Modulation of CB1 cannabinoid receptor alters the electrophysiological properties of cerebellar Purkinje cells in harmaline-induced essential tremor.

Essential tremor (ET) is one of the most common motor disorders with debilitating effects on the affected individuals. The endocannabinoid system is widely involved in cerebellar signaling. Therefore, modulation of cannabinoid-1 receptors (CB1Rs) has emerged as a novel target for motor disorders. In this study, we aimed to assess whether modulation of cannabinoid receptors (CBRs) could alter the electrophysiological properties of Purkinje cells (PCs) in the harmaline-induced ET model. Male Wistar rats were assigned to control, harmaline (30 mg/kg), CBR agonist WIN 55,212-2 (WIN; 1 mg/kg), CB1R antagonists AM251 (1 mg/kg) and rimonabant (10 mg/kg). Spontaneous activity and positive and negative evoked potentials of PCs were evaluated using whole-cell patch clamp recording. Findings demonstrated that harmaline exposure induced alterations in the spontaneous and evoked firing behavior of PCs, as evidenced by a significant decrease in the mean number of spikes and half-width of action potential in spontaneous activity. WIN administration exacerbated the electrophysiological function of PCs, particularly in the spontaneous activity of PCs. However, CB1R antagonists provided protective effects against harmaline-induced electrophysiological changes in the spontaneous activity of PCs. Our findings reinforce the pivotal role of the endocannabinoid system in the underlying electrophysiological mechanisms of cerebellar disorders and suggest that antagonism of CB1R might provide therapeutic utility.

Mice lacking Astn2 have ASD-like behaviors and altered cerebellar circuit properties.

Astrotactin 2 (ASTN2) is a transmembrane neuronal protein highly expressed in the cerebellum that functions in receptor trafficking and modulates cerebellar Purkinje cell (PC) synaptic activity. Individuals with ASTN2 mutations exhibit neurodevelopmental disorders, including autism spectrum disorder (ASD), attention-deficit/hyperactivity disorder (ADHD), learning difficulties, and language delay. To provide a genetic model for the role of the cerebellum in ASD-related behaviors and study the role of ASTN2 in cerebellar circuit function, we generated global and PC-specific conditional Astn2 knockout (KO and cKO, respectively) mouse lines. Astn2 KO mice exhibit strong ASD-related behavioral phenotypes, including a marked decrease in separation-induced pup ultrasonic vocalization calls, hyperactivity, repetitive behaviors, altered behavior in the three-chamber test, and impaired cerebellar-dependent eyeblink conditioning. Hyperactivity and repetitive behaviors are also prominent in Astn2 cKO animals, but they do not show altered behavior in the three-chamber test. By Golgi staining, Astn2 KO PCs have region-specific changes in dendritic spine density and filopodia numbers. Proteomic analysis of Astn2 KO cerebellum reveals a marked upregulation of ASTN2 family member, ASTN1, a neuron-glial adhesion protein. Immunohistochemistry and electron microscopy demonstrate a significant increase in Bergmann glia volume in the molecular layer of Astn2 KO animals. Electrophysiological experiments indicate a reduced frequency of spontaneous excitatory postsynaptic currents (EPSCs), as well as increased amplitudes of both spontaneous EPSCs and inhibitory postsynaptic currents in the Astn2 KO animals, suggesting that pre- and postsynaptic components of synaptic transmission are altered. Thus, ASTN2 regulates ASD-like behaviors and cerebellar circuit properties.

Heterogeneity in Slow Synaptic Transmission Diversifies Purkinje Cell Timing.

The cerebellum plays an important role in diverse brain functions, ranging from motor learning to cognition. Recent studies have suggested that molecular and cellular heterogeneity within cerebellar lobules contributes to functional differences across the cerebellum. However, the specific relationship between molecular and cellular heterogeneity and diverse functional outputs of different regions of the cerebellum remains unclear. Here, we describe a previously unappreciated form of synaptic heterogeneity at parallel fiber synapses to Purkinje cells in the mouse cerebellum (both sexes). In contrast to uniform fast synaptic transmission, we found that the properties of slow synaptic transmission varied by up to threefold across different lobules of the mouse cerebellum, resulting in surprising heterogeneity. Depending on the location of a Purkinje cell, the time of peak of slow synaptic currents varied by hundreds of milliseconds. The duration and decay time of these currents also spanned hundreds of milliseconds, based on lobule. We found that, as a consequence of the heterogeneous synaptic dynamics, the same brief input stimulus was transformed into prolonged firing patterns over a range of timescales that depended on Purkinje cell location.

