Apoptosis in the Dentate Nucleus Following Kindling-induced Seizures in Rats.

BACKGROUND: Epilepsy is a common neurological disorder characterized by abnormal and recurrent neuronal discharges that result in epileptic seizures. The dentate nuclei of the cerebellum receive excitatory input from different brain regions. Purkinje cell loss due to chronic seizures could lead to decreased inhibition of these excitatory neurons, resulting in the activation of apoptotic cascades in the dentate nucleus. OBJECTIVE: The present study was designed to determine whether there is a presence of apoptosis (either intrinsic or extrinsic) in the dentate nucleus, the final relay of the cerebellar circuit, following kindling-induced seizures. METHODS: In order to determine this, seizures were triggered via the amygdaloid kindling model. Following 0, 15, or 45 stimuli, rats were sacrificed, and the cerebellum was extracted. It was posteriorly prepared for the immunohistochemical analysis with cell death biomarkers: TUNEL, Bcl-2, truncated Bid (tBid), Bax, cytochrome C, and cleaved caspase 3 (active form). Our findings reproduce results obtained in other parts of the cerebellum. RESULTS: We found a decrease of Bcl-2 expression, an anti-apoptotic protein, in the dentate nucleus of kindled rats. We also determined the presence of TUNEL-positive neurons, which confirms the presence of apoptosis in the dentate nucleus. We observed the expression of tBid, Bax, as well as cytochrome C and cleaved caspase-3, the main executor caspase of apoptosis. CONCLUSION: There is a clear activation of both the intrinsic and extrinsic apoptotic pathways in the cells of the dentate nucleus of the cerebellum of rats subjected to amygdaloid kindling.

Cerebellar Fastigial Nucleus Stimulation in a Chronic Unpredictable Mild Stress Rat Model Reduces Post-Stroke Depression by Suppressing Brain Inflammation via the microRNA-29c/TNFRSF1A Signaling Pathway.

BACKGROUND We previously reported that cerebellar fastigial nucleus stimulation reduced post-stroke depression in a rat model by reducing inflammation. This study aimed to investigate the molecular inflammatory signaling pathways associated with cerebellar fastigial nucleus stimulation in an established rat model of post-stroke depression. MATERIAL AND METHODS Twenty-four Sprague-Dawley rats included a sham group (N=6), an untreated stroke group (N=6), an untreated post-stroke depression model group (PSD) (N=6), and the model group treated with cerebellar fastigial nucleus stimulation (FNS) (N=6). The rat stroke model involved occlusion of the middle cerebral artery occlusion (MCAO). Post-stroke depression model was established using chronic unpredictable mild stress treatment and was verified using an open field test. Real-time polymerase chain reaction (PCR) and Western blot compared expression levels of microRNA-29c (miR-29c), miR-676, TNFRSF1A, tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6 and IL-1ß in cerebellar tissue. U251 human glioblastoma cells and SH-SY5Y human neuroblastoma cells were studied in vitro. RESULTS Cerebellar fastigial nucleus stimulation reduced behaviors associated with depression in the rat model, upregulated the expression of miR-29c, and reduced the expression of TNFRSF1A and inflammatory cytokines, and mildly reduced neuronal apoptosis. Bioinformatics data analysis identified a regulatory relationship between miR-29c and TNFRSF1A. SH-SY5Y cells treated with a miR-29c mimic, or TNFRSF1A short interfering RNA (siRNA), identified a negative regulatory relationship between TNFRSF1A and miR-29c. CONCLUSIONS In a rat model, cerebellar fastigial nucleus stimulation reduced the expression of TNFRSF1A by upregulating miR-29c expression, which suppressed the expression of inflammatory cytokines, resulting in reduced severity of post-stroke depression.

Longitudinal evaluation of iron concentration and atrophy in the dentate nuclei in friedreich ataxia.

