Dopamine receptors of the rodent fastigial nucleus support skilled reaching for goal-directed action.

The dopaminergic (DA) system regulates both motor function, and learning and memory. The cerebellum supports motor control and the acquisition of procedural memories, including goal-directed behavior, and is subjected to DA control. Its fastigial nucleus (FN) controls and interprets body motion through space. The expression of dopamine receptors has been reported in the deep cerebellar nuclei of mice. However, the presence of dopamine D1-like (D1R) and D2-like (D2R) receptors in the rat FN has not yet been verified. In this study, we first confirmed that DA receptors are expressed in the FN of adult rats and then targeted these receptors to explore to what extent the FN modulates goal-directed behavior. Immunohistochemical assessment revealed expression of both D1R and D2R receptors in the FN, whereby the medial lateral FN exhibited higher receptor expression compared to the other FN subfields. Bilateral treatment of the FN with a D1R antagonist, prior to a goal-directed pellet-reaching task, significantly impaired task acquisition and decreased task engagement. D2R antagonism only reduced late performance post-acquisition. Once task acquisition had occurred, D1R antagonism had no effect on successful reaching, although it significantly decreased reaching speed, task engagement, and promoted errors. Motor coordination and ambulation were, however, unaffected as neither D1R nor D2R antagonism altered rotarod latencies or distance and velocity in an open field. Taken together, these results not only reveal a novel role for the FN in goal-directed skilled reaching, but also show that D1R expressed in FN regulate this process by modulating motivation for action.

Cytokine enrichment in deep cerebellar nuclei is contributed by multiple glial populations and linked to reduced amyloid plaque pathology.

Alzheimer's disease (AD) pathology and amyloid-beta (Aß) plaque deposition progress slowly in the cerebellum compared to other brain regions, while the entorhinal cortex (EC) is one of the most vulnerable regions. Using a knock-in AD mouse model (App KI), we show that within the cerebellum, the deep cerebellar nuclei (DCN) has particularly low accumulation of Aß plaques. To identify factors that might underlie differences in the progression of AD-associated neuropathology across regions, we profiled gene expression in single nuclei (snRNAseq) across all cell types in the DCN and EC of wild-type (WT) and App KI male mice at age 7 months. We found differences in expression of genes associated with inflammatory activation, PI3K-AKT signalling, and neuron support functions between both regions and genotypes. In WT mice, the expression of interferon-response genes in microglia is higher in the DCN than the EC and this enrichment is confirmed by RNA in situ hybridisation, and measurement of inflammatory cytokines by protein array. Our analyses also revealed that multiple glial populations are responsible for establishing this cytokine-enriched niche. Furthermore, homogenates derived from the DCN induced inflammatory gene expression in BV2 microglia. We also assessed the relationship between the DCN microenvironment and Aß pathology by depleting microglia using a CSF1R inhibitor PLX5622 and saw that, surprisingly, the expression of a subset of inflammatory cytokines was increased while plaque abundance in the DCN was further reduced. Overall, our study revealed the presence of a cytokine-enriched microenvironment unique to the DCN that when modulated, can alter plaque deposition.

Therapeutic effect of fastigial nucleus stimulation is mediated by the microRNA-182 & microRNA-382/BDNF signaling pathways in the treatment of post-stroke depression.

