Activation of Presynaptic GABAAReceptors Induces Glutamate Release from Parallel Fiber Synapses

The number of synaptic AMPA receptors (AMPARs) is the major determinant of synaptic strength and is differently regulated in input pathway-dependent and target cell type-dependent manners. In cerebellar Purkinje cells (PCs), the density of synaptic AMPARs is approximately five times lower at parallel fiber (PF) synapses than at climbing fiber (CF) synapses. However, molecular mechanisms underlying this biased synaptic distribution remain unclear. As a candidate molecule, we focused on glutamate receptor δ2 (GluRδ2 or GluD2), which is known to be efficiently trafficked to and selectively expressed at PF synapses in PCs. We applied postembedding immunogold electron microscopy to GluRδ2 knock-out (KO) and control mice, and measured labeling density for GluA1-4 at three excitatory synapses in the cerebellar molecular layer. In both control and GluRδ2-KO mice, GluA1-3 were localized at PF and CF synapses in PCs, while GluA2-4 were at PF synapses in interneurons. In control mice, labeling density for each of GluA1-3 was four to six times lower at PF-PC synapses than at CF-PC synapses. In GluRδ2-KO mice, however, their labeling density displayed a three- to fivefold increase at PF synapses, but not at CF synapses, thus effectively eliminating input pathway-dependent disparity between the two PC synapses. Furthermore, we found an unexpected twofold increase in labeling density for GluA2 and GluA3, but not GluA4, at PF-interneuron synapses, where we identified low but significant expression of GluRδ2. These results suggest that GluRδ2 is involved in a common mechanism that restricts the number of synaptic AMPARs at PF synapses in PCs and molecular layer interneurons.

Parallel-fibre synaptic membranes were examined by freeze-fracture and ethanolic-phosphotungstic acid methods in the cerebellum of homozygous (j/j) Gunn rats with hereditary jaundice. Parallel-fibre synapses with dendritic spines of Purkinje cell were severely affected since many Purkinje cells degenerated during the early postnatal period. Some parallel-fibre synaptic terminals lacked their postsynaptic partners and faced astrocytic processes from 18 days of age to the adult stage. These parallel-fibre terminals contained clusters of synaptic vesicles adjacent to synaptic membranes, and synaptic membranes and synaptic cleft materials were identical to those of parallel fibres with postsynaptic partners, as visualized by conventional electron microscopy with osmium tetroxide postfixation and staining of sections with uranyl acetate and lead citrate. In freeze-fractured specimens, the presynaptic membrane of parallel fibres had diffusely distributed large particles and tiny pits on the P-face and protuberances on the E-face, together representing synaptic vesicle attachment sites. Such vesicle attachment sites were present on the presynaptic membranes of parallel fibres without postsynaptic partners from day 18 to the adult stage. After ethanolic-phosphotungstic acid staining, parallel-fibre terminals displayed presynaptic dense projections, intercleft materials and postsynaptic thickening, but some parallel fibres lacked postsynaptic thickening. These observations suggest that the presynaptic membrane structure of the parallel fibre is preserved, even in the absence of a postsynaptic partner, in j/j cerebella. A mechanism for persistence of presynaptic membrane structures without postsynaptic partners in j/j cerebella is discussed.

The cerebellum is crucial for different types of motor learning. Established theories of cerebellar learning posit that the cerebellum learns by adjusting the weights of Parallel Fiber (PF) to Purkinje cells (PC) synapses, thanks to teaching signals provided by Climbing Fiber inputs. While these theories are consistent with a large body of experimental data, in particular on synaptic plasticity in PF to PC synapses, they cannot easily explain a growing body of experimental work, which seems to indicate a significant role of other sites of plasticity. Recent advances in the development of a large number in transgenic animals, as well as behavioral and electrophysiogical comparative studies between these animals and wild-type animals, have opened an unprecedented window into the mechanisms underlying learning in this structure. In particular, it has been shown that specific knock-outs are impaired selectively on difficult variants of the vestibulo-ocular reflex (VOR) adaptation task, one of the most studied cerebellar-dependent motor learning tasks. These impairments can occur even though the classical plasticity mechanisms are left untouched. These data pose significant new challenges for established models of cerebellar learning. To better understand the mechanisms of learning in the cerebellum, we built a model that can reproduce the available data on VOR adaptation, in both wild-type and transgenic animals. The model includes some of the main cell types involved in this task: granule cells (GCs), the input layer of cerebellar cortex, that receives vestibular information from the mossy fibers (MFs); Purkinje cells (PCs), as well as molecular layer interneurons (INs); and two cell populations in the medial vestibular nuclei (MVN), one excitatory and one inhibitory, that together control eye movement. The model also includes two sites of learning: the classical GC to PF plasticity site, as well as plasticity in the MF to MVN synapses. We provide a mechanistic understanding on how the system learns VOR adaptation in normal conditions, as well as how the system is impaired by specific knock-outs, which selectively suppress inhibition onto PCs, or increase the excitability of GCs. Finally, we show that our model is consistent with behavioral, as well as in vivo electrophysiological recordings.