BACKGROUND: Friedreich ataxia is a recessively inherited, progressive neurological disease characterized by impaired mitochondrial iron metabolism. The dentate nuclei of the cerebellum are characteristic sites of neurodegeneration in the disease, but little is known of the longitudinal progression of abnormalities in these structures. METHODS: Using in vivo magnetic resonance imaging, including quantitative susceptibility mapping, we investigated changes in iron concentration and volume in the dentate nuclei in individuals with Friedreich ataxia (n = 20) and healthy controls (n = 18) over a 2-year period. RESULTS: The longitudinal rate of iron concentration was significantly elevated bilaterally in participants with Friedreich ataxia relative to healthy controls. Atrophy rates did not differ significantly between groups. Change in iron concentration and atrophy both correlated with baseline disease severity or duration, indicating sensitivity of these measures to disease stage. Specifically, atrophy was maximal in individuals early in the disease course, whereas the rate of iron concentration increased with disease progression. CONCLUSIONS: Progressive dentate nucleus abnormalities are evident in vivo in Friedreich ataxia, and the rates of change of iron concentration and atrophy in these structures are sensitive to the disease stage. The findings are consistent with an increased rate of iron concentration and atrophy early in the disease, followed by iron accumulation and stable volume in later stages. This pattern suggests that iron dysregulation persists after loss of the vulnerable neurons in the dentate. The significant changes observed over a 2-year period highlight the utility of quantitative susceptibility mapping as a longitudinal biomarker and staging tool. © 2019 International Parkinson and Movement Disorder Society.

Absence of dentate nucleus resting-state functional connectivity changes in nonneurological patients with gadolinium-related hyperintensity on T1 -weighted images.

BACKGROUND: The dentate nuclei of the cerebellum are the areas where gadolinium predominantly accumulates. It is not yet known whether gadolinium deposition affects brain functions. PURPOSE/HYPOTHESIS: To assess whether gadolinium-dependent high signal intensity of the cerebellum on T1 -weighted images of nonneurological adult patients with Crohn's disease is associated with modifications of resting-state functional connectivity (RSFC) of the cerebellum and dentate nucleus. STUDY TYPE: Observational, cross-sectional. POPULATION: Fifteen patients affected by Crohn's disease were compared with 16 healthy age- and gender-matched control subjects. All participants underwent neurological, neurocognitive-psychological assessment, and blood sampling. FIELD STRENGTH/SEQUENCE: 1.5-T magnet blood oxygenation level-dependent (BOLD) functional MRI. ASSESSMENT: High signal intensity on T1 -weighted images, cerebellum functional connectivity, neurocognitive performance, and blood circulating gadolinium levels. STATISTICAL TESTS: An unpaired two-sample t-test (age and sex were nuisance variables) was used to investigate between-group differences in cerebellar and dentate nucleus functional connectivity. Z-statistical images were set using clusters determined by Z > 2.3 and a familywise error (FWE)-corrected cluster significance threshold of P = 0.05. RESULTS: Dentate nuclei RSFC was not different (P = n.s.) between patients with gadolinium-dependent high signal intensity on T1 -weighted images and controls. Pre- and postcentral gyrus bilaterally and the right supplementary motor cortex showed a decrease of RSFC with the cerebellum hemispheres (P < 0.05 FWE-corrected) and was related to disease duration but not to gadodiamide cumulative doses (P = n.s.). DATA CONCLUSION: Crohn's disease patients with gadolinium-dependent hyperintense dentate nuclei on unenhanced T1 -weighted images do not show dentate nucleus RSFC changes. LEVEL OF EVIDENCE: 2 Technical Efficacy Stage: 5 J. Magn. Reson. Imaging 2019;50:445-455.

Phosphorylation of the neurogenic transcription factor SOX11 on serine 133 modulates neuronal morphogenesis.

The intellectual disability gene, Sox11, encodes for a critical neurodevelopmental transcription factor with functions in precursor survival, neuronal fate determination, migration and morphogenesis. The mechanisms regulating SOX11's activity remain largely unknown. Mass spectrometric analysis uncovered that SOX11 can be post-translationally modified by phosphorylation. Here, we report that phosphorylatable serines surrounding the high-mobility group box modulate SOX11's transcriptional activity. Through Mass Spectrometry (MS), co-immunoprecipitation assays and in vitro phosphorylation assays followed by MS we verified that protein kinase A (PKA) interacts with SOX11 and phosphorylates it on S133. In vivo replacement of SoxC factors in developing adult-generated hippocampal neurons with SOX11 S133 phospho-mutants indicated that phosphorylation on S133 modulates dendrite development of adult-born dentate granule neurons, while reporter assays suggested that S133 phosphorylation fine-tunes the activation of select target genes. These data provide novel insight into the control of the critical neurodevelopmental regulator SOX11 and imply SOX11 as a mediator of PKA-regulated neuronal development.