The deregulation of Brain-Derived Neurotrophic Factor (BDNF) was reported to be responsible for the development of post-stroke depression (PSD), while the stimulation of the fastigial nucleus (FN) can be used to treat PDS by down-regulating the expression of miR-182 and miR-382. Therefore, we aim to test the hypothesis that the therapeutic effect of FN stimulation obtained in the treatment of PSD is mediated by the miR-382&miR-182/BDNF mRNA signaling pathways. Rat models of PSD were established and divided into sham, stroke, PSD and PSD + FNS groups to receive different treatments. Post-stroke depression-like behaviors were observed after the initiation of the treatments. TUNEL assay, Western Blot, IHC assay, real-time PCR, bioinformatics tools and luciferase assays were performed to examine the effect of FN stimulation on the expression of miR-182, miR-382 and BDNF mRNA/protein, as well as to further clarify the role of miR-382&miR-182/BDNF mRNA signaling pathways in FN stimulation. Post-stroke depression-like behaviors were significantly reduced in PSD rats. In contrary, the treatment by FN stimulation alleviated the symptoms of PSD and reduced the apoptosis index in the PSD group. Furthermore, in the PSD group, BDNF mRNA/protein levels were suppressed while the miR-382/miR-182 levels were both significantly up-regulated. After the treatment of FN stimulation, BDNF mRNA/protein levels were partly recovered, while miR-382/miR-182 levels was decreased. Furthermore, BDNF was identified as a virtual target of miR-382 and miR-182. In conclusion, FN stimulation increases the expression of BDNF via down-regulating the expression of miR-382/miR-182, thus exhibiting a positive effect in the management of PSD.

The involvement of Pellino-1 downregulation in the modulation of visceral hypersensitivity via the TLR4/NF-κB pathway in the rat fastigial nucleus.

Irritable bowel syndrome (IBS) is a common functional bowel disorder whose key characteristics include chronic visceral hypersensitivity (CVH) and abnormal brain-gut interactions. Pellino-1 is an E3 ubiquitin ligase, mediating the degradation or modification of targeted proteins. Some brain regions, such as the fastigial nucleus (FN), may play important roles in CVH; however, the molecular mechanism underlying this phenomenon is not clear. In this study, we assessed the roles of Pellino-1 within the FN in modulating VH by generating a colorectal distention (CRD) model in male Sprague-Dawley rats. Our results showed that the downregulation of Pellino-1 in the fastigial nucleus (FN) was involved in the modulation of visceral hypersensitivity. The expression of Pellino-1 was downregulated in the FN of adult CRD rats compared with control rats, whereas TLR4 and NF-κB were upregulated in the CRD model. To overexpress Pellino-1, a lentivirus specifically expressing Pellino-1 and green fluorescent protein was administered into the FN. The overexpression of Pellino-1 increased the visceral sensitivity of CRD rats, and the expression of TLR4 and NF-κB increased further. After administration of TAK-242 (a specific TLR4 inhibitor), the visceral response to overexpression of Pellino-1 was reversed. Overall, the findings indicated the involvement of the FN in the development of CVH; the downregulation of Pellino-1 in the FN acted through the TLR4/NF-κB pathway to protect against CVH in a CRD rat model.

Gamma-aminobutyric acid and glutamate/glutamine levels in the dentate nucleus and periaqueductal gray with episodic and chronic migraine: a proton magnetic resonance spectroscopy study.

BACKGROUND: The pathogenesis of migraine chronification remains unclear. Functional and structural magnetic resonance imaging studies have shown impaired functional and structural alterations in the brains of patients with chronic migraine. The cerebellum and periaqueductal gray (PAG) play pivotal roles in the neural circuits of pain conduction and analgesia in migraine. However, few neurotransmitter metabolism studies of these migraine-associated regions have been performed. To explore the pathogenesis of migraine chronification, we measured gamma-aminobutyric acid (GABA) and glutamate/glutamine (Glx) levels in the dentate nucleus (DN) and PAG of patients with episodic and chronic migraine and healthy subjects. METHODS: Using the MEGA-PRESS sequence and a 3-Tesla magnetic resonance scanner (Signa Premier; GE Healthcare, Chicago, IL, USA), we obtained DN and PAG metabolite concentrations from patients with episodic migraine (n = 25), those with chronic migraine (n = 24), and age-matched and sex-matched healthy subjects (n = 16). Patients with chronic migraine were further divided into those with (n = 12) and without (n = 12) medication overuse headache. All scans were performed at the Beijing Tiantan Hospital, Capital Medical University. RESULTS: We found that patients with chronic migraine had significantly lower levels of GABA/water (p = 0.011) and GABA/creatine (Cr) (p = 0.026) in the DN and higher levels of Glx/water (p = 0.049) in the PAG than healthy controls. In all patients with migraine, higher GABA levels in the PAG were significantly associated with poorer sleep quality (GABA/water: r = 0.515, p = 0.017, n = 21; GABA/Cr: r = 0.522, p = 0.015, n = 21). Additionally, a lower Glx/Cr ratio in the DN may be associated with more severe migraine disability (r = -0.425, p = 0.055, n = 20), and lower GABA/water (r = -0.424, p = 0.062, n = 20) and Glx/Water (r = -0.452, p = 0.045, n = 20) may be associated with poorer sleep quality. CONCLUSIONS: Neurochemical levels in the DN and PAG may provide evidence of the pathological mechanisms of migraine chronification. Correlations between migraine characteristics and neurochemical levels revealed the pathological mechanisms of the relevant characteristics.

