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Within the cerebellar cortex, mossy fibers (MFs) excite granule cells (GCs) that excite Purkinje cells (PCs), which provide outputs to the deep cerebellar nuclei (DCNs). It is well established that PC disruption produces motor deficits such as ataxia. This could arise from either decreases in ongoing PC-DCN inhibition, increases in the variability of PC firing, or disruption of the flow of MF-evoked signals. Remarkably, it is not known whether GCs are essential for normal motor function. Here we address this issue by selectively eliminating calcium channels that mediate transmission (Ca(V)2.1, Ca(V)2.2, and Ca(V)2.3) in a combinatorial manner. We observe profound motor deficits but only when all Ca(V)2 channels are eliminated. In these mice, the baseline rate and variability of PC firing are unaltered, and locomotion-dependent increases in PC firing are eliminated. We conclude that GCs are indispensable for normal motor performance and that disruption of MF-induced signals impairs motor performance.

Synaptotagmin 7 (Syt7) is a multifunctional calcium sensor expressed throughout the body. Its high calcium affinity makes it well suited to act in processes triggered by modest calcium increases within cells. In synaptic transmission, Syt7 has been shown to mediate asynchronous neurotransmitter release, facilitation, and vesicle replenishment. In this review we provide an update on recent developments, and the newly emerging roles of Syt7 in frequency invariant synaptic transmission and in suppressing spontaneous release. Additionally, we discuss Syt7's regulation of membrane fusion in non-neuronal cells, and its involvement in disease. How such diversity of functions is regulated remains an open question. We discuss several potential factors including temperature, presynaptic calcium signals, the localization of Syt7, and its interaction with other Syt isoforms.

The output of the cerebellar cortex is conveyed to the deep cerebellar nuclei (DCN) by Purkinje cells (PCs). Here we characterize the properties of the PC-DCN synapse in juvenile and adult mice and find that prolonged high-frequency stimulation leads to steady-state responses that become increasingly frequency-independent within the physiological firing range of PCs in older animals, resulting in a linear relationship between charge transfer and activation frequency. We use a low affinity antagonist to show that GABAA receptor saturation occurs at this synapse, but it does not underlie frequency invariant transmission. We propose that PC-DCN synapses have two components of release: one prominent early in trains, and another specialized to maintain transmission during prolonged activation. Short-term facilitation offsets partial vesicle depletion to produce frequency-independent transmission.

Purkinje cells (PCs) provide the sole output from the cerebellar cortex. Although PCs are well characterized on many levels, surprisingly little is known about their axon collaterals and their target neurons within the cerebellar cortex. It has been proposed that PC collaterals transiently control circuit assembly in early development, but it is thought that PC-to-PC connections are subsequently pruned. Here, we find that all PCs have collaterals in young, juvenile, and adult mice. Collaterals are restricted to the parasagittal plane, and most synapses are located in close proximity to PCs. Using optogenetics and electrophysiology, we find that in juveniles and adults, PCs make synapses onto other PCs, molecular layer interneurons, and Lugaro cells, but not onto Golgi cells. These findings establish that PC output can feed back and regulate numerous circuit elements within the cerebellar cortex and is well suited to contribute to processing in parasagittal zones.

SUMMARY Granule cells (GCs) of the cerebellar input layer express high-affinity δ GABAA subunit-containing GABAA receptors (δGABAARs) that respond to ambient GABA levels and context-dependent neuromodulators like steroids. We find that GC-specific deletion of δGABAA (cerebellar [cb] δ knockout [KO]) decreases tonic inhibition, makes GCs hyperexcitable, and in turn, leads to differential activation of cb output regions as well as many cortical and subcortical brain areas involved in cognition, anxiety-like behaviors, and the stress response. Cb δ KO mice display deficits in many behaviors, but motor function is normal. Strikingly, δGABAA deletion alters maternal behavior as well as spontaneous, stress-related, and social behaviors specifically in females. Our findings establish that δGABAARs enable the cerebellum to control diverse behaviors not previously associated with the cerebellum in a sex-dependent manner. These insights may contribute to a better understanding of the mechanisms that underlie behavioral abnormalities in psychiatric and neurodevelopmental disorders that display a gender bias., Graphical Abstract, In Brief Rudolph et al. show that deletion of the neuromodulator and hormone-sensitive δGABAA receptor subunit from cerebellar granule cells results in anxiety-like behaviors and female-specific deficits in social behavior and maternal care. δGABAA deletion is associated with hyperexcitability of the cerebellar input layer and altered activation of many stress-related brain regions.