Postnatal changes in the concentration and distribution of cholinesterase in the cerebellar cortex of rats

Genetic variation in CACNA1D, the gene that encodes the pore-forming subunit of the L-type calcium channel CaV1.3, has been associated with increased risk for neuropsychiatric disorders that display abnormalities in cerebellar structures. We sought to clarify if deletion of CaV1.3 in mice would induce abnormalities in cerebellar cortex cytoarchitecture or synapse morphology. Since CaV1.3 is highly expressed in cerebellar molecular layer interneurons (MLIs) and L-type channels appear to regulate GABA release from MLIs, we hypothesized that loss of CaV1.3 would alter GABAergic synapses between MLIs and Purkinje cells (PCs) without altering MLI density or PC structure. As expected, we did not observe changes in the density of MLIs or PCs. Surprisingly, CaV1.3 KO mice do have decreased complexity of PC dendritic arbors without differences in the number or structure of GABAergic synapses onto PCs. Loss of CaV1.3 was not associated with impaired acquisition of delay eyeblink conditioning. Therefore, our data suggest that CaV1.3 expression is important for PC structure but does not affect other measures of cerebellar cortex morphology or cerebellar function as assessed by delay eyeblink conditioning.

1. Potential fields and unitary spikes in the cat cerebellar cortex were generated specifically by mossy fibre volleys and recorded by means of microelectrodes. The mossy fibres were excited by trans-folial (T. F.) stimulation which was compared with juxtafastigial (J.F.) stimulation. Both were conditioned by local stimuli of parallel fibres. 2. In the granular layer, an incoming mossy fibre volley evoked a small diphasic potential (P1 N1) and about 0.4 msec later a second negative wave (N2) due to the excitatory synaptic current generated by synapses of mossy fibres with granule cells and Golgi cells. In the typical configuration the N2 wave usually had a superimposed double spike potential, which is due to impulses discharged first by Golgi cells and then, about 0.5 msec later, by granule cells. 3. The transmission of impulses along the perpendicular axons of the granule cells and thence along the parallel fibres gave the fairly sharp positive potential (P2) in the granular layer, and simultaneously the negative wave (N3) in the molecular layer. The parallel fibre impulses, in turn, synaptically excited and so evoked local responses and action potentials in the dendrites of Purkinje and other cells, which aided in the production of the latter part of the N3 wave. 4. The impulses in the Purkinje cell dendrites propagate into the granular layer via the Purkinje cell somata and axons so producing the negative wave (N4) in the Purkinje and the granular layer. 5. The late and prolonged positive wave (P3) may be attributable to the deep active sources produced by postsynaptic inhibition of Purkinje cells and of granule cells by basket and Golgi cells respectively. 6. There has been good correlation between the physiological findings and the anatomical structures of the various types of cells and the synaptic connections, even to the synapses of mossy fibres on Golgi cell dendrites that have been recently described by HAmori and SzentAgothai.

Eph receptor tyrosine kinases are involved in many cellular processes. In the developing brain, they act as migratory and cell adhesive cues while in the adult brain they regulate dendritic spine plasticity. Here we show a new role for Eph receptor signalling in the cerebellar cortex. Cerebellar Purkinje cells are innervated by two different excitatory inputs. The climbing fibres contact the proximal dendritic domain of Purkinje cells, where synapse and spine density is low; the parallel fibres contact the distal dendritic domain, where synapse and spine density is high. Interestingly, Purkinje cells have the intrinsic ability to generate a high number of spines over their entire dendritic arborisations, which can be innervated by the parallel fibres. However, the climbing fibre input continuously exerts an activity-dependent repression on parallel fibre synapses, thus confining them to the distal Purkinje cell dendritic domain. Such repression persists after Eph receptor activation, but is overridden by Eph receptor inhibition with EphA4/Fc in neonatal cultured cerebellar slices as well as mature acute cerebellar slices, following in vivo infusion of the EphA4/Fc inhibitor and in EphB receptor-deficient mice. When electrical activity is blocked in vivo by tetrodotoxin leading to a high spine density in Purkinje cell proximal dendrites, stimulation of Eph receptor activation recapitulates the spine repressive effects of climbing fibres. These results suggest that Eph receptor signalling mediates the repression of spine proliferation induced by climbing fibre activity in Purkinje cell proximal dendrites. Such repression is necessary to maintain the correct architecture of the cerebellar cortex.