Up to 52 administrations of macrocyclic ionic MR contrast agent are not associated with intracranial gadolinium deposition: Multifactorial analysis in 385 patients.

PURPOSE: To determine whether multiple repeated administrations of gadolinium-based macrocyclic ionic MR contrast agent (MICA) are associated with intracranial gadolinium deposition and identify the predisposing factors for deposition in various clinical situations. MATERIALS AND METHODS: In this institutional review board-approved retrospective study, 385 consecutive patients who underwent MICA-enhanced MR imaging were enrolled. The dentate nucleus-to-pons (DN/P) and globus pallidus-to-thalamus (GP/Th) signal intensity (SI) ratios on unenhanced T1-weighted images were recorded by 2 independent readers and averaged. The mean DN/P and GP/Th SI ratio difference between the last and the first examinations were tested using the one-sample t-test. Student's t-test and stepwise regression analysis were used to identify the predisposing factors for deposition based on the number of administrations, time interval, hepatic and renal function, magnetic field strength, and chemo- or radiation therapy. RESULTS: The mean DN/P SI ratio difference was not different from zero (P = .697), even in patients with ≥20 administrations (n = 33). Only patients with abnormal renal function showed an increase in the mean DN/P SI ratio difference (P = .019). The mean DN/P SI ratio difference was not associated with any predisposing factors. However, the mean GP/Th SI ratio difference showed decrease (P < .001), which was associated with age (P = .007), number of administrations (P = .01) and number of radiation therapy sessions (P = .022) on multivariate analysis. CONCLUSION: Multiple repeated administrations of MICA were not associated with increased T1 signal intensity in deep brain nuclei suggestive of Gd deposition in patients with normal renal function.

Signal intensity change on unenhanced T1-weighted images in dentate nucleus and globus pallidus after multiple administrations of gadoxetate disodium: an intraindividual comparative study.

PURPOSE: To investigate whether there is an increased signal intensity (SI) of dentate nucleus (DN) and globus pallidus (GP) on unenhanced T1-weighted magnetic resonance imaging (MRI), in patients who had undergone multiple administrations of gadoxetate disodium. MATERIALS AND METHODS: We retrospectevely included stage III melanoma patients, who had been previously enrolled in a trial of adjuvant therapy and who had undergone whole-body contrast-enhanced MRIs with gadoxetate disodium every three months for their follow-up. The SI ratios of DN-to-pons and GP-to-thalamus on unenhanced T1-weighted images were calculated. The difference in SI ratios between the first and the last MRI examinations was assessed and a linear mixed model was performed to detect how SI ratios varied with the number of administrations. RESULTS: Eighteen patients were included in our study. The number of gadoxetate disodium administrations ranged from 2 to 18. Paired t-test did not show any significant difference in DN-to-pons (p=0.21) and GP-to-thalamus (p=0.09) SI ratios by the end of the study. DN-to-pons SI ratio and GP-to-thalamus SI ratio did not significantly increase with increasing the number of administrations (p=0.14 and p=0.06, respectively). CONCLUSION: Multiple administrations of gadoxetate disodium are not associated with increased SI in DN and GP in the brain. KEY POINTS: • Gadolinium may deposit in the human brain after multiple GBCA administrations. • Gadolinium deposition is associated with increased T1W signal intensity • Increase in signal intensity is most apparent within the DN and GP • Multiple administrations of gadoxetate disodium do not increase T1W signal.

Gadolinium deposition in the brain: association with various GBCAs using a generalized additive model.