Investigation of the neuroprotective effect of crocin against electromagnetic field-induced cerebellar damage in male Balb/c mice.

Objective: Mobile devices are sources of electromagnetic fields (EMFs) that cause increasing concern among scientists about human health, especially with the long-term use of mobile phones. With regard to this issue, the potential adverse health effects, particularly on brain function have raised public concern. There is considerable evidence that natural compounds have neuro-protective effects due to their antioxidant and anti-inflammatory properties. Growing evidence suggests that crocin as a natural bioactive compound can be considered a potential therapeutic agent against various neurologic disorders. Therefore, the present study investigated the effects of crocin on the cerebellum after exposure to EMF. Materials and Methods: Twenty-four Male Balb/c mice were divided into control group, EMF group (2100 MHZ), EMF +Crocin group (2100 MHZ+50 mg/kg), and crocin group (50 mg/kg). The animals in the EMF and EMF+Crocin groups were exposed continuously for 30 days to an EMF 120 min/day. After 30 days, cerebellar cortex was evaluated by histomorphometric and immunohistochemical methods. Results: The results showed that 30 days of exposure to EMF had no significant effect on Purkinje cell size. However, EMF reduced significantly the diameter of astrocytes and increased Glial fibrillary acidic protein (GFAP) expression compared to the controls (p<0.05). Our findings also indicated that crocin treatment could improve the diameter of astrocytes and normalize GFAP expression (p<0.05). Conclusion: This study concluded that 2100-MHz EMF caused adverse effects on the cerebellum through astrocyte damage and crocin could partially reverse the EMF-related adverse effects.

Different Numbers of Conjunctive Stimuli Induce LTP or LTD in Mouse Cerebellar Purkinje Cell.

Long-term depression (LTD) of synaptic transmission at parallel fiber (PF)-Purkinje cell (PC) synapses plays an important role in cerebellum-related motor coordination and learning. LTD is induced by the conjunction of PF stimulation and climbing fiber (CF) stimulation or somatic PC depolarization, while long-term potentiation (LTP) is induced by PF stimulation alone. Therefore, it is considered that different types of stimulation induce different types of synaptic plasticity. However, we found that a small number of conjunctive stimulations (PF + somatic depolarization of PC) induced LTP, but did not induce LTD of a small size. This LTP was not associated with changes in paired-pulse ratio, suggesting postsynaptic origin. Additionally this LTP was dependent on nitric oxide. This LTP was also induced by a smaller number of physiological conjunctive PF and CF stimuli. These results suggested that a larger number or longer period of conjunctive stimulation is required to induce LTD by overcoming LTP. Ca2+ transients at the PC dendritic region was measured by calcium imaging during LTD-inducing conjunctive stimulation. Peak amplitude of Ca2+ transients increased gradually during repetitive conjunctive stimulation. Instantaneous peak amplitude was not different between the early phase and late phase, but the average amplitude was larger in the later phase than in the early phase. These results show that LTD overcomes LTP, and increased Ca2+ integration or a number of stimulations is required for LTD induction.

Altered prefrontal and cerebellar parvalbumin neuron counts are associated with cognitive changes in male rats.

Exposure to valproic acid (VPA), a common anti-seizure medication, in utero is a risk factor for autism spectrum disorder (ASD). People with ASD often display changes in the cerebellum, including volume changes, altered circuitry, and changes in Purkinje cell populations. ASD is also characterized by changes in the medial prefrontal cortex (mPFC), where excitatory/inhibitory balance is often altered. This study exposed rats to a high dose of VPA during gestation and assessed cognition and anxiety-like behaviors during young adulthood using a set-shifting task and the elevated plus maze. Inhibitory parvalbumin-expressing (PV +) neuron counts were assessed in the mPFC and cerebellar lobules VI and VII (Purkinje cell layers), which are known to modulate cognition. VPA males had increased PV + counts in crus I and II of lobule VII. VPA males also had decreased parvalbumin-expressing neuron counts in the mPFC. It was also found that VPA-exposed rats, regardless of sex, had increased parvalbumin-expressing Purkinje cell counts in lobule VI. In males, this was associated with impaired intra-dimensional shifting on a set-shifting task. Purkinje cell over proliferation may be contributing to the previously observed increase in volume of Lobule VI. These findings suggest that altered inhibitory signaling in cerebellar-frontal circuits may contribute to the cognitive deficits that occur within ASD.