Climbing fiber (CF) inputs to Purkinje cells (PCs) instruct plasticity and learning in the cerebellum1–3. Paradoxically, CFs also excite molecular layer interneurons (MLIs)4,5, a cell-type that inhibits PCs and can restrict plasticity and learning6,7. However, two types of MLIs with opposing influences have recently been identified: MLI1s inhibit PCs, reduce dendritic calcium signals, and suppress plasticity of granule cell to PC synapses2,6–9, whereas MLI2s inhibit MLI1s and disinhibit PCs8. To determine how CFs can activate MLIs without also suppressing the PC calcium signals necessary for plasticity and learning, we investigated the specificity of CF inputs onto MLIs. Serial EM reconstructions indicate that CFs contact both MLI subtypes without making conventional synapses, but more CFs contact each MLI2 via more sites with larger contact areas. Slice experiments indicate that CFs preferentially excite MLI2s via glutamate spillover4,5. In agreement with these anatomical and slice experiments, in vivo Neuropixels recordings show that spontaneous CF activity excites MLI2s, inhibits MLI1s, and disinhibits PCs. In contrast, learning-related sensory stimulation produced more complex responses, driving convergent CF and granule cell inputs that could either activate or suppress MLI1s. This balance was robustly shifted toward MLI1 suppression when CFs were synchronously active, in turn elevating the PC dendritic calcium signals necessary for LTD. These data provide mechanistic insight into why CF synchrony can be highly effective at inducing cerebellar learning2,3 by revealing a critical disinhibitory circuit that allows CFs to act through MLIs to enhance PC dendritic calcium signals necessary for plasticity.

Climbing fibre (CF) inputs to Purkinje cells (PCs) instruct plasticity and learning in the cerebellum1-3. Paradoxically, CFs also excite molecular layer interneurons (MLIs)4,5, a cell type that inhibits PCs and can restrict plasticity and learning6,7. However, two types of MLI with opposing influences have recently been identified: MLI1s inhibit PCs, reduce dendritic calcium signals and suppress plasticity of granule cell to PC synapses2,6-9, whereas MLI2s inhibit MLI1s and disinhibit PCs8. To determine how CFs can activate MLIs without also suppressing the PC calcium signals necessary for plasticity and learning, we investigated the specificity of CF inputs onto MLIs. Serial electron microscopy reconstructions indicate that CFs contact both MLI subtypes without making conventional synapses, but more CFs contact each MLI2 through more sites with larger contact areas. Slice experiments indicate that CFs preferentially excite MLI2s through glutamate spillover4,5. In agreement with these anatomical and slice experiments, in vivo Neuropixels recordings show that spontaneous CF activity excites MLI2s, inhibits MLI1s and disinhibits PCs. By contrast, learning-related sensory stimulation produces more complex responses, driving convergent CF and granule cell inputs that could either activate or suppress MLI1s. This balance was robustly shifted towards MLI1 suppression when CFs were synchronously active, in turn elevating the PC dendritic calcium signals necessary for long-term depression. These data provide mechanistic insight into why CF synchrony can be highly effective at inducing cerebellar learning2,3 by revealing a critical disinhibitory circuit that allows CFs to act through MLIs to enhance PC dendritic calcium signals necessary for plasticity.

Inhibition from molecular layer interneurons (MLIs) is thought to play an important role in cerebellar function by sharpening the precision of Purkinje cell spike output. Yet the coding features of MLIs during behavior are poorly understood. To study MLI activity, we used in vivo Ca2+ imaging in head-fixed mice during the performance of a rhythmic motor behavior, licking during water consumption. MLIs were robustly active during lick-related movement across a lobule-specific region of the cerebellum showing high temporal correspondence within their population. Average MLI Ca2+ activity strongly correlated with movement rate but not to the intentional, or unexpected, adjustment of lick position or to sensory feedback that varied with task condition. Chemogenetic suppression of MLI output reduced lick rate and altered tongue movements, indicating that activity of these interneurons not only encodes temporal aspects of movement kinematics but also influences motor outcome pointing to an integral role in online control of rhythmic behavior.SIGNIFICANCE STATEMENT The cerebellum helps fine-tune coordinated motor actions via signaling from projection neurons called Purkinje cells. Molecular layer interneurons (MLIs) provide powerful inhibition onto Purkinje cells, but little is understood about how this inhibitory circuit is engaged during behavior or what type of information is transmitted through these neurons. Our work establishes that MLIs in the lateral cerebellum are broadly activated during movement with calcium activity corresponding to movement rate. We also show that suppression of MLI output slows and disorganizes the precise movement pattern. Therefore, MLIs are an important circuit element in the cerebellum allowing for accurate motor control.