OBJECTIVES: To determine the relationship between the number of administrations of various gadolinium-based contrast agents (GBCAs) and increased T1 signal intensity in the globus pallidus (GP) and dentate nucleus (DN). METHODS: This retrospective study included 122 patients who underwent double-dose GBCA-enhanced magnetic resonance imaging. Two radiologists calculated GP-to-thalamus (TH) signal intensity ratio, DN-to-pons signal intensity ratio and relative change (Rchange) between the baseline and final examinations. Interobserver agreement was evaluated. The relationships between Rchange and several factors, including number of each GBCA administrations, were analysed using a generalized additive model. RESULTS: Six patients (4.9%) received linear GBCAs (mean 20.8 number of administration; range 15-30), 44 patients (36.1%) received macrocyclic GBCAs (mean 26.1; range 14-51) and 72 patients (59.0%) received both types of GBCAs (mean 31.5; range 12-65). Interobserver agreement was almost perfect (0.99; 95% CI: 0.99-0.99). Rchange (DN:pons) was associated with gadodiamide (p = 0.006) and gadopentetate dimeglumine (p < 0.001), but not with other GBCAs. Rchange (GP:TH) was not associated with GBCA administration. CONCLUSIONS: Previous administration of linear agents gadoiamide and gadopentetate dimeglumine is associated with increased T1 signal intensity in the DN, whereas macrocyclic GBCAs do not show an association. KEY POINTS: • Certain linear GBCAs are associated with T1 signal change in the dentate nucleus. • The signal change is related to the administration number of certain linear GBCAs. • Difference in signal change may reflect differences in stability of agents.

Electrical Stimulation Normalizes c-Fos Expression in the Deep Cerebellar Nuclei of Depressive-like Rats: Implication of Antidepressant Activity.

The electrical stimulation of specific brain targets has been shown to induce striking antidepressant effects. Despite that recent data have indicated that cerebellum is involved in emotional regulation, the mechanisms by which stimulation improved mood-related behaviors in the cerebellum remained largely obscure. Here, we investigated the stimulation effects of the ventromedial prefrontal cortex (vmPFC), nucleus accumbens (NAc), and lateral habenular nucleus on the c-Fos neuronal activity in various deep cerebellar and vestibular nuclei using the unpredictable chronic mild stress (CMS) animal model of depression. Our results showed that stressed animals had increased number of c-Fos cells in the cerebellar dentate and fastigial nuclei, as well as in the spinal vestibular nucleus. To examine the stimulation effects, we found that vmPFC stimulation significantly decreased the c-Fos activity within the cerebellar fastigial nucleus as compared to the CMS sham. Similarly, there was also a reduction of c-Fos expression in the magnocellular part of the medial vestibular nucleus in vmPFC- and NAc core-stimulated animals when compared to the CMS sham. Correlational analyses showed that the anxiety measure of home-cage emergence escape latency was positively correlated with the c-Fos neuronal activity of the cerebellar fastigial and magnocellular and parvicellular parts of the interposed nuclei in CMS vmPFC-stimulated animals. Interestingly, there was a strong correlation among activation in these cerebellar nuclei, indicating that the antidepressant-like behaviors were possibly mediated by the vmPFC stimulation-induced remodeling within the forebrain-cerebellar neurocircuitry.

Dentate nucleus iron deposition is a potential biomarker for tremor-dominant Parkinson's disease.

Parkinson's disease (PD) is a heterogeneous neurodegenerative disorder with variable clinicopathologic phenotypes and underlying neuropathologic mechanisms. Each clinical phenotype has a unique set of motor symptoms. Tremor is the most frequent initial motor symptom of PD and is the most difficult symptom to treat. The dentate nucleus (DN) is a deep iron-rich nucleus in the cerebellum and may be involved in PD tremor. In this study, we test the hypothesis that DN iron may be elevated in tremor-dominant PD patients using quantitative susceptibility mapping. Forty-three patients with PD [19 tremor dominant (TD)/24 akinetic rigidity (AR) dominant] and 48 healthy gender- and age-matched controls were recruited. Multi-echo gradient echo data were collected for each subject on a 3.0-T MR system. Inter-group susceptibility differences in the bilateral DN were investigated and correlations of clinical features with susceptibility were also examined. In contrast with the AR-dominant group, the TD group was found to have increased susceptibility in the bilateral DN when compared with healthy controls. In addition, susceptibility was positively correlated with tremor score in drug-naive PD patients. These findings indicate that iron load within the DN may make an important contribution to motor phenotypes in PD. Moreover, our results suggest that TD and AR-dominant phenotypes of PD can be differentiated on the basis of the susceptibility of the DN, at least at the group level. Copyright © 2016 John Wiley & Sons, Ltd.

Broad distribution of ataxin 1 silencing in rhesus cerebella for spinocerebellar ataxia type 1 therapy.

