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The cerebellar cortex exhibits a strikingly high expression of type 1 cannabinoid receptor (CB1), the cannabinoid binding protein responsible for the psychoactive effects of marijuana. CB1 is primarily found in presynaptic elements in the molecular layer. While the functional importance of cerebellar CB1 is supported by the effect of gene deletion or exogenous cannabinoids on animal behavior, evidence for a role of endocannabinoids in synaptic signaling is provided by in vitro experiments on superfused acute rodent cerebellar slices. These studies have demonstrated that endocannabinoids can be transiently released by Purkinje cells and signal at synapses in a direction opposite to information transfer (retrograde). Here, following a description of the reported expression pattern of the endocannabinoid system in the cerebellum, I review the accumulated in vitro data, which have addressed the mechanism of retrograde endocannabinoid signaling and identified 2-arachidonoylglycerol as the mediator of this signaling. The mechanisms leading to endocannabinoid release, the effects of CB1 activation, and the associated synaptic plasticity mechanisms are discussed and the remaining unknowns are pointed. Notably, it is argued that the spatial specificity of this signaling and the physiological conditions required for its induction need to be determined in order to understand endocannabinoid function in the cerebellar cortex.

Cerebellar Purkinje neurons integrate information transmitted at excitatory synapses formed by granule cells. Although these synapses are considered essential sites for learning, most of them appear not to transmit any detectable electrical information and have been defined as silent. It has been proposed that silent synapses are required to maximize information storage capacity and ensure its reliability, and hence to optimize cerebellar operation. Such optimization is expected to occur once the cerebellar circuitry is in place, during its maturation and the natural and steady improvement of animal agility. We therefore investigated whether the proportion of silent synapses varies over this period, from the third to the sixth postnatal week in mice. Selective expression of a calcium indicator in granule cells enabled quantitative mapping of presynaptic activity, while postsynaptic responses were recorded by patch clamp in acute slices. Through this approach and the assessment of two anatomical features (the distance that separates adjacent planar Purkinje dendritic trees and the synapse density), we determined the average excitatory postsynaptic potential per synapse. Its value was four to eight times smaller than responses from paired recorded detectable connections, consistent with over 70% of synapses being silent. These figures remained remarkably stable across maturation stages. According to the proposed role for silent synapses, our results suggest that information storage capacity and reliability are optimized early during cerebellar maturation. Alternatively, silent synapses may have roles other than adjusting the information storage capacity and reliability.

Climbing fibers, the projections from the inferior olive to the cerebellar cortex, carry sensorimotor error and clock signals that trigger motor learning by controlling cerebellar Purkinje cell synaptic plasticity and discharge. Purkinje cells target the deep cerebellar nuclei, which are the output of the cerebellum and include an inhibitory GABAergic projection to the inferior olive. This pathway identifies a potential closed loop in the olivo-cortico-nuclear network. Therefore, sets of Purkinje cells may phasically control their own climbing fiber afferents. Here, using in vitro and in vivo recordings, we describe a genetically modified mouse model that allows the specific optogenetic control of Purkinje cell discharge. Tetrode recordings in the cerebellar nuclei demonstrate that focal stimulations of Purkinje cells strongly inhibit spatially restricted sets of cerebellar nuclear neurons. Strikingly, such stimulations trigger delayed climbing-fiber input signals in the stimulated Purkinje cells. Therefore, our results demonstrate that Purkinje cells phasically control the discharge of their own olivary afferents and thus might participate in the regulation of cerebellar motor learning.

The brain's endocannabinoid retrograde messenger system decreases presynaptic transmitter release, but its physiological function is uncertain. We show that endocannabinoid signaling is absent when spatially dispersed synapses are activated on rodent cerebellar Purkinje cells but that it reduces presynaptic glutamate release when nearby synapses are active. This switching of signaling according to the spatial pattern of activity is controlled by postsynaptic type I metabotropic glutamate receptors, which are activated disproportionately when glutamate spillover between synapses produces synaptic crosstalk. When spatially distributed synapses are activated, endocannabinoid inhibition of transmitter release can be rescued by inhibiting glutamate uptake to increase glutamate spillover. Endocannabinoid signaling initiated by type I metabotropic glutamate receptors is a homeostatic mechanism that detects synaptic crosstalk and downregulates glutamate release in order to promote synaptic independence.