Enceladus has a young, tectonically active south polar region, which is erupting material from a prominent set of fractures called Tiger Stripes. No comparable activity is observed at the north pole, which is heavily cratered with limited tectonism. Given the many lines of evidence supporting a global ocean under Enceladus' icy shell, the reason for the dichotomy in geologic activity is unclear. We model the formation of the Tiger Stripes as tidally-driven fractures and examine the magnitudes of tidal stresses with different ice shell structures in order to explore whether and how tidal stress might explain Enceladus' distribution of tectonic activity. We find that eccentricity-driven tidal stresses would produce fractures of nearly identical orientations to the observed Tiger Stripe Fractures and that a 10-km difference in ice shell thickness between the north and south poles can result in substantially different tidal stress magnitudes, providing a natural explanation for the hemispheric dichotomy in tectonic activity on Enceladus. Finally, we synthesize these results with Enceladus' global geologic record to offer insight into the evolution of this enigmatic moon.

Clinical trials have provided guidance in developing triggers for transfusing in the hemodynamically stable patient. These studies have identified that improved outcomes can be obtained in the massively transfused patient when platelets and fresh frozen plasma are transfused with packed red blood cells. Studies that characterize the complications of transfusions, such as transfusion-related acute lung injury and poor cancer-related outcomes, are discussed. Emerging data that characterize the risk factors associated with transfusion-related acute lung injury and suggest metastasis and local recurrence occur at a higher rate in the transfused patient are discussed. Hematologic disorders commonly encountered by surgeons are discussed.

Pluto and its largest satellite, Charon, currently orbit in a mutually synchronous state; both bodies continuously show the same face to one another. This orbital configuration is a natural end-state for bodies that have undergone tidal dissipation. In order to achieve this state, both bodies would have experienced tidal heating and stress, with the extent of tidal activity controlled by the orbital evolution of Pluto and Charon and by the interior structure and rheology of each body. As the secondary, Charon would have experienced a larger tidal response than Pluto, which may have manifested as observable tectonism. Unfortunately, there are few constraints on the interiors of Pluto and Charon. In addition, the pathway by which Charon came to occupy its present orbital state is uncertain. If Charon's orbit experienced a high-eccentricity phase, as suggested by some orbital evolution models, tidal effects would have likely been more significant. Therefore, we determine the conditions under which Charon could have experienced tidally-driven geologic activity and the extent to which upcoming New Horizons spacecraft observations could be used to constrain Charon's internal structure and orbital evolution. Using plausible interior structure models that include an ocean layer, we find that tidally-driven tensile fractures would likely have formed on Charon if its eccentricity were on the order of 0.01, especially if Charon were orbiting closer to Pluto than at present. Such fractures could display a variety of azimuths near the equator and near the poles, with the range of azimuths in a given region dependent on longitude; east-west-trending fractures should dominate at mid-latitudes. The fracture patterns we predict indicate that Charon's surface geology could provide constraints on the thickness and viscosity of Charon's ice shell at the time of fracture formation.

Rat models have emerged as a common tool to study neuroinflammation to intracortical microelectrodes. While a number of studies have attempted to understand the factors resulting in neuroinflammation using rat models, a complete understanding of key mechanistic pathways remains elusive. Transgenic mouse models, however, could facilitate a deeper understanding of mechanistic pathways due to an ease of genetic alteration. Therefore, the goal of the present study is to compare neuroinflammation following microelectrode implantation s between the rat and the mouse model. Our study suggests that subtle differences in the classic neuroinflammatory markers exist between the animal models at both two and sixteen weeks post implantation. Most notably, neuronal densities surrounding microelectrodes were significantly lower in the rat model at two weeks, while similar densities were observed between the animal models at sixteen weeks. Physiological differences between the species and slight alterations in surgical methods are likely key contributors to the observed differences. Moving forward, we propose that differences in the time course of neuroinflammation between the animal models should be considered when trying to understand and prevent intracortical microelectrode failure.