Increase of vesicular glutamate transporter 2 co-expression in the deep cerebellar nuclei related to skilled reach learning.

Motor learning induces plasticity in multiple brain regions involving the cerebellum as a crucial player. Synaptic plasticity in the excitatory collaterals to the cerebellar output, the deep cerebellar nuclei (DCN), have recently been shown to be an important part of motor learning. These synapses are composed of climbing fiber (CF) and mossy fiber synapses, with the former conveying unconditioned and the latter conditioned responses in classical conditioning paradigms. The CF synapse on to the cerebellar cortex and the DCN express vesicular transporter 2 (vGluT2), whereas mossy fibers express vGluT1 and /or vGluT2 in their terminals. However, the underlying regulatory mechanism of vGluT expression in the DCN remains unknown. Here we confirm the increase of vGluT2 in a specific part of the DCN during the acquisition of a skilled reaching task in mice. Furthermore, our findings show that this is due to an increase in co-expression of vGluT2 in vGluT1 presynapses instead of the formation of new vGluT2 synapses. Our data indicate that remodeling of synapses - in contrast to synaptogenesis - also plays an important role in motor learning and may explain the presence of both vGluT's in some mossy fiber synapses.

A Distinct Metabolically Defined Central Nucleus Circuit Bidirectionally Controls Anxiety-Related Behaviors.

Anxiety disorders are debilitating psychiatric diseases that affect ∼16% of the world's population. Although it has been proposed that the central nucleus of the amygdala (CeA) plays a role in anxiety, the molecular and circuit mechanisms through which CeA neurons modulate anxiety-related behaviors are largely uncharacterized. Soluble epoxide hydrolase (sEH) is a key enzyme in the metabolism of polyunsaturated fatty acids (PUFAs), and has been shown to play a role in psychiatric disorders. Here, we reported that sEH was enriched in neurons in the CeA and regulated anxiety-related behaviors in adult male mice. Deletion of sEH in CeA neurons but not astrocytes induced anxiety-like behaviors. Mechanistic studies indicated that sEH was required for maintaining the the excitability of sEH positive neurons (sEHCeA neurons) in the CeA. Using chemogenetic manipulations, we found that sEHCeA neurons bidirectionally regulated anxiety-related behaviors. Notably, we identified that sEHCeA neurons directly projected to the bed nucleus of the stria terminalis (BNST; sEHCeA-BNST). Optogenetic activation and inhibition of the sEHCeA-BNST pathway produced anxiolytic and anxiogenic effects, respectively. In summary, our studies reveal a set of molecular and circuit mechanisms of sEHCeA neurons underlying anxiety.SIGNIFICANCE STATEMENT Soluble epoxide hydrolase (sEH), a key enzyme that catalyzes the degradation of EETs, is shown to play a key role in mood disorders. It is well known that sEH is mostly localized in astrocytes in the prefrontal cortex and regulates depressive-like behaviors. Notably, sEH is also expressed in central nucleus of the amygdala (CeA) neurons. While the CeA has been studied for its role in the regulation of anxiety, the molecular and circuit mechanism is quite complex. In the present study, we explored a previously unknown cellular and circuitry mechanism that guides sEHCeA neurons response to anxiety. Our findings reveal a critical role of sEH in the CeA, sEHCeA neurons and CeA-bed nucleus of the stria terminalis (BNST) pathway in regulation of anxiety-related behaviors.