Metabotropic glutamate receptor (mGluR) activation has been extensively studied under steady-state conditions. However, at central synapses, mGluRs are exposed to brief submillisecond glutamate transients and may not reach steady-state. The lack of information on the kinetics of mGluR activation impairs accurate predictions of their operation during synaptic transmission. Here, we report experiments designed to investigate mGluR kinetics in real-time. We inserted either CFP or YFP into the second intracellular loop of mGluR1β. When these constructs were coexpressed in PC12 cells, glutamate application induced a conformational change that could be monitored, using fluorescence resonance energy transfer (FRET), with an EC₅₀ of 7.5 μM. The FRET response was mimicked by the agonist DHPG, abolished by the competitive antagonist MCPG, and partially inhibited by mGluR1-selective allosteric modulators. These results suggest that the FRET response reports active conformations of mGluR1 dimers. The solution exchange at the cell membrane was optimized for voltage-clamped cells by recording the current induced by co-application of 30 mM potassium. When glutamate was applied at increasing concentrations up to 2 mM, the activation time course decreased to a minimum of approximately 10 ms, whereas the deactivation time course remained constant (~50 ms). During long-lasting applications, no desensitization was observed. In contrast, we observed a robust sensitization of the FRET response that developed over approximately 400 ms. Activation, deactivation, and sensitization time courses and amplitudes were used to derive a kinetic scheme and rate constants, from which we inferred the EC₅₀ and frequency dependence of mGluR1 activation under non-steady-state conditions, as occurs during synaptic transmission.

The great demand for long-wavelength and high signal-to-noise Ca2+ indicators has led us to develop CaRuby-Nano, a new functionalizable red calcium indicator with nanomolar affinity for use in cell biology and neuroscience research. In addition, we generated CaRuby-Nano dextran conjugates and an AM-ester variant for bulk loading of tissue. We tested the new indicator using in vitro and in vivo experiments demonstrating the high sensitivity of CaRuby-Nano as well as its power in dual color imaging experiments. The movement of calcium ions within cells controls many vital biological processes, ranging from cell growth to muscle contraction and brain activity. These calcium signals are triggered by stimuli, such as nerve impulses, which drive calcium entry into cells or release calcium from internal stores. These changes in calcium levels can span several orders of magnitude, and can be either localized to very small parts of the cell or span the entire cell. Scientists have developed numerous indicators or ‘probes’ that can detect even very low levels of calcium. One common method uses proteins that fluoresce when viewed under a fluorescence microscope each time the protein senses increases of calcium. Most of these probes fluoresce green, and so to view a second signal that occurs in the cell at the same time it's easier to use a probe that fluoresces with a different color, such as red. However, the red-shifted probes that are currently available either produce unreliable results because they tend to leak through cell membranes, or are not very sensitive to calcium ions. New types of red-shifted probes are therefore urgently needed. In 2012, researchers developed a family of red fluorescent probes known as Calcium Ruby (CaRuby for short) that were more versatile than earlier red probes. Now, Collot, Wilms et al.—including several of the researchers involved in the 2012 research—have enhanced the properties of CaRuby by modifying the chemical structure of the probes. This increased the ability of CaRuby to bind calcium ions, making it more sensitive to small calcium changes. Testing the usefulness of the newly developed probes—called CaRuby Nano—in mouse nerve cells revealed the probes are highly sensitive and can even detect the calcium signal resulting from a single nerve impulse. Collot, Wilms et al. then went on to demonstrate that CaRuby-Nano can be used alongside a green-fluorescing probe to record two signals at the same time. In one experiment, the release of chemical messengers known as neurotransmitters was stimulated, which caused calcium ions to flow into the observed nerve cells. The researchers succeeded in simultaneously detecting a green signal indicating an increase in neurotransmitter levels and a red signal produced by the corresponding release of calcium. Such dual-color imaging was not possible with previous probes. Finally, it was shown that CaRuby-Nano can also be used to produce dual-color images of the brain activity of live mice. In summary, these results demonstrate that CaRuby-Nano is a highly sensitive and versatile indicator and can be used together with other probes to observe two simultaneous events in cells.

In the brain, glutamate is an extracellular transmitter that mediates cell-to-cell communication. Prior to synaptic release it is pumped into vesicles by vesicular glutamate transporters (VGLUTs). To inactivate glutamate receptor responses after release, glutamate is taken up into glial cells or neurons by excitatory amino acid transporters (EAATs). In the pancreatic islets of Langerhans, glutamate is proposed to act as an intracellular messenger, regulating insulin secretion from β-cells, but the mechanisms involved are unknown. By immunogold cytochemistry we show that insulin containing secretory granules express VGLUT3. Despite the fact that they have a VGLUT, the levels of glutamate in these granules are low, indicating the presence of a protein that can transport glutamate out of the granules. Surprisingly, in β-cells the glutamate transporter EAAT2 is located, not in the plasma membrane as it is in brain cells, but exclusively in insulin-containing secretory granules, together with VGLUT3. In EAAT2 knock out mice, the content of glutamate in secretory granules is higher than in wild type mice. These data imply a glutamate cycle in which glutamate is carried into the granules by VGLUT3 and carried out by EAAT2. Perturbing this cycle by knocking down EAAT2 expression with a small interfering RNA, or by over-expressing EAAT2 or a VGLUT in insulin granules, significantly reduced the rate of granule exocytosis. Simulations of granule energetics suggest that VGLUT3 and EAAT2 may regulate the pH and membrane potential of the granules and thereby regulate insulin secretion. These data suggest that insulin secretion from β-cells is modulated by the flux of glutamate through the secretory granules.