SUMMARY Activation of CaV2.1 voltage-gated calcium channels is facilitated by preceding calcium entry. Such self-modulatory facilitation is thought to contribute to synaptic facilitation. Using knockin mice with mutated CaV2.1 channels that do not facilitate (Ca IM-AA mice), we surprisingly found that, under conditions of physiological calcium and near-physiological temperatures, synaptic facilitation at hippocampal CA3 to CA1 synapses was not attenuated in Ca IM-AA mice and facilitation was paradoxically more prominent at two cerebellar synapses. Enhanced facilitation at these synapses is consistent with a decrease in initial calcium entry, suggested by an action-potential-evoked CaV2.1 current reduction in Purkinje cells from Ca IM-AA mice. In wild-type mice, CaV2.1 facilitation during high-frequency action potential trains was very small. Thus, for the synapses studied, facilitation of calcium entry through CaV2.1 channels makes surprisingly little contribution to synaptic facilitation under physiological conditions. Instead, CaV2.1 facilitation offsets CaV2.1 inactivation to produce remarkably stable calcium influx during high-frequency activation., Graphical Abstract, In Brief Weyrer et al. use Ca IM-AA mice in which CaV2.1 calcium channel facilitation is eliminated to study synaptic facilitation at hippocampal and cerebellar synapses. Under conditions of physiological temperature, external calcium, and presynaptic waveforms, facilitation of CaV2.1 channels is small and does not contribute to synaptic facilitation at these synapses.

Delayed maturation and age-biased dispersal work in concert under past selection to maximize individual fitness and contribute to population viability; however, these life history attributes can become dysfunctional in a dynamic, anthropogenic landscape when important population demographics cannot redistribute in response to novel landscape change. We used long-term monitoring data to estimate age at maturity, potential longevity, survival, and fidelity for the declining eastern diamondback rattlesnake (EDB), Crotalus adamanteus. We used radio telemetry data and known-fate models to examine adult survival, and we combined mark-recapture and radio telemetry data to examine survival and fidelity using a combined recapture/recovery model. Monthly adult survival was higher during the active season (April–November; 99.5%) as compared to the inactive season (December–March; 96.3%), despite a higher probability of detecting human-caused mortalities during the active season. Rattlesnakes matured in 7.1 years and potential longevity exceeded 20 years. Fidelity estimates indicated mature EDBs had a low probability of dispersing from the study area, while younger, sexually immature individuals were more likely to emigrate. The combination of a slow life history and an ontogenetic shift in emigration suggests EDB life history limits the species’ ability to respond to landscape change, shedding further light on EDB imperilment. Management efforts will benefit from activities that maintain high adult survival. Furthermore, EDB fidelity should be considered in conservation plans, particularly in light of the species’ longevity, as these characteristics suggest that mature EDBs may not readily redistribute at the landscape scale in response to habitat degradation.

The nucleus of the thorium-229 isotope possesses a first excited nuclear state ( 229m Th) at an exceptionally low energy of 7.8±0.5 eV above the nuclear ground state ( 229g Th), as determined by earlier indirect measurements. This is the only nuclear excited state known that is within the range of optical spectroscopy. This paper reports progress toward detecting the 229m Th state directly by luminescence spectroscopy in the vacuum ultraviolet spectral region. The estimated natural linewidth of the 229g Th↔ 229m Th isomer transition of 2 π ×0.1 to 2 π ×10 mHz is expected to broaden to ∼10 kHz for 229 Th 4+ doped into a suitable crystal. The factors governing the choice of crystal system and the substantial challenges in acquiring a sufficiently large quantity of 229 Th are discussed. We show that the 229g Th↔ 229m Th transition energy can be identified to within 0.1 nm by luminescence excitation and luminescence spectroscopy using the Advanced Light Source (ALS) at Lawrence Berkeley National Laboratory. This would open the door for subsequent laser-based measurements of the isomer transition and future applications of 229 Th in nuclear clocks. We also show that 233 U-doped materials should produce an intrinsic, continuous, and sufficiently high rate of 229m Th→ 229g Th luminescence and could be a useful aid in the initial direct search of the isomer transition.