Advances in the Differentiation of hiPSCs into Cerebellar Neuronal Cells.

The cerebellum has historically been primarily associated with the regulation of precise motor functions. However, recent findings suggest that it also plays a pivotal role in the development of advanced cognitive functions, including learning, memory, and emotion regulation. Pathological changes in the cerebellum, whether congenital hereditary or acquired degenerative, can result in a diverse spectrum of disorders, ranging from genetic spinocerebellar ataxias to psychiatric conditions such as autism, and schizophrenia. While studies in animal models have significantly contributed to our understanding of the genetic networks governing cerebellar development, it is important to note that the human cerebellum follows a protracted developmental timeline compared to the neocortex. Consequently, employing animal models to uncover human-specific molecular events in cerebellar development presents significant challenges. The emergence of human induced pluripotent stem cells (hiPSCs) has provided an invaluable tool for creating human-based culture systems, enabling the modeling and analysis of cerebellar physiology and pathology. hiPSCs and their differentiated progenies can be derived from patients with specific disorders or carrying distinct genetic variants. Importantly, they preserve the unique genetic signatures of the individuals from whom they originate, allowing for the elucidation of human-specific molecular and cellular processes involved in cerebellar development and related disorders. This review focuses on the technical advancements in the utilization of hiPSCs for the generation of both 2D cerebellar neuronal cells and 3D cerebellar organoids.

Purkinje cell models: past, present and future.

The investigation of the dynamics of Purkinje cell (PC) activity is crucial to unravel the role of the cerebellum in motor control, learning and cognitive processes. Within the cerebellar cortex (CC), these neurons receive all the incoming sensory and motor information, transform it and generate the entire cerebellar output. The relatively homogenous and repetitive structure of the CC, common to all vertebrate species, suggests a single computation mechanism shared across all PCs. While PC models have been developed since the 70's, a comprehensive review of contemporary models is currently lacking. Here, we provide an overview of PC models, ranging from the ones focused on single cell intracellular PC dynamics, through complex models which include synaptic and extrasynaptic inputs. We review how PC models can reproduce physiological activity of the neuron, including firing patterns, current and multistable dynamics, plateau potentials, calcium signaling, intrinsic and synaptic plasticity and input/output computations. We consider models focusing both on somatic and on dendritic computations. Our review provides a critical performance analysis of PC models with respect to known physiological data. We expect our synthesis to be useful in guiding future development of computational models that capture real-life PC dynamics in the context of cerebellar computations.

Graded control of Purkinje cell outputs by cAMP through opposing actions on axonal action potential and transmitter release.

All-or-none signalling by action potentials (APs) in neuronal axons is pivotal for the precisely timed and identical size of outputs to multiple distant targets. However, technical limitations with respect to measuring the signalling in small intact axons have hindered the evaluation of high-fidelity signal propagation. Here, using direct recordings from axonal trunks and/or terminals of cerebellar Purkinje cells in slice and culture, we demonstrate that the timing and amplitude of axonal outputs are gradually modulated by cAMP depending on the length of axon. During the propagation in long axon, APs were attenuated and slowed in conduction by cAMP via specifically decreasing axonal Na+ currents. Consequently, the Ca2+ influx and transmitter release at distal boutons are reduced by cAMP, counteracting its direct facilitating effect on release machinery as observed at various CNS synapses. Together, our tour de force functional dissection has unveiled the axonal distance-dependent graded control of output timing and strength by intracellular signalling. KEY POINTS: The information processing in the nervous system has been classically thought to rely on the axonal faithful and high-speed conduction of action potentials (APs). We demonstrate that the strength and timing of axonal outputs are weakened and delayed, respectively, by cytoplasmic cAMP depending on the axonal length in cerebellar Purkinje cells (PCs). Direct axonal patch clamp recordings uncovered axon-specific attenuation of APs by cAMP through reduction of axonal Na+ currents. cAMP directly augments transmitter release at PC terminals without changing presynaptic Ca2+ influx or readily releasable pool of vesicles, although the extent is weaker compared to other CNS synapses. Two opposite actions of cAMP on PC axons, AP attenuation and release augmentation, together give rise to graded control of synaptic outputs in a manner dependent on the axonal length.