Comprehensive Speciation Analysis of Residual Gadolinium in Deep Cerebellar Nuclei in Rats Repeatedly Administered With Gadoterate Meglumine or Gadodiamide.

PURPOSE: Several preclinical studies have reported the presence of gadolinium (Gd) in different chemical forms in the brain, depending on the class (macrocyclic versus linear) of Gd-based contrast agent (GBCA) administered. The aim of this study was to identify, with a special focus on insoluble species, the speciation of Gd retained in the deep cerebellar nuclei (DCN) of rats administered repeatedly with gadoterate or gadodiamide 4 months after the last injection. METHODS: Three groups (N = 6/group) of healthy female Sprague-Dawley rats (SPF/OFA rats; Charles River, L'Arbresle, France) received a cumulated dose of 50 mmol/kg (4 daily intravenous administrations of 2.5 mmol/kg, for 5 weeks, corresponding to 80-fold the usual clinical dose if adjusted for man) of gadoterate meglumine (macrocyclic) or gadodiamide (linear) or isotonic saline for the control group (4 daily intravenous administrations of 5 mL/kg, for 5 weeks). The animals were sacrificed 4 months after the last injection. Deep cerebellar nuclei were dissected and stored at -80°C before sample preparation. To provide enough tissue for sample preparation and further analysis using multiple techniques, DCN from each group of 6 rats were pooled. Gadolinium species were extracted in 2 consecutive steps with water and urea solution. The total Gd concentrations were determined by inductively coupled plasma mass spectrometry (ICP-MS). Soluble Gd species were analyzed by size-exclusion chromatography coupled to ICP-MS. The insoluble Gd species were analyzed by single-particle (SP) ICP-MS, nanoscale secondary ion mass spectroscopy (NanoSIMS), and scanning transmission electron microscopy with energy-dispersive X-ray spectroscopy (STEM-EDX) for elemental detection. RESULTS: The Gd concentrations in pooled DCN from animals treated with gadoterate or gadodiamide were 0.25 and 24.3 nmol/g, respectively. For gadoterate, the highest amount of Gd was found in the water-soluble fractions. It was present exclusively as low-molecular-weight compounds, most likely as the intact GBCA form. In the case of gadodiamide, the water-soluble fraction of DCN was composed of high-molecular-weight Gd species of approximately 440 kDa and contained only a tiny amount (less than 1%) of intact gadodiamide. Furthermore, the column recovery calculated for this fraction was incomplete, which suggested presence of labile complexes of dissociated Gd3+ with endogenous molecules. The highest amount of Gd was detected in the insoluble residue, which was demonstrated, by SP-ICP-MS, to be a particulate form of Gd. Two imaging techniques (NanoSIMS and STEM-EDX) allowed further characterization of these insoluble Gd species. Amorphous, spheroid structures of approximately 100-200 nm of sea urchin-like shape were detected. Furthermore, Gd was consistently colocalized with calcium, oxygen, and phosphorous, strongly suggesting the presence of structures composed of mixed Gd/Ca phosphates. No or occasional colocalization with iron and sulfur was observed. CONCLUSION: A dedicated analytical workflow produced original data on the speciation of Gd in DCN of rats repeatedly injected with GBCAs. The addition, in comparison with previous studies of Gd speciation in brain, of SP element detection and imaging techniques allowed a comprehensive speciation analysis approach. Whereas for gadoterate the main fraction of retained Gd was present as intact GBCA form in the soluble fractions, for linear gadodiamide, less than 10% of Gd could be solubilized and characterized using size-exclusion chromatography coupled to ICP-MS. The main Gd species detected in the soluble fractions were macromolecules of 440 kDa. One of them was speculated to be a Gd complex with iron-binding protein (ferritin). However, the major fraction of residual Gd was present as insoluble particulate species, very likely composed of mixed Gd/Ca phosphates. This comprehensive Gd speciation study provided important evidence for the dechelation of linear GBCAs and offered a deeper insight into the mechanisms of Gd deposition in the brain.

HnRNP K mislocalisation in neurons of the dentate nucleus is a novel neuropathological feature of neurodegenerative disease and ageing.