The function of a feedback inhibitory circuit between cerebellar Purkinje cells and molecular layer interneurons (MLIs) was defined by combining optogenetics, neuronal activity recordings both in cerebellar slices and in vivo, and computational modeling. Purkinje cells inhibit a subset of MLIs in the inner third of the molecular layer. This inhibition is non-reciprocal, short-range (less than 200 μm) and is based on convergence of one to two Purkinje cells onto MLIs. During learning-related eyelid movements in vivo, the activity of a subset of MLIs progressively increases as Purkinje cell activity decreases, with Purkinje cells usually leading the MLIs. Computer simulations indicate that these relationships are best explained by the feedback circuit from Purkinje cells to MLIs and that this feedback circuit plays a central role in making cerebellar learning efficient.

Dendritic NMDA Receptors Activate Axonal Calcium Channels

The present study is devoted to a detailed analysis of the structural and synaptic organization of mormyrid Purkinje cells in order to evaluate the possible functional significance of their dendritic palisade pattern. For this purpose, the properties of Golgi-impregnated as well as unimpregnated Purkinje cells in lobe C1 and C3 of the cerebellum of Gnathonemus petersii were light and electron microscopically analyzed, quantified, reconstructed, and mutually compared. Special attention was paid to the degree of regularity of their dendritic trees, their relations with Bergmann glia, and the distribution and numerical properties of their synaptic connections with parallel fibers, stellate cells, "climbing" fibers, and Purkinje axonal boutons. The highest degree of palisade specialization was encountered in lobe C1, where Purkinje cells have on average 50 palisade dendrites with a very regular distribution in a sagittal plane. Their spine density decreases from superficial to deep (from 14 to 6 per micron dendritic length), a gradient correlated with a decreasing parallel fiber density but an increasing parallel fiber diameter. Each Purkinje cell makes on average 75,000 synaptic contacts with parallel fibers, some of which are rather coarse (0.45 microns), and provided with numerous short collaterals. Climbing fibers do not climb, since their synaptic contacts are restricted to the ganglionic layer (i.e., the layer of Purkinje and eurydendroid projection cells), where they make about 130 synaptic contacts per cell with 2 or 3 clusters of thorns on the proximal dendrites. These clusters contain also a type of "shunting" elements that make desmosome-like junctions with both the climbing fiber boutons and the necks of the thorns. The axons of Purkinje cells in lobe C1 make small terminal arborizations, with about 20 boutons, that may be substantially (up to 500 microns) displaced rostrally or caudally with respect to the soma. Purkinje axonal boutons were observed to make synaptic contacts with eurydendroid projection cells and with the proximal dendritic and somatic receptive surface of Purkinje cells, where about 15 randomly distributed boutons per neuron occur. The organization of Purkinje cells in lobe C3 differs markedly from that in C1 and seems to be less regular and specialized, although the overall palisade pattern is even more regular than in lobe C1 because of the absence of large eurydendroid neurons. However, individual neurons have a less regular dendritic tree, there is no apical-basal gradient in spine density or parallel fiber density and diameter, and there are no "shunting" elements in the climbing fiber glomeruli.(ABSTRACT TRUNCATED AT 400 WORDS)

Precise spike timing of tactile-evoked cerebellar golgi cell responses: a reflection of combined mossy fiber and parallel fiber activation?

Cerebellar Purkinje cells are excited by two afferent pathways: climbing and mossy fibers. Climbing fibers evoke large "complex spikes" (CSs) that discharge at low frequencies. Mossy fibers synapse on granule cells whose parallel fibers excite Purkinje cells and may contribute to the genesis of "simple spikes" (SSs). Both afferent systems convey vestibular information to folia 9c-10. After making a unilateral labyrinthectomy (UL) in mice, we tested how the discharge of CSs and SSs was changed by the loss of primary vestibular afferent mossy fibers during sinusoidal roll tilt. We recorded from cells identified by juxtacellular neurobiotin labeling. The UL preferentially reduced vestibular modulation of CSs and SSs in folia 8-10 contralateral to the UL. The effects of a UL on Purkinje cell discharge were similar in folia 9c-10, to which vestibular primary afferents project, and in folia 8-9a, to which they do not project, suggesting that vestibular primary afferent mossy fibers were not responsible for the UL-induced alteration of SS discharge. UL also induced reduced vestibular modulation of stellate cell discharge contralateral to the UL. We attribute the decreased modulation to reduced vestibular modulation of climbing fibers. In summary, climbing fibers modulate CSs directly and SSs indirectly through activation of stellate cells. Whereas vestibular primary afferent mossy fibers cannot account for the modulated discharge of SSs or stellate cells, the nonspecific excitation of Purkinje cells by parallel fibers may set an operating point about which the discharges of SSs are sculpted by climbing fibers.