterior gradient of density, with the highest concentration in the medial prefrontal, frontal, and cingulate cortices. Double NOS-calbindin-immunoreactive neurons prevailed in the deep cortical layers and they were relatively numerous in the cingulate cortex. The present data indicate a selectivity in the expression of NOS vs. calbindin and parvalbumin in cortical cells, and further support the chemical heterogeneity of GABAergic interneurons in the cerebral cortex.

Fos-related protein expression in the midline paraventricular nucleus of the rat thalamus: basal oscillation and relationship with limbic efferents.

The expression of Fos-related protein, encoded by the proto-oncogene c-fos, was investigated by means of immunohistochemistry in the paraventricular nucleus of the thalamic midline (PV) during nighttime and daytime in rats entrained to a 12-h light/12-h dark cycle. In the first step of this study the animal's physiological state preceding perfusion was monitored with electro-encephalographic recording. It was thus detected that the PV contained a considerable number of Fos-like-immunostained neurons during the hours of darkness, when the rats had been awake, and that the number of Fos-like-immunoreactive neurons was significantly lower during the hours of light, after a period of sleep. In the second step of this study Fos immunohistochemistry was combined with the retrograde transport of a gold-labeled tracer injected either in the amygdala or in the nucleus accumbens. This strategy enabled us to determine that in the rats perfused during nighttime Fos-related protein was spontaneously induced in PV cells projecting to these targets, with a significant prevalence of neurons projecting to the amygdala in the anterior portion of the PV and of neurons projecting to the nucleus accumbens in the posterior part of the nucleus. In addition, a significant reduction of Fos-like-immunoreactive cells was detected in the PV ipsilaterally to the injection, indicating that tracer administration and axonal transport may interfere with c-fos expression in neurons. Altogether the present data indicate that Fos-related protein expression undergoes a marked oscillation in the PV during 24 h in basal conditions, and that c-fos is induced in the PV relay neuronal subsets when the animal is awake.(ABSTRACT TRUNCATED AT 250 WORDS)

A sensitive double immunostaining protocol for Fos-immunoreactive neurons.

Immunostaining of Fos, the nuclear protein encoded by the immediate early gene c-fos, is widely used to reveal the functional activation of neurons. The chemical identity of cells that express c-fos can be investigated with double immunohistochemistry. We report the usefulness of a sequential two-color avidin-biotin-immunoperoxidase method that provides a highly sensitive double immunostaining and allows long-term storage of the sections. In this protocol, metal intensification of diaminobenzidine (int-DAB) resulted in dark brown/black Fos immunostaining of the neuronal nucleus. The use of alpha-naphthol/pyronin reaction product yielded pink immunostaining of a second antigen in the cytoplasm. This combination produced higher contrast than that produced by int-DAB Fos immunostaining combined with conventional DAB light brown cytoplasmic staining. The sensitivity of the use of int-DAB and alpha-naphthol/pyronin was verified in different experimental paradigms, combining the immunocytochemical detection of Fos with that of the p75 nerve growth factor receptor, or parvalbumin, or calbindin D28k.

Neuronal IFN-gamma in tuberomammillary neurones.

Neuronal interferon-gamma (N-IFN-gamma), recently isolated from the nervous system, has a molecular weight distinct from that of lymphocyte-derived IFN-gamma, but crossreacts immunologically and shares certain bioactivities with this cytokine. In the rat brain N-IFN-gamma-immunoreactive perikarya were concentrated in the hypothalamic tuberomammillary nuclei; some immunostained neurones were also detected in the dorsal pontine tegmentum. Immunopositive nerve fibres were profusely distributed through the periventricular hypothalamus and midline thalamus. Scattered fibres occurred diffusely through the brain, ramified in the subpial layer and also surrounded intrathecal vessels. A dense concentration of puncta was detected in the suprachiasmatic nuclei and in the molecular layer of the hippocampal dentate gyrus. A role of N-IFN-gamma in immunological reactions and in modulation of selective brain functions is suggested.