Spinocerebellar ataxia type 1 is one of nine polyglutamine expansion diseases and is characterized by cerebellar ataxia and neuronal degeneration in the cerebellum and brainstem. Currently, there are no effective therapies for this disease. Previously, we have shown that RNA interference mediated silencing of ATXN1 mRNA provides therapeutic benefit in mouse models of the disease. Adeno-associated viral delivery of an engineered microRNA targeting ATXN1 to the cerebella of well-established mouse models improved motor phenotypes, neuropathy, and transcriptional changes. Here, we test the translatability of this approach in adult rhesus cerebella. Nine adult male and three adult female rhesus macaque were unilaterally injected with our therapeutic vector, a recombinant adeno-associated virus type 1 (rAAV1) expressing our RNAi trigger (miS1) and co-expressing enhanced green fluorescent protein (rAAV1.miS1eGFP) into the deep cerebellar nuclei using magnetic resonance imaging guided techniques combined with a Stealth Navigation system (Medtronics Inc.). Transduction was evident in the deep cerebellar nuclei, cerebellar Purkinje cells, the brainstem and the ventral lateral thalamus. Reduction of endogenous ATXN1 messenger RNA levels were ≥30% in the deep cerebellar nuclei, the cerebellar cortex, inferior olive, and thalamus relative to the uninjected hemisphere. There were no clinical complications, and quantitative and qualitative analyses suggest that this therapeutic intervention strategy and subsequent reduction of ATXN1 is well tolerated. Collectively the data illustrate the biodistribution and tolerability of rAAV1.miS1eGFP administration to the adult rhesus cerebellum and are supportive of clinical application for spinocerebellar ataxia type 1.

Age and sex related differences in subcortical brain iron concentrations among healthy adults.

Age and sex can influence brain iron levels. We studied the influence of these variables on deep gray matter magnetic susceptibilities. In 183 healthy volunteers (44.7 ± 14.2 years, range 20-69, ♀ 49%), in vivo quantitative susceptibility mapping (QSM) at 1.5T was performed to estimate brain iron accumulation in the following regions of interest (ROIs): caudate nucleus (Cd), putamen (Pt), globus pallidus (Gp), thalamus (Th), pulvinar (Pul), red nucleus (Rn), substantia nigra (Sn) and the cerebellar dentate nuclei (Dn). We gauged the influence of age and sex on magnetic susceptibility by specifying a series of structural equation models. The distributions of susceptibility varied in degree across the structures, conforming to histologic findings (Hallgren and Sourander, 1958), with the highest degree of susceptibility in the Gp and the lowest in the Th. Iron increase correlated across several ROIs, which may reflect an underlying age-related process. Advanced age was associated with a particularly strong linear rise of susceptibility in the striatum. Nonlinear age trends were found in the Rn, where they were the most pronounced, followed by the Pul and Sn, while minimal nonlinear trends were observed for the Pt, Th, and Dn. Moreover, sex related variations were observed, so that women showed lower levels of susceptibility in the Sn after accounting for age. Regional susceptibility of the Pul increased linearly with age in men but exhibited a nonlinear association with age in women with a leveling off starting from midlife. Women expected to be post menopause (+51 years) showed lower total magnetic susceptibility in the subcortical gray matter. The current report not only is consistent with previous reports of age related variations of brain iron, but also adds to the current knowledge by reporting age-related changes in less studied, smaller subcortical nuclei. This is the first in-vivo report to show lower total subcortical brain iron levels selectively in women from midlife, compared to men and younger women. These results encourage further assessment of sex differences in brain iron. We anticipate that age and sex are important co-factors to take into account when establishing a baseline level for differentiating pathologic neurodegeneration from healthy aging. The variations in regional susceptibility reported herein should be evaluated further using a longitudinal study design to determine within-person changes in aging.

Stress-induced inhibition of LH pulses in female rats: role of GABA in arcuate nucleus.