Nuclear depletion and cytoplasmic mislocalisation of the RNA-binding protein heterogeneous ribonucleoprotein K (hnRNP K) within pyramidal neurons of the frontal cortex have been shown to be a common neuropathological feature in frontotemporal lobar degeneration (FTLD) and elderly control brain. Here, we describe a second neuronal subtype vulnerable to mislocalisation within the dentate nucleus of the cerebellum. In contrast to neurons within the cerebellar cortex that typically exhibited normal, nuclear staining, many neurons of the dentate nucleus exhibited striking mislocalisation of hnRNP K to the cytoplasm within neurodegenerative disease brain. Mislocalisation frequency in this region was found to be significantly higher in both FTLD-TDP A and Alzheimer's disease (AD) brain than in age-matched controls. However, within control (but not disease) subjects, mislocalisation frequency was significantly associated with age-at-death with more elderly controls typically exhibiting greater levels of the pathology. This study provides further evidence for hnRNP K mislocalisation being a more anatomically diverse pathology than previously thought and suggests that potential dysfunction of the protein may be more broadly relevant to the fields of neurodegeneration and ageing.

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.

Epigenetic Regulation of Neural Transmission after Cerebellar Fastigial Nucleus Lesions in Juvenile Rats.

Structural and functional abnormalities in the cerebellar midline region, including the fastigial nucleus, have been reported in neuropsychiatric disorders, also comprising the cerebellar cognitive affecting syndrome. In rats, early fastigial lesions reduce social interaction during development and lead to cognitive and emotional deficits in adults, accompanied by compromised neuronal network activity. Since epigenetic mechanisms are implicated in the etiology of neuropsychiatric disorders, we investigated whether fastigial nucleus lesions in juvenile rats would impact epigenetic regulation of neural transmission. The fastigial nucleus was lesioned bilaterally in 23-day-old male rats. Sham-lesion and naïve rats served as controls. DNA methylation was investigated for target genes of the GABAergic, dopaminergic, glutamatergic and oxytocinergic systems in brain regions with anatomic connections to the fastigial nucleus, i.e., medial prefrontal cortex, nucleus accumbens, striatum, thalamus, and sensorimotor cortex. Protein expression was examined for the respective target genes in case of altered DNA methylation between lesion and control groups. Lesioning of the fastigial nucleus led to significant differences in the epigenetic regulation of glutamate decarboxylase 1 and the oxytocin receptor in the nucleus accumbens and the prefrontal cortex. No differences were found for the other target genes and brain regions. Our findings indicate that epigenetic dysregulation after lesioning of the fastigial nucleus may influence long-term recovery and the emergence of behavioral changes. Together with previous behavioral and electrophysiological investigations of this rat model, these observations can play a role in the cerebellar cognitive affective syndrome and other neuropsychiatric disorders.

Catecholaminergic Innervation of the Lateral Nucleus of the Cerebellum Modulates Cognitive Behaviors.