Plasticity at the cerebellar parallel fiber to Purkinje cell synapse may underlie information processing and motor learning. In vivo, parallel fibers appear to fire in short high frequency bursts likely to activate sparsely distributed synapses over the Purkinje cell dendritic tree. Here, we report that short parallel fiber tetanic stimulation evokes a ∼7-15% depression which develops over 2 min and lasts for at least 20 min. In contrast to the concomitantly evoked short-term endocannabinoid-mediated depression, this persistent posttetanic depression (PTD) does not exhibit a dependency on the spatial pattern of synapse activation and is not caused by any detectable change in presynaptic calcium signaling. This persistent PTD is however associated with increased paired-pulse facilitation and coefficient of variation of synaptic responses, suggesting that its expression is presynaptic. The chelation of postsynaptic calcium prevents its induction, suggesting that post- to presynaptic (retrograde) signaling is required. We rule out endocannabinoid signaling since the inhibition of type 1 cannabinoid receptors, monoacylglycerol lipase or vanilloid receptor 1, or incubation with anandamide had no detectable effect. The persistent PTD is maximal in pre-adolescent mice, abolished by adrenergic and dopaminergic receptors block, but unaffected by adrenergic and dopaminergic agonists. Our data unveils a novel form of plasticity at parallel fiber synapses: a persistent PTD induced by physiologically relevant input patterns, age-dependent, and strongly modulated by the monoaminergic system. We further provide evidence supporting that the plasticity mechanism involves retrograde signaling and presynaptic diacylglycerol.

The glutamate—glutamine shuttle requires a flux of fixed N from neurons to astrocytes. The suggestion that some or all of this N is ammonium has received support from reports that ammonium (as NH+4) rapidly enters astrocytes. Ammonium might also help control astrocyte energy metabolism by increasing lactate production. If ammonium has these functions, then its effect on brain metabolism must be rapid and reversible. To make a minimal test of this requirement, we have followed the time courses of the changes induced by a 4 min venous infusion of 1 mol/L NH4Cl, 2.5 mmol/kg body weight, in rat. Extracellular [NH+4] in cortex, monitored with ion-selective microelectrodes, reached a peak of approximately 0.7 mmol/L 1.65 mins after the end of the infusion, then recovered. Brain metabolites were monitored non-invasively every 4 mins by 1H magnetic resonance spectroscopy. Lactate peak area during the 3.2 min acquisition starting at the end of the infusion was 1.84 ± 0.24 times baseline (± s.e.m., P = 0.009, n = 9). Lactate increased until 13.2 ± 2.1 mins after the end of the infusion and recovered halfway to baseline by 31.2 mins. Glutamate decreased by at least 7.1% (P = 0.0026). Infusion of NaCl caused no change in lactate signal. Cerebral blood flow, measured by arterial magnetization labeling, more than doubled, suggesting that the lactate increase was not caused by hypoxia. At least three consecutive ammonium-induced increases in lactate signal could be evoked. The results are compatible with an intercellular trafficking/signaling function for ammonium.

Cerebellar Purkinje neurons integrate information transmitted at excitatory synapses formed by granule cells. Although these synapses are considered essential sites for learning, most of them appear not to transmit any detectable electrical information and have been defined as silent. It has been proposed that silent synapses are required to maximize information storage capacity and ensure its reliability, and hence to optimize cerebellar operation. Such optimization is expected to occur once the cerebellar circuitry is in place, during its maturation and the natural and steady improvement of animal agility. We therefore investigated whether the proportion of silent synapses varies over this period, from the third to the sixth postnatal week in mice. Selective expression of a calcium indicator in granule cells enabled quantitative mapping of presynaptic activity, while postsynaptic responses were recorded by patch clamp in acute slices. Through this approach and the assessment of two anatomical features (the distance that separates adjacent planar Purkinje dendritic trees and the synapse density), we determined the average excitatory postsynaptic potential per synapse. Its value was four to eight times smaller than responses from paired recorded detectable connections, consistent with over 70% of synapses being silent. These figures remained remarkably stable across maturation stages. According to the proposed role for silent synapses, our results suggest that information storage capacity and reliability are optimized early during cerebellar maturation. Alternatively, silent synapses may have roles other than adjusting the information storage capacity and reliability.