Stress exerts profound inhibitory effects on reproductive function by suppression of the pulsatile release of GnRH and therefore LH. Besides the corticotrophin-releasing factor (CRF), this effect also might be mediated via GABAergic signaling within the arcuate nucleus (ARC) since its inhibitory effects on LH pulses and increased activity during stress. In the present study, we investigated the role of endogenous GABAergic signaling within the ARC in stress-induced suppression of LH pulses. Ovariectomised oestradiol-replaced rats were implanted with bilateral and unilateral cannulae targeting toward the ARC and lateral cerebral ventricle respectively. Blood samples (25 µl) were taken via chronically implanted cardiac catheters every 5 min for 6 h for measurement of LH pulses. Intra-ARC infusion of GABAA receptor antagonist, bicuculline (0.2 pmol in 200 nl artificial cerebrospinal fluid (aCSF) each side, three times at 20-min intervals) markedly attenuated the inhibitory effect of lipopolysaccharide (LPS; 25 µg/kg i.v.) but not restraint (1 h) stress on pulsatile LH secretion. In contrast, restraint but not LPS stress-induced suppression of LH pulse frequency was reversed by intra-ARC administration of GABABR antagonist, CGP-35348 (1.5 nmol in 200 nl aCSF each side, three times at 20-min intervals). Moreover, intra-ARC application of either bicuculline or CGP-35348 attenuated the inhibitory effect of CRF (1 nmol in 4 µl aCSF, i.c.v.) on the LH pulses. These data indicate a pivotal and differential role of endogenous GABAA and GABAB signaling mechanisms in the ARC with respect to mediating immunological and psychological stress-induced suppression of the GnRH pulse generator respectively.

Cerebellar pathology in Friedreich's ataxia: atrophied dentate nuclei with normal iron content.

BACKGROUND: In Friedreich's ataxia (FA) the genetically decreased expression of the mitochondrial protein frataxin leads to disturbance of the mitochondrial iron metabolism. Within the cerebellum the dentate nuclei (DN) are primarily affected. Histopathological studies show atrophy and accumulation of mitochondrial iron in DN. Dentate iron content has been suggested as a biomarker to measure the effects of siderophores/antioxidant treatment of FA. We assessed the iron content and the volume of DN in FA patients and controls based on ultra-high-field MRI (7 Tesla) images. METHODS: Fourteen FA patients (mean age 38.1 yrs) and 14 age- and gender-matched controls participated. Multi-echo gradient echo and susceptibility weighted imaging (SWI) sequences were acquired on a 7 T whole-body scanner. For comparison SWI images were acquired on a 1.5 T MR scanner. Volumes of the DN and cerebellum were assessed at 7 and 1.5 T, respectively. Parametric maps of T2 and T2* sequences were created and proton transverse relaxation rates were estimated as a measure of iron content. RESULTS: In FA, the DN and the cerebellum were significantly smaller compared to controls. However, proton transverse relaxation rates of the DN were not significantly different between both groups. CONCLUSIONS: Applying in vivo MRI methods we could demonstrate significant atrophy of the DN in the presence of normal iron content. The findings suggest that relaxation rates are not reliable biomarkers in clinical trials evaluating the potential effect of FA therapy.

Distribution of SNAP25, VAMP1 and VAMP2 in mature and developing deep cerebellar nuclei after estrogen administration.

Synaptosomal-associated protein of 25kDa (SNAP25), vesicle-associated membrane protein 1 (VAMP1) and 2 (VAMP2) are components of soluble N-ethylmaleimide-sensitive fusion attachment protein receptors (SNARE) complex which is involved in synaptic vesicle exocytosis, a fundamental step in neurotransmitter release. SNARE expression in cerebellum correlates with specific neurotransmitter pathways underlying synaptic diversification and defined synaptic properties. In this study we firstly characterized the distribution of SNAP25, VAMP1 and VAMP2 in the nerve terminals of a defined cerebellar region, the deep cerebellar nuclei (DCN), of adult and newborn rats. Then, given the pivotal role of estradiol (E2) in the synaptic organization of the cerebellar circuitry in early postnatal life, we examined whether administration of E2 in the newborn DCN affected synaptic density and changed the distribution of the presynaptic proteins SNAP25, VAMP1 and VAMP2, together with post synaptic density protein 95 (PSD95). Results showed that: (1) distribution of SNAP25, VAMP1 and VAMP2 in adult DCN differs significantly from that found in newborn DCN; (2) administration of E2 in the newborn DCN affected synaptic density and also changed the distribution of the pre- and postsynaptic proteins. The differential distribution of SNAP25, VAMP1 and VAMP2 in nerve terminals of adult and newborn rats may correlate with specific stages of neuronal phenotypic differentiation. The effects of E2 on SNAP25, VAMP1, VAMP2, PDS95 and synaptic density suggest that pre- and postsynaptic proteins are under estrogenic control during development and that synaptic maturation can also be related with the activity of this steroid.

Electrical stimulation of cerebellar fastigial nucleus promotes the expression of growth arrest and DNA damage inducible gene ß and motor function recovery in cerebral ischemia/reperfusion rats.