Dysregulation of photic induction of Fos-related protein in the biological clock during experimental trypanosomiasis.

The mammalian suprachiasmatic nuclei of the hypothalamus (SCN) serve as pacemaker for circadian rhythms and the immediate-early gene c-fos is known to be induced by photic stimulation in the SCN of rodents. We studied the induction of Fos-related protein following a light pulse in rats infected with Trypanosoma brucei. This parasite causes in humans African sleeping sickness, a neuropsychiatric syndrome that involves changes of endogenous biological rhythms. Fos-like immunoreactivity after photic stimulation was dramatically reduced in the SCN of trypanosome- infected rats during the subjective night. These findings indicate that the photic entrainment of the biological clock may be altered during the infection.

Co-induction of neuronal interferon-gamma and nitric oxide synthase in rat motor neurons after axotomy: a role in nerve repair or death?

Induction of an interferon-gamma-like molecule, previously isolated from neurons (N-IFN-gamma), and of the neuronal isoform I of the synthetic enzyme of the free radical nitric oxide, nitric oxide synthase I, as well as of NADPH-diaphorase, were examined in axotomized dorsal motor vagal and hypoglossal neurons. Unilateral transection of the vagal and hypoglossal nerves was performed in the same rat and an induction of N-IFN-gamma and nitric oxide synthase I immunostaining as well as NADPH-diaphorase histochemical positivity was observed in the ipsilateral motoneurons after 2-4 days. The immuno- and enzyme-histochemical positivities were much stronger in the dorsal motor vagal neurons than in hypoglossal neurons. Two and 4 weeks after axotomy N-IFN-gamma immunoreactivity and NADPH-diaphorase positivity persisted in the former, but started to decrease in the latter neurons. Previous data have shown that 23 weeks after nerve transection the majority of the dorsal motor vagal neurons are lost, while the majority of the hypoglossal neurons survive. The high and persistent expression of N-IFN-gamma and nitric oxide synthase I after axotomy in the dorsal motor vagal neurons, that are largely destined to die, indicates that the co-induction of these two molecules may be implicated in the pathogenesis of neuronal degeneration.

Interferon-gamma and a factor derived from trypanosomes cause behavioural changes in the rat.

A newly isolated interferon-gamma (IFN-gamma) immunoreactive molecule, "neuronal IFN-gamma", and recombinant lymphocyte-derived IFN-gamma were injected intracerebroventricularly (i.c.v.) through a previously implanted cannula into adult male rats during both the light and the dark phases of the light/dark cycle. The two molecules caused a reduction in both frequency and duration of rearing and locomotion during the dark, but not the light, phase. A molecule isolated from Trypanosoma brucei brucei, a parasite of the same subspecies of trypanosomes which causes African sleeping sickness, can induce production and release of IFN-gamma and "neuronal IFN-gamma" from lymphocytes and neurons, respectively. I.c.v. injection of this factor also reduced rearing during the dark period, but to a less extent. Thus, "neuronal IFN-gamma" appears to have effects on animal behaviour in common with lymphocyte-derived IFN-gamma. This study highlights the potential role of these cytokines in behaviour disturbances.

Trypanosomes cause dysregulation of c-fos expression in the rat suprachiasmatic nucleus.

Rats infected with the parasite Trypanosoma brucei brucei showed selective changes of c-fos expression in the suprachiasmatic nucleus of the hypothalamus (SCN) during spontaneous sleep (S) and wakefulness (W) under a basal 12 h/12 h light-dark (L-D) cycle. In the vast majority of W (D phase) control animals the SCN was devoid of cells displaying Fos-related immunopositivity, while Fos-like-immunoreactive (ir) neurones were detected in most S (L phase) control rats. In most infected animals, on the other hand, Fos-ir neurones were detected in the SCN during W, but not during the S period, with a significant difference between control and infected S rats. Thus, these data indicate that the basal c-fos expression in the SCN during the L-D and S-W cycles is considerably altered in experimental trypanosomiasis. This is the first observation of a selective change in the SCN in trypanosome-infected rat brains. Since the SCN plays an important role as a pace-maker for biological rhythms, this finding may provide a basis for understanding the pathogenesis behind endogenous rhythm dyregulation and changes in sleeping pattern in human trypanosomiasis (African sleeping sickness).