The cerebellum processes neural signals related to rewarding and aversive stimuli, suggesting that the cerebellum supports nonmotor functions in cognitive and emotional domains. Catecholamines are a class of neuromodulatory neurotransmitters well known for encoding such salient stimuli. Catecholaminergic modulation of classical cerebellar functions have been demonstrated. However, a role for cerebellar catecholamines in modulating cerebellar nonmotor functions is unknown. Using biochemical methods in male mice, we comprehensively mapped TH+ fibers throughout the entire cerebellum and known precerebellar nuclei. Using electrochemical (fast scan cyclic voltammetry), and viral/genetic methods to selectively delete Th in fibers innervating the lateral cerebellar nucleus (LCN), we interrogated sources and functional roles of catecholamines innervating the LCN, which is known for its role in supporting cognition. The LCN has the most TH+ fibers in cerebellum, as well as the most change in rostrocaudal expression among the cerebellar nuclei. Norepinephrine is the major catecholamine measured in LCN. Distinct catecholaminergic projections to LCN arise only from locus coeruleus, and a subset of Purkinje cells that are positive for staining of TH. LC stimulation was sufficient to produce catecholamine release in LCN. Deletion of Th in fibers innervating LCN (LCN-Th-cKO) resulted in impaired sensorimotor integration, associative fear learning, response inhibition, and working memory in LCN-Th-cKO mice. Strikingly, selective inhibition of excitatory LCN output neurons with inhibitory designer receptor exclusively activated by designer drugs led to facilitation of learning on the same working memory task impaired in LCN-Th-cKO mice. Collectively, these data demonstrate a role for LCN catecholamines in cognitive behaviors.SIGNIFICANCE STATEMENT Here, we report on interrogating sources and functional roles of catecholamines innervating the lateral nucleus of the cerebellum (LCN). We map and quantify expression of TH, the rate-limiting enzyme in catecholamine synthesis, in the entire cerebellar system, including several precerebellar nuclei. We used cyclic voltammetry and pharmacology to demonstrate sufficiency of LC stimulation to produce catecholamine release in LCN. We used advanced viral techniques to map and selectively KO catecholaminergic neurotransmission to the LCN, and characterized significant cognitive deficits related to this manipulation. Finally, we show that inhibition of excitatory LCN neurons with designer receptor exclusively activated by designer drugs, designed to mimic Gi-coupled catecholamine GPCR signaling, results in facilitation of a working memory task impaired in LCN-specific TH KO mice.

Impact of Kidney Function on CNS Gadolinium Deposition in Patients Receiving Repeated Doses of Gadobutrol.

BACKGROUND AND PURPOSE: Studies associate repeat gadolinium-based contrast agent administration with T1 shortening in the dentate nucleus and globus pallidus, indicating CNS gadolinium deposition, most strongly with linear agents but also reportedly with macrocyclics. Renal impairment effects on long-term CNS gadolinium deposition remain underexplored. We investigated the relationship between signal intensity changes and renal function in patients who received ≥10 administrations of the macrocyclic agent gadobutrol. MATERIALS AND METHODS: Patients who underwent ≥10 brain MR imaging examinations with administration of intravenous gadobutrol between February 1, 2014, and January 1, 2018, were included in this retrospective study. Dentate nucleus-to-pons and globus pallidus-to-thalamus signal intensity ratios were calculated, and correlations were calculated between the estimated glomerular filtration rate (minimum and mean) and the percentage change in signal intensity ratios from the first to last scan. Partial correlations were calculated to control for potential confounders. RESULTS: One hundred thirty-one patients (73 women; mean age at last scan, 55.9 years) showed a mean percentage change of the dentate nucleus-to-pons of 0.31%, a mean percentage change of the globus pallidus-to-thalamus of 0.15%, a mean minimum estimated glomerular filtration rate of 69.65 (range, 10.16-132.26), and a mean average estimated glomerular filtration rate at 89.48 (range, 38.24-145.93). No significant association was found between the estimated glomerular filtration rate and percentage change of the dentate nucleus-to-pons (minimum estimated glomerular filtration rate, r = -0.09, P = .28; average estimated glomerular filtration rate, r = -0.09, P = .30,) or percentage change of the globus pallidus-to-thalamus (r = 0.07, P = .43; r = 0.07, P = .40). When we controlled for age, sex, number of scans, and total dose, there were no significant associations between the estimated glomerular filtration rate and the percentage change of the dentate nucleus-to-pons (r = 0.16, P = .07; r = 0.15, P = .08) or percentage change of the globus pallidus-to-thalamus (r = -0.14, P = .12; r = -0.15, P = .09). CONCLUSIONS: In patients receiving an average of 12 intravenous gadobutrol administrations, no correlation was found between renal function and signal intensity ratio changes, even in those with mild or moderate renal impairment.

Cerebellar nuclei evolved by repeatedly duplicating a conserved cell-type set.