This study focused on the effects of electrical stimulation of cerebellar fastigial nucleus on the expression of growth arrest and DNA damage inducible gene ß (Gadd45ß) and on motor function recovery after focal cerebral ischemia/reperfusion (I/R) in rats. Sprague-Dawley (SD) rats were randomly divided into 4 groups: sham I/R (control group), I/R (I/R group), I/R with sham stimulation and I/R with electrical stimulation at 6h, 12h, 24h, 2d and 3d after I/R. Cerebral ischemia and reperfusion was established by nylon monofilament occlusion method. Fastigial nucleus (FN) electrical stimulation was applied at 2h after ischemia for 1h. The changes in the expression of Gadd45ß were analyzed by immunohistochemistry, real-time polymerase chain reaction (PCR) and Western-blot respectively. Another group of rats were divided into the same 4 groups. Montoya staircase test score was used to test the motor function of affected forelimb. The levels of Gadd45ß were significantly elevated after I/R injury. FN electrical stimulation treatment elevated the expression of Gadd45ß further and improved motor function recovery. These results suggest that FN electrical stimulation can promote the expression of Gadd45ß and motor function recovery after focal cerebral ischemia.

Friedreich's ataxia causes redistribution of iron, copper, and zinc in the dentate nucleus.

Friedreich's ataxia (FRDA) causes selective atrophy of the large neurons of the dentate nucleus (DN). High iron (Fe) concentration and failure to clear the metal from the affected brain tissue are potential risk factors in the progression of the lesion. The DN also contains relatively high amounts of copper (Cu) and zinc (Zn), but the importance of these metals in FRDA has not been established. This report describes nondestructive quantitative X-ray fluorescence (XRF) and "mapping" of Fe, Cu, and Zn in polyethylene glycol-dimethylsulfoxide (PEG/DMSO)-embedded DN of 10 FRDA patients and 13 controls. Fe fluorescence arose predominantly from the hilar white matter, whereas Cu and Zn were present at peak levels in DN gray matter. Despite collapse of the DN in FRDA, the location of the peak Fe signal did not change. In contrast, the Cu and Zn regions broadened and overlapped extensively with the Fe-rich region. Maximal metal concentrations did not differ from normal (in micrograms per milliliter of solid PEG/DMSO as means ± S.D.): Fe normal, 364 ± 117, FRDA, 344 ± 159; Cu normal, 33 ± 13, FRDA, 33 ± 18; and Zn normal, 32 ± 16, FRDA, 33 ± 19. Tissues were recovered from PEG/DMSO and transferred into paraffin for matching with immunohistochemistry of neuron-specific enolase (NSE), glutamic acid decarboxylase (GAD), and ferritin. NSE and GAD reaction products confirmed neuronal atrophy and grumose degeneration that coincided with abnormally diffuse Cu and Zn zones. Ferritin immunohistochemistry matched Fe XRF maps, revealing the most abundant reaction product in oligodendroglia of the DN hilus. In FRDA, these cells were smaller and more numerous than normal. In the atrophic DN gray matter of FRDA, anti-ferritin labeled mostly hypertrophic microglia. Immunohistochemistry and immunofluorescence of the Cu-responsive proteins Cu,Zn-superoxide dismutase and Cu(++)-transporting ATPase α-peptide did not detect specific responses to Cu redistribution in FRDA. In contrast, metallothionein (MT)-positive processes were more abundant than normal and contributed to the gliosis of the DN. The isoforms of MT, MT-1/2, and brain-specific MT-3 displayed only limited co-localization with glial fibrillary acidic protein. The results suggest that MT can provide effective protection against endogenous Cu and Zn toxicity in FRDA, similar to the neuroprotective sequestration of Fe in holoferritin.

Neonatal corticosterone administration impairs adult eyeblink conditioning and decreases glucocorticoid receptor expression in the cerebellar interpositus nucleus.