Sleep and timekeeping changes, and dysregulation of the biological clock in experimental trypanosomiasis.

The rest-activity and body temperature 24 hours cycles, as well as the pattern of spontaneous sleep, were investigated in rats after infection with Trypanosoma brucei brucei. In the infected rats, which were entrained to a 12 hours/12 hours photoperiod, a considerable hypokinesia was detected during the hours of darkness. In most of the infected animals, the body temperature cycle displayed a lower amplitude and an advance of about 3 hours in respect to control rats; in addition, the body temperature rhythm was not significant in some infected rats. The relative proportion of slow wave synchronized sleep, as well as the rapid-eye movement (REM) latency, were significantly reduced in the infected animals, in which sleep was considerably fragmented. The induction of Fos (the protein encoded by the immediate early gene c-fos), in response to light stimulation during the early subjective night, was severely impaired in the hypothalamic suprachiasmatic nuclei in trypanosome-infected rats. Altogether these data point out a disruption of locomotor activity and body temperature 24 hours cycle and a major disorganization of sleep during experimental trypanosomiasis. In addition, our findings indicate that the molecular and functional correlates of the synchronizing action of the suprachiasmatic nuclei, which play a major role of biological clock of endogenous biological rhythms, could be altered during trypanosome infection.

Co-localization of nitric oxide synthase and NGF receptor in neurons in the medial septal and diagonal band nuclei of the rat.

The relationship of neurons that express nitric oxide synthase type I (NOS-I), the synthetic enzyme of the free radical nitric oxide (NO), with cells that contain the low affinity p75 nerve growth factor receptor (NGFr) was examined in the basal forebrain region of the rat using double immunohistofluorescence. NOS and NGFr were found to be co-localized in a neuronal population that displayed a selective distribution. Double immunopositive neurons were evident in the medial septum and in rostral levels of the diagonal band nuclei where the vast majority (more than 80%) of NOS-immunoreactive cells were also NGFr-positive; the two cell populations were separate at more caudal levels of the basal forebrain. These findings support the chemical heterogeneity of septal and basal forebrain neurons which contain NGFr and indicate that in the rat a subset of neurons of the septum-diagonal band complex may release NO.

Neural substrate of a cerebellar movement disorder induced by intracerebroventricular injection of propidium iodide in the rat: a Fos immunocytochemical study.

Intracerebroventricular (icv) injection of propidium iodide (PI) in the rat results in a transient movement disorder characterized by nystagmus, ataxia, and shaking. In the present study we used c-Fos as a marker for neuronal activation to investigate the neural substrate underlying this movement disorder. PI was injected into the lateral cerebral ventricle of freely moving rats through a previously implanted cannula. Animals were perfused 3 h after the injection and the brains were processed for c-Fos immunocytochemistry. Paired control animals were injected with saline. After PI injection, a significant Fos expression was seen in the cerebral cortex, thalamic midline nuclei, thalamic intralaminar nuclei, hypothalamus, central gray, pontine nuclei, locus coeruleus, vestibular complex, inferior olive, ventrolateral medulla, nucleus of solitary tract, and deep cerebellar nuclei. Few or no Fos immunoreactive cells were seen in the above structures of the control animals. The present study indicates that a large number of neurons located in many different neural structures are activated following icv injection of PI. Second, consistent with the cerebellar feature of the movement disorder, a major Fos expression was found in the cerebellar circuitry (deep cerebellar nuclei, pontine nuclei, vestibular complex, and inferior olive). It reinforces further the assumption that the movement disorder is due to cerebellar dysfunction caused by PI.