Production of Spinocerebellar Ataxia Type 3 Model Mice by Intravenous Injection of AAV-PHP.B Vectors.

We aimed to produce a mouse model of spinocerebellar ataxia type 3 (SCA3) using the mouse blood-brain barrier (BBB)-penetrating adeno-associated virus (AAV)-PHP.B. Four-to-five-week-old C57BL/6 mice received injections of high-dose (2.0 × 1011 vg/mouse) or low-dose (5.0 × 1010 vg/mouse) AAV-PHP.B encoding a SCA3 causative gene containing abnormally long 89 CAG repeats [ATXN3(Q89)] under the control of the ubiquitous chicken ß-actin hybrid (CBh) promoter. Control mice received high doses of AAV-PHP.B encoding ATXN3 with non-pathogenic 15 CAG repeats [ATXN3(Q15)] or phosphate-buffered saline (PBS) alone. More than half of the mice injected with high doses of AAV-PHP.B encoding ATXN3(Q89) died within 4 weeks after the injection. No mice in other groups died during the 12-week observation period. Mice injected with low doses of AAV-PHP.B encoding ATXN3(Q89) exhibited progressive motor uncoordination starting 4 weeks and a shorter stride in footprint analysis performed at 12 weeks post-AAV injection. Immunohistochemistry showed thinning of the molecular layer and the formation of nuclear inclusions in Purkinje cells from mice injected with low doses of AAV-PHP.B encoding ATXN3(Q89). Moreover, ATXN3(Q89) expression significantly reduced the number of large projection neurons in the cerebellar nuclei to one third of that observed in mice expressing ATXN3(Q15). This AAV-based approach is superior to conventional methods in that the required number of model mice can be created simply by injecting AAV, and the expression levels of the responsible gene can be adjusted by changing the amount of AAV injected. Moreover, this method may be applied to produce SCA3 models in non-human primates.

Kainate receptors regulate synaptic integrity and plasticity by forming a complex with synaptic organizers in the cerebellum.

Kainate (KA)-type glutamate receptors (KARs) are implicated in various neuropsychiatric and neurological disorders through their ionotropic and metabotropic actions. However, compared to AMPA- and NMDA-type receptor functions, many aspects of KAR biology remain incompletely understood. Our study demonstrates an important role of KARs in organizing climbing fiber (CF)-Purkinje cell (PC) synapses and synaptic plasticity in the cerebellum, independently of their ion channel or metabotropic functions. The amino-terminal domain (ATD) of the GluK4 KAR subunit binds to C1ql1, provided by CFs, and associates with Bai3, an adhesion-type G protein-coupled receptor expressed in PC dendrites. Mice lacking GluK4 exhibit no KAR-mediated responses, reduced C1ql1 and Bai3 levels, and fewer CF-PC synapses, along with impaired long-term depression and oculomotor learning. Remarkably, introduction of the ATD of GluK4 significantly improves all these phenotypes. These findings demonstrate that KARs act as synaptic scaffolds, orchestrating synapses by forming a KAR-C1ql1-Bai3 complex in the cerebellum.

Cerebellar damage with inflammation upregulates oxytocin receptor expression in Bergmann Glia.

The cerebellum plays an important role in cognitive and social functioning. Childhood damage in the cerebellum increases the risk of autism spectrum disorder. Cerebellar inflammation induces social avoidance in mice. Oxytocin regulates social relationship and expression pattern of the oxytocin receptor in the brain is related to social behaviors. However, the expression patterns of the oxytocin receptor in the cerebellum remain controversial. Here, we report that the expression patterns of the oxytocin receptor in the cerebellum are highly variable among knock-in transgenic lines. We used Oxtr-Cre knock-in mice combined with a fluorescent reporter line and found that oxytocin receptor expression in Bergmann glia was more variable than that in Purkinje cells. We found that physical damage with inflammation induced the selective upregulation of the oxytocin receptor in Bergmann glia. Our findings indicate high variability in oxytocin receptor expression in the cerebellum and suggest that the oxytocin receptor can affect neural processing in pathological conditions, such as inflammation.