How have complex brains evolved from simple circuits? Here we investigated brain region evolution at cell-type resolution in the cerebellar nuclei, the output structures of the cerebellum. Using single-nucleus RNA sequencing in mice, chickens, and humans, as well as STARmap spatial transcriptomic analysis and whole-central nervous system projection tracing, we identified a conserved cell-type set containing two region-specific excitatory neuron classes and three region-invariant inhibitory neuron classes. This set constitutes an archetypal cerebellar nucleus that was repeatedly duplicated to form new regions. The excitatory cell class that preferentially funnels information to lateral frontal cortices in mice becomes predominant in the massively expanded human lateral nucleus. Our data suggest a model of brain region evolution by duplication and divergence of entire cell-type sets.

Kv1 potassium channels control action potential firing of putative GABAergic deep cerebellar nuclear neurons.

Low threshold voltage activated Kv1 potassium channels play key roles in regulating action potential (AP) threshold, neural excitability, and synaptic transmission. Kv1 channels are highly expressed in the cerebellum and mutations of human Kv1 genes are associated to episodic forms of ataxia (EAT-1). Besides the well-established role of Kv1 channels in controlling the cerebellar basket-Purkinje cells synapses, Kv1 channels are expressed by the deep cerebellar nuclear neurons (DCNs) where they regulate the activity of principal DCNs carrying the cerebellar output. DCNs include as well GABAergic neurons serving important functions, such as those forming the inhibitory nucleo-olivary pathway, the nucleo-cortical DCNs providing feed-back inhibition to the cerebellar cortex, and those targeting principal DCNs, but whether their function is regulated by Kv1 channels remains unclear. Here, using cerebellar slices from mature GAD67-GFP mice to identify putative GABAergic-DCNs (GAD + DCN) we show that specific Kv1 channel blockers (dendrotoxin-alpha/I/K, DTXs) hyperpolarized the threshold of somatic action potentials, increased the spontaneous firing rate and hampered evoked high frequency repetitive responses of GAD + DCNs. Moreover, DTXs induced somatic depolarization and tonic firing in previously silent, putative nucleo-cortical DCNs. These results reveal a novel role of Kv1 channels in regulating GABAergic-DCNs activity and thereby, cerebellar function at multiple levels.

Hyper-Formation of GABA and Glycine Co-Releasing Terminals in the Mouse Cerebellar Nuclei after Deprivation of GABAergic Inputs from Purkinje Cells.

GABA and glycine are inhibitory neurotransmitters. However, the mechanisms underlying the formation of GABAergic and glycinergic synapses remain unclear. The influence of GABAergic input deprivation on inhibitory terminal formation was investigated using Purkinje cell (PC)-specific vesicular GABA transporter (VGAT) knockout (L7-VGAT) mice, in which GABA release from PCs diminishes in an age-dependent manner. We compared the late development of GABAergic and glycinergic terminals in the cerebellar nucleus (CN) between control and L7-VGAT mice. In the control CN, the density of glutamate decarboxylase (GAD)-positive dots remained unchanged between postnatal 2 months (P2M) and 13 months (P13M), whereas glycine transporter 2 (GlyT2)-positive dots increased in density during this time frame. No difference in the density of GlyT2-positive dots was observed between control and L7-VGAT mice at P2M, but the density was significantly higher in the L7-VGAT fastigial nuclei (FN) than the control FN at P13M. When VGAT was absent from PC terminals, GlyT2-positive dots included GAD and VGAT and formed synapses. These results indicated that GABAergic terminals were formed by P2M, glycinergic terminals were actively formed after P2M, and more glycinergic terminals were formed in the L7-VGAT FN than in the control FN, suggesting that the increased glycinergic terminals may derive from interneurons within the FN and may also release GABA. These results suggest that the deprivation of GABAergic inputs from PCs may accelerate the formation of co-releasing terminals derived from interneurons and that the inhibitory terminal numbers and types may be regulated by the quantity of functional GABAergic inputs.

Effect of Long-Term Retention of Gadolinium on Metabolism of Deep Cerebellar Nuclei After Repeated Injections of Gadodiamide in Rats.