Neonatal maternal separation alters adult learning and memory. Previously, we showed that neonatal separation impaired eyeblink conditioning in adult rats and increased glucocorticoid receptor (GR) expression in the cerebellar interpositus nucleus, a critical site of learning-related plasticity. Daily neonatal separation (1 h/day on postnatal days 2-14) increases neonatal plasma corticosterone levels. Therefore, effects of separation on GR expression in the interpositus and consequently adult eyeblink conditioning may be mediated by neonatal increases in corticosterone. As a first step in exploring a potential role for corticosterone in the neonatal separation effects we observed, we assessed whether systemic daily (postnatal days 2-14) corticosterone injections mimic neonatal separation effects on adult eyeblink conditioning and GR expression in the interpositus. Control uninjected animals were compared to animals receiving either daily corticosterone injections or daily injections of an equal volume of vehicle. Plasma corticosterone values were measured in a separate group of control, neonatally separated, vehicle injected, or corticosterone injected pups. In adulthood, rats underwent surgery for implantation of recording and stimulating electrodes. After recovery from surgery, rats underwent 10 daily sessions of eyeblink conditioning. Then, brains were processed for GR immunohistochemistry and GR expression in the interpositus nucleus was assessed. Vehicle and corticosterone injections both produced much larger increases in neonatal plasma corticosterone than did daily maternal separation, with the largest increases occurring in the corticosterone-injected group. Neonatal corticosterone injections impaired adult eyeblink conditioning and decreased GR expression in the interpositus nucleus, while the effects of vehicle injections were intermediate. Thus, while neonatal injections and maternal separation both produce adult impairments in learning and memory, these manipulations produce opposite changes in GR expression. This suggests an inverted U-shaped relationship may exist between both neonatal corticosterone levels and adult GR expression in the interpositus nucleus, and adult GR expression in the interpositus and eyeblink conditioning.

Glucocorticoid receptor blockade in the posterior interpositus nucleus reverses maternal separation-induced deficits in adult eyeblink conditioning.

Previously, we showed that neonatal maternal separation impaired eyeblink conditioning in adult rats. This impairment is correlated with increased glucocorticoid receptor (GR) expression in the cerebellar posterior interpositus nucleus, a critical site of learning-related plasticity. To assess whether increased GR expression is responsible for the separation-induced learning impairment, we infused a GR antagonist (mifepristone) or vehicle into the posterior interpositus during eyeblink conditioning in adult male Long-Evans rats that had undergone control rearing or neonatal maternal separation (1h/day, postnatal days 2-14). Rats received standard rearing (control) or neonatal maternal separation (separated; 1h/day on postnatal days 2-14). In adulthood, rats underwent surgery for implantation of recording electrodes in the orbicularis oculi of the left eyelid, a bipolar stimulating electrode dorsocaudal to the left eye, and an infusion guide cannula positioned over the posterior interpositus. Then, rats underwent 10 daily sessions of eyeblink conditioning. Rats in each group received either 0.2microl of mifepristone (2ng in 2% EtOH) or vehicle infusion prior to each eyeblink conditioning session. Mifepristone infusions improved conditioning in separated rats, but impaired control rats' performance. Thus, separation-induced increases in GRs may mediate the learning deficit seen in adult neonatally separated rats.

Otolith stimulation induces c-Fos expression in vestibular and precerebellar nuclei in cats and squirrel monkeys.

Vestibular information is critical for the control of balance, posture, and eye movements. Signals from the receptors, the semicircular canals and otoliths, are carried by the eighth nerve and distributed to the four nuclei of the vestibular nuclear complex, the VNC. However, anatomical and physiological data suggest that many additional brainstem nuclei are engaged in the processing of vestibular signals and generation of motor responses. To assess the role of these structures in vestibular functions, we have used the expression of the immediate early gene c-Fos as a marker for neurons activated by stimulation of the otoliths or the semicircular canals. Excitation of the otolith organs resulted in widespread c-Fos expression in the VNC, but also in other nuclei, including the external cuneate nucleus, the postpyramidal nucleus of the raphé, the nucleus prepositus hypoglossi, the subtrigeminal nucleus, the pontine nuclei, the dorsal tegmental nucleus, the locus coeruleus, and the reticular formation. Rotations that excited the semicircular canals were much less effective in inducing c-Fos expression. The large number of brainstem nuclei that showed c-Fos expression may reflect the multiple functions of the vestibular system. Some of these neurons may be involved in sensory processing of the vestibular signals, while others provide input to the vestibulo-ocular, vestibulocollic, and vestibulospinal reflexes or mediate changes in autonomic function. The data show that otolith stimulation engages brainstem structures both within and outside of the VNC, many of which project to the cerebellum.