Here we provide evidence that revises the inhibitory circuit diagram of the cerebellar cortex. It was previously thought that Golgi cells, interneurons that are the sole source of inhibition onto granule cells, were exclusively coupled via gap junctions. Moreover, Golgi cells were believed to receive GABAergic inhibition from molecular layer interneurons (MLIs). Here we challenge these views by optogenetically activating the cerebellar circuitry to determine the timing and pharmacology of inhibition onto Golgi cells, and by performing paired recordings to directly assess synaptic connectivity. In contrast with current thought, we find that Golgi cells, not MLIs, make inhibitory GABAergic synapses onto other Golgi cells. As a result, MLI feedback does not regulate the Golgi cell network, and Golgi cells are inhibited approximately two milliseconds before Purkinje cells following a mossy fiber input. Hence, Golgi cells and Purkinje cells receive unique sources of inhibition, and can differentially process shared granule cell inputs.

Lingual and labial frenulectomy are commonly performed as an outpatient procedure, either in an office setting or under general anesthesia. Frenulectomy is generally regarded by both otolaryngologists and dentists as a straightforward and low-risk procedure with limited evidence-based indications and similarly few contraindications. We describe two cases of hypovolemic shock occurring after outpatient frenulectomy requiring emergent interventions of cardiopulmonary resuscitation and blood transfusion. These rare, but life-threatening outcomes warrant recognition as potential complications for the presumed benign labial and lingual frenulectomy. We additionally briefly review indications for upper labial and lingual frenulectomy.

Synapses exhibit several forms of short-term plasticity that play a multitude of computational roles. Short-term depression suppresses neurotransmitter release for hundreds of milliseconds to tens of seconds; facilitation and post-tetanic potentiation lead to synaptic enhancement lasting hundreds of milliseconds to minutes. Recent advances have provided insight into the mechanisms underlying these forms of plasticity. Vesicle depletion, as well as inactivation of both release sites and calcium channels, contribute to synaptic depression. Mechanisms of short-term enhancement include calcium channel facilitation, local depletion of calcium buffers, increases in the probability of release downstream of calcium influx, altered vesicle pool properties, and increases in quantal size. Moreover, there is a growing appreciation of the heterogeneity of vesicles and release sites and how they can contribute to use-dependent plasticity.

SUMMARY Despite a prevalence exceeding 1%, mechanisms underlying autism spectrum disorders (ASDs) are poorly understood, and targeted therapies and guiding parameters are urgently needed. We recently demonstrated that cerebellar dysfunction is sufficient to generate autistic-like behaviors in a mouse model of tuberous sclerosis complex (TSC). Here, using the mechanistic target of rapamycin (mTOR)-specific inhibitor rapamycin, we define distinct sensitive periods for treatment of autistic-like behaviors with sensitive periods extending into adulthood for social behaviors. We identify cellular and electro-physiological parameters that may contribute to behavioral rescue, with rescue of Purkinje cell survival and excitability corresponding to social behavioral rescue. In addition, using anatomic and diffusion-based MRI, we identify structural changes in cerebellar domains implicated in ASD that correlate with sensitive periods of specific autism-like behaviors. These findings thus not only define treatment parameters into adulthood, but also support a mechanistic basis for the targeted rescue of autism-related behaviors., In Brief A mechanistic understanding of and establishment of time windows for effective therapy (sensitive periods) for autism-related behaviors remain unknown. Tsai et al. delineate specific time windows for treatment of specific autism-relevant behaviors and evaluate underlying cellular, electrophysiological, and anatomic mechanisms for these sensitive periods., Graphical Abstract