The Spinocerebellar Ataxia 34-Causing W246G ELOVL4 Mutation Does Not Alter Cerebellar Neuron Populations in a Rat Model.

Spinocerebellar ataxia 34 (SCA34) is an autosomal dominant disease that arises from point mutations in the fatty acid elongase, Elongation of Very Long Chain Fatty Acids 4 (ELOVL4), which is essential for the synthesis of Very Long Chain-Saturated Fatty Acids (VLC-SFA) and Very Long Chain-Polyunsaturated Fatty Acids (VLC-PUFA) (28-34 carbons long). SCA34 is considered a neurodegenerative disease. However, a novel rat model of SCA34 (SCA34-KI rat) with knock-in of the W246G ELOVL4 mutation that causes human SCA34 shows early motor impairment and aberrant synaptic transmission and plasticity without overt neurodegeneration. ELOVL4 is expressed in neurogenic regions of the developing brain, is implicated in cell cycle regulation, and ELOVL4 mutations that cause neuroichthyosis lead to developmental brain malformation, suggesting that aberrant neuron generation due to ELOVL4 mutations might contribute to SCA34. To test whether W246G ELOVL4 altered neuronal generation or survival in the cerebellum, we compared the numbers of Purkinje cells, unipolar brush cells, molecular layer interneurons, granule and displaced granule cells in the cerebellum of wildtype, heterozygous, and homozygous SCA34-KI rats at four months of age, when motor impairment is already present. An unbiased, semi-automated method based on Cellpose 2.0 and ImageJ was used to quantify neuronal populations in cerebellar sections immunolabeled for known neuron-specific markers. Neuronal populations and cortical structure were unaffected by the W246G ELOVL4 mutation by four months of age, a time when synaptic and motor dysfunction are already present, suggesting that SCA34 pathology originates from synaptic dysfunction due to VLC-SFA deficiency, rather than aberrant neuronal production or neurodegeneration.

Identification and Organization of a Postural Anti-Gravity Module in the Cerebellar Vermis.

The cerebellum is known to control the proper balance of isometric muscular contractions that maintain body posture. Current optogenetic manipulations of the cerebellar cortex output, however, have focused on ballistic body movements, examining movement initiation or perturbations. Here, by optogenetic stimulations of cerebellar Purkinje cells, which are the output of the cerebellar cortex, we evaluate body posture maintenance. By sequential analysis of body movement, we dissect the effect of optogenetic stimulation into a directly induced movement that is then followed by a compensatory reflex to regain body posture. We identify a module in the medial part of the anterior vermis which, through multiple muscle tone regulation, is involved in postural anti-gravity maintenance of the body. Moreover, we report an antero-posterior and medio-lateral functional segregation over the vermal lobules IV/V/VI. Taken together our results open new avenues for better understanding of the modular functional organization of the cerebellar cortex and its role in postural anti-gravity maintenance.

Specialized connectivity of molecular layer interneuron subtypes leads to disinhibition and synchronous inhibition of cerebellar Purkinje cells.

Molecular layer interneurons (MLIs) account for approximately 80% of the inhibitory interneurons in the cerebellar cortex and are vital to cerebellar processing. MLIs are thought to primarily inhibit Purkinje cells (PCs) and suppress the plasticity of synapses onto PCs. MLIs also inhibit, and are electrically coupled to, other MLIs, but the functional significance of these connections is not known. Here, we find that two recently recognized MLI subtypes, MLI1 and MLI2, have a highly specialized connectivity that allows them to serve distinct functional roles. MLI1s primarily inhibit PCs, are electrically coupled to each other, fire synchronously with other MLI1s on the millisecond timescale in vivo, and synchronously pause PC firing. MLI2s are not electrically coupled, primarily inhibit MLI1s and disinhibit PCs, and are well suited to gating cerebellar-dependent behavior and learning. The synchronous firing of electrically coupled MLI1s and disinhibition provided by MLI2s require a major re-evaluation of cerebellar processing.

Tools for Cre-Mediated Conditional Deletion of Floxed Alleles from Developing Cerebellar Purkinje Cells.