OBJECTIVES: The aim of this study was to determine potential metabolism and histological modifications due to gadolinium retention within deep cerebellar nuclei (DCN) after linear gadolinium-based contrast agent injection (gadodiamide) in rats at 1 year after the last injection. MATERIALS AND METHODS: Twenty female rats received 20 doses of gadodiamide (0.6 mmol of gadolinium per kilogram each) over 5 weeks. They were followed at 1 week (M0), 6 weeks (M1), and 54 to 55 weeks (M13) postinjections to evaluate hypersignal on unenhanced T1-weighted magnetic resonance imaging and metabolic alterations by H magnetic resonance spectroscopy (H-MRS). At 1 year postinjections, brains were sampled to determine the localization of gadolinium within cerebellum by laser ablation inductively coupled mass spectroscopy and to evaluate morphological changes by semiquantitative immunofluorescence analysis. RESULTS: There is a significant increase of the ratio DCN/brainstem for the gadodiamide group at M0 (+7.2% vs control group = 0.989 ± 0.01), M1 (+7.6% vs control group = 1.002 ± 0.018), and it lasted up to M13 (+4.7% vs control group = 0.9862 ± 0.008). No variation among metabolic markers (cellular homeostasis [creatine, choline, taurine], excitatory neurotransmitter [glutamate], and metabolites specific to a cellular compartment [N-acetyl aspartate for neurons and myo-inositol for glial cells]) were detected by H-MRS between gadodiamide and saline groups at M0, M1, and M13. At M13, laser ablation inductively coupled mass spectroscopy demonstrated that long-term gadolinium retention occurred preferentially in DCN. No histological abnormalities (including analysis of astrocytes, neurons, and microglial cells) were found in the rostral part of DCN. CONCLUSIONS: Repeated administration of gadodiamide lead to a retention of gadolinium preferentially within DCN at 1 year postinjections. This retention did not lead to any detectable changes of the measured metabolic biomarkers nor histological alterations.

Intrinsic Functional Connectivity of Dentate Nuclei in Autism Spectrum Disorder.

Cerebellar abnormalities are commonly reported in autism spectrum disorder (ASD). Dentate nuclei (DNs) are key structures in the anatomical circuits linking the cerebellum to the extracerebellum. Previous resting-state functional connectivity (RsFc) analyses reported DN abnormalities in high-functioning ASD (HF-ASD). This study examined the RsFc of the DN in young adults with HF-ASD compared with healthy controls (HCs) with the aim to expand upon previous findings of DNs in a dataset using advanced, imaging acquisition methods that optimize spatiotemporal resolution and statistical power. Additional seed-to-voxel analyses were carried out using motor and nonmotor DN coordinates reported in previous studies as seeds. We report abnormal dentato-cerebral and dentato-cerebellar functional connectivity in ASD. Our results expand and, in part, replicate previous descriptions of DN RsFc abnormalities in this disorder and reveal correlations between DN-cerebral RsFc and ASD symptom severity.

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.

Early trigeminal ganglion afferents enter the cerebellum before the Purkinje cells are born and target the nuclear transitory zone.

In the standard model for the development of climbing and mossy fiber afferent pathways to the cerebellum, the ingrowing axons target the embryonic Purkinje cell somata (around embryonic ages (E13-E16 in mice). In this report, we describe a novel earlier stage in afferent development. Immunostaining for a neurofilament-associated antigen (NAA) reveals the early axon distributions with remarkable clarity. Using a combination of DiI axon tract tracing, analysis of neurogenin1 null mice, which do not develop trigeminal ganglia, and mouse embryos maintained in vitro, we show that the first axons to innervate the cerebellar primordium as early as E9 arise from the trigeminal ganglion. Therefore, early trigeminal axons are in situ before the Purkinje cells are born. Double immunostaining for NAA and markers of the different domains in the cerebellar primordium reveal that afferents first target the nuclear transitory zone (E9-E10), and only later (E10-E11) are the axons, either collaterals from the trigeminal ganglion or a new afferent source (e.g., vestibular ganglia), seen in the Purkinje cell plate. The finding that the earliest axons to the cerebellum derive from the trigeminal ganglion and enter the cerebellar primordium before the Purkinje cells are born, where they seem to target the cerebellar nuclei, reveals a novel stage in the development of the cerebellar afferents.