SummaryThroughout the brain, multiple interneuron types influence distinct aspects of synaptic processing. Interneuron diversity can thereby promote differential firing from neurons receiving common excitation. In contrast, Golgi cells are the sole interneurons regulating granule cell spiking evoked by mossy fibers, thereby gating inputs to the cerebellar cortex. Here, we examine how this single interneuron class modifies activity in its targets. We find that GABAA-mediated transmission at unitary Golgi cell → granule cell synapses consists of varying contributions of fast synaptic currents and sustained inhibition. Fast IPSCs depress and slow IPSCs gradually build during high-frequency Golgi cell activity. Consequently, fast and slow inhibition differentially influence granule cell spike timing during persistent mossy fiber input. Furthermore, slow inhibition reduces the gain of the mossy fiber → granule cell input-output curve, while fast inhibition increases the threshold. Thus, a lack of interneuron diversity need not prevent flexible inhibitory control of synaptic processing.

SummaryTools for suppressing synaptic transmission gain power when able to target highly selective neuron subtypes, thereby sharpening attainable links between neuron type, behavior, and disease; and when able to silence most any neuron subtype, thereby offering broad applicability. Here, we present such a tool, RC::PFtox, that harnesses breadth in scope along with high cell-type selection via combinatorial gene expression to deliver tetanus toxin light chain (tox), an inhibitor of vesicular neurotransmission. When applied in mice, we observed cell-type-specific disruption of vesicle exocytosis accompanied by loss of excitatory postsynaptic currents and commensurately perturbed behaviors. Among various test populations, we applied RC::PFtox to silence serotonergic neurons, en masse or a subset defined combinatorially. Of the behavioral phenotypes observed upon en masse serotonergic silencing, only one mapped to the combinatorially defined subset. These findings provide evidence for separability by genetic lineage of serotonin-modulated behaviors; collectively, these findings demonstrate broad utility of RC::PFtox for dissecting neuron functions.

SummaryThroughout the brain, postsynaptic neurons release substances from their cell bodies and dendrites that regulate the strength of the synapses they receive. Diverse chemical messengers have been implicated in retrograde signaling from postsynaptic neurons to presynaptic boutons. Here, we provide an overview of the signaling systems that lead to rapid changes in synaptic strength. We consider the capabilities, specializations, and physiological roles of each type of signaling system.

SummarySynapses throughout the brain are modified through associative mechanisms in which one input provides an instructive signal for changes in the strength of a second coactivated input. In cerebellar Purkinje cells, climbing fiber synapses provide an instructive signal for plasticity at parallel fiber synapses. Here, we show that noradrenaline activates α2-adrenergic receptors to control short-term and long-term associative plasticity of parallel fiber synapses. This regulation of plasticity does not reflect a conventional direct modulation of the postsynaptic Purkinje cell or presynaptic parallel fibers. Instead, noradrenaline reduces associative plasticity by selectively decreasing the probability of release at the climbing fiber synapse, which in turn decreases climbing fiber-evoked dendritic calcium signals. These findings raise the possibility that targeted presynaptic modulation of instructive synapses could provide a general mechanism for dynamic context-dependent modulation of associative plasticity.

SummaryInhibitory interneurons in the dorsal lateral geniculate nucleus (dLGN) process visual information by precisely controlling spike timing and by refining the receptive fields of thalamocortical (TC) neurons. Previous studies indicate that dLGN interneurons inhibit TC neurons by releasing GABA from both axons and dendrites. However, the mechanisms controlling GABA release are poorly understood. Here, using simultaneous whole-cell recordings from interneurons and TC neurons and two-photon calcium imaging, we find that synchronous activation of multiple retinal ganglion cells (RGCs) triggers sodium spikes that propagate throughout interneuron axons and dendrites, and calcium spikes that invade dendrites but not axons. These distinct modes of interneuron firing can trigger both a rapid and a sustained component of inhibition onto TC neurons. Our studies suggest that active conductances make LGN interneurons flexible circuit-elements that can shift their spatial and temporal properties of GABA release in response to coincident activation of functionally related subsets of RGCs.

Long-term depression (LTD) of the granule cell to Purkinje cell synapse is thought to contribute to motor learning. According to the Marr/Albus/Ito model, sensory inputs drive granule cells to fire, thereby exciting Purkinje cells and influencing motor output. Inappropriate motor output causes neurons in the inferior olive to fire and activate Purkinje cells