The Cre-lox system is an indispensable tool in neuroscience research for targeting gene deletions to specific cellular populations. Here we assess the utility of several transgenic Cre lines, along with a viral approach, for targeting cerebellar Purkinje cells (PCs) in mice. Using a combination of a fluorescent reporter line (Ai14) to indicate Cre-mediated recombination and a floxed Dystroglycan line (Dag1flox ), we show that reporter expression does not always align precisely with loss of protein. The commonly used Pcp2Cre line exhibits a gradual mosaic pattern of Cre recombination in PCs from Postnatal Day 7 (P7) to P14, while loss of Dag1 protein is not complete until P30. Ptf1aCre drives recombination in precursor cells that give rise to GABAergic neurons in the embryonic cerebellum, including PCs and molecular layer interneurons. However, due to its transient expression in precursors, Ptf1aCre results in stochastic loss of Dag1 protein in these neurons. NestinCre , which is often described as a "pan-neuronal" Cre line for the central nervous system, does not drive Cre-mediated recombination in PCs. We identify a Calb1Cre line that drives efficient and complete recombination in embryonic PCs, resulting in loss of Dag1 protein before the period of synaptogenesis. AAV8-mediated delivery of Cre at P0 results in gradual transduction of PCs during the second postnatal week, with loss of Dag1 protein not reaching appreciable levels until P35. These results characterize several tools for targeting conditional deletions in cerebellar PCs at different developmental stages and illustrate the importance of validating the loss of protein following recombination.

Ultrasound, Histomorphologic, and Immunohistochemical Analysis of a Cardiac Tumor with Increased Purkinje Cells Detected in a Canine Fetus 42 Days into Pregnancy.

A seven-year-old healthy female Chow Chow was referred for pregnancy monitoring. Ultrasonography was used to evaluate all pregnancy and fetus parameters, and they were found to be normal. During the examination of the 42 day pregnant bitch, an unusual mass was seen in a fetus's heart. This fetus had a cardiac frequency of 273-300 beats, while the others had heart rates of 220-240 beats. Natural vaginal birth occurred at 63 days pregnant: the first two puppies were stillborn but perfectly formed, and the other three were alive and had optimal APGAR. In one of two deceased puppies, an unusual, reddish, smooth mass occupying the space in the heart was found through necroscopy. The organ was submitted for histological examination. Histopathology, immunohistochemical, and histochemical analyses all indicated a cardiac tumor with increased Purkinje cells. This type of tumor has been described in infants, swine, bearded seals, and deer but never in fetuses and neonates of dogs. To our knowledge, this is the first such case reported in veterinary medicine.

Plasticity mechanisms of genetically distinct Purkinje cells.

Despite its uniform appearance, the cerebellar cortex is highly heterogeneous in terms of structure, genetics and physiology. Purkinje cells (PCs), the principal and sole output neurons of the cerebellar cortex, can be categorized into multiple populations that differentially express molecular markers and display distinctive physiological features. Such features include action potential rate, but also their propensity for synaptic and intrinsic plasticity. However, the precise molecular and genetic factors that correlate with the differential physiological properties of PCs remain elusive. In this article, we provide a detailed overview of the cellular mechanisms that regulate PC activity and plasticity. We further perform a pathway analysis to highlight how molecular characteristics of specific PC populations may influence their physiology and plasticity mechanisms.

Cerebellum Purkinje cell vulnerability in aged rats with memory impairment.

The cerebellum is involved in higher order cognitive function and is susceptible to age-related atrophy. However, limited evidence has directly examined the cerebellum's role in cognitive aging. To interrogate potential substrates of the relationship between cerebellar structure and memory in aging, here we target the Purkinje cells (PCs). The sole output neurons of the cerebellum, PC loss and/or degeneration underlie a variety of behavioral abnormalities. Using a rat model of normal cognitive aging, we immunostained sections through the cerebellum for the PC-specific protein, calbindin-D28k. Although morphometric quantification revealed no significant difference in total PC number as a function of age or cognitive status, regional cell number was a more robust correlate of memory performance in the young cerebellum than in aged animals. Parallel biochemical analysis of PC-specific protein levels in whole cerebellum additionally revealed that calbindin-D28k and Purkinje cell protein-2 (pcp-2) levels were lower selectively in aged rats with spatial memory impairment compared to both young animals and aged rats with intact memory. These results suggest that cognitive aging is associated with cerebellum vulnerability, potentially reflecting disruption of the cerebellum-medial temporal lobe network.