We report a type of synaptic modulation that involves retrograde signaling from postsynaptic metabotropic glutamate receptors (mGluRs) to presynaptic cannabinoid receptors. Activation of mGluR subtype 1 (mGluR1) expressed in cerebellar Purkinje cells (PCs) reduced neurotransmitter release from excitatory climbing fibers. This required activation of G proteins but not Ca2+ elevation in postsynaptic PCs. This effect was occluded by a cannabinoid agonist and totally abolished by cannabinoid antagonists. Depolarization-induced Ca2+ transients in PCs also caused cannabinoid receptor-mediated presynaptic inhibition. Thus, endocannabinoid production in PCs can be initiated by two distinct stimuli. Activation of mGluR1 by repetitive stimulation of parallel fibers, the other excitatory input to PCs, caused transient cannabinoid receptor-mediated depression of climbing fiber input. Our data highlight a signaling mechanism whereby activation of postsynaptic mGluR retrogradely influences presynaptic functions via endocannabinoid system.

To faithfully recreate the features of the human neurodegenerative disease spinocerebellar ataxia type 1 (SCA1) in the mouse, we targeted 154 CAG repeats into the endogenous mouse locus. Sca1(154Q/2Q) mice developed a progressive neurological disorder that resembles human SCA1, featuring motor incoordination, cognitive deficits, wasting, and premature death, accompanied by Purkinje cell loss and age-related hippocampal synaptic dysfunction. Mutant ataxin-1 solubility varied with brain region, being most soluble in the neurons most vulnerable to degeneration. Solubility decreased overall as the mice aged; Purkinje cells, the most affected in SCA1, did not form aggregates of mutant protein until an advanced stage of disease. It appears that those neurons that cannot sequester the mutant protein efficiently and thereby curb its toxicity suffer the worst damage from polyglutamine-induced toxicity.

During cerebellar development, the main portion of the cerebellar plate neuroepithelium gives birth to Purkinje cells and interneurons, whereas the rhombic lip, the germinal zone at its dorsal edge, generates granule cells and cerebellar nuclei neurons. However, it remains elusive how these components cooperate to form the intricate cerebellar structure. Here, we found that a polarized cerebellar structure self-organizes in 3D human embryonic stem cell (ESC) culture. The self-organized neuroepithelium differentiates into electrophysiologically functional Purkinje cells. The addition of fibroblast growth factor 19 (FGF19) promotes spontaneous generation of dorsoventrally polarized neural-tube-like structures at the level of the cerebellum. Furthermore, addition of SDF1 and FGF19 promotes the generation of a continuous cerebellar plate neuroepithelium with rhombic-lip-like structure at one end and a three-layer cytoarchitecture similar to the embryonic cerebellum. Thus, human-ESC-derived cerebellar progenitors exhibit substantial self-organizing potential for generating a polarized structure reminiscent of the early human cerebellum at the first trimester.

To study the function of GLAST, a glutamate transporter highly expressed in the cerebellar Bergmann astrocytes, the mouse GLAST gene was inactivated. GLAST-deficient mice developed normally and could manage simple coordinated tasks, such as staying on a stationary or a slowly rotating rod, but failed more challenging task such as staying on a quickly rotating rod. Electrophysiological examination revealed that Purkinje cells in the mutant mice remained to be multiply innervated by climbing fibres even at the adult stage. We also found that oedema volumes in the mutant mice increased significantly after cerebellar injury. These results indicate that GLAST plays active roles both in the cerebellar climbing fibre synapse formation and in preventing excitotoxic cerebellar damage after acute brain injury.

PKC gamma is highly expressed in Purkinje cells (PCs) but not in other types of neurons in the cerebellum. The expression of PKC gamma changes markedly during cerebellar development, being very low at birth and reaching a peak around the third postnatal week. This temporal pattern of PKC gamma expression coincides with the developmental transition from multiple to single climbing fiber innervation onto each PC. In adult mutant mice deficient in PKC gamma, we found that 41% of PCs are still innervated by multiple climbing fibers, while other aspects of the cerebellum including the morphology and excitatory synaptic transmission of PCs appear normal. Thus, elimination of multiple climbing fiber innervation appears to be specifically impaired in the mutant cerebellum. We suggest that the developmental role of PKC gamma may be to act as a downstream element in the signal cascade necessary for the elimination of surplus climbing fiber synapses.

Targeted deletion of metabotropic glutamate receptor-subtype 1 (mGluR1) gene can cause defects in development and function in the cerebellum. We introduced the mGluR1alpha transgene into mGluR1-null mutant [mGluR1 (-/-)] mice with a Purkinje cell (PC)-specific promoter. mGluR1-rescue mice showed normal cerebellar long-term depression and regression of multiple climbing fiber innervation, events significantly impaired in mGluR1 (-/-) mice. The impaired motor coordination was rescued by this transgene, in a dose-dependent manner. We propose that mGluR1 in PCs is a key molecule for normal synapse formation, synaptic plasticity, and motor control in the cerebellum.

We studied how physiological properties of cerebellar climbing fiber (CF) to Purkinje cell (PC) synapses change during developmental transition from multiple to mono CF innervation onto each PC. From P3 to P6, differences in the strengths of multiple CFs became larger. Around P10, each PC was either monoinnervated by one strong CF (CF-mono) or multiply innervated by one strong CF (CF-multi-S) plus a few weaker CFs (CF-multi-W). We show that simultaneous release of multiple vesicles per site occurs normally from CF-multi-S, CF-mono, and mature CFs, but less frequently from CF-multi-W and neonatal CFs. We also present evidence suggesting that weaker CFs with lower probability of multivesicular release would be withdrawn preferentially. The results suggest that differentiation into strong and weak CFs with high and low probabilities of multivesicular release precedes developmental CF synapse elimination.

Most of the cerebellar Purkinje cells (PCs) of an adult animal are innervated individually by a single climbing fiber (CF) that forms strong excitatory synapses with the PCs. This one-to-one relationship between a PC and a CF is a consequence of a developmentally regulated regression of the innervation of PCs by CFs. We found that, in mice deficient in the type 1 metabotropic glutamate receptor (mGluR1), the regression of supernumerary CFs ceases by the end of the second postnatal week, which is about one week earlier than in normal mice. Consequently, about one third of PCs in the mGluR1 mutant mice are innervated by multiple CFs in adulthood. We conclude that the regression of CFs normally occurs in two developmental phases and that mGluR1 plays a crucial role in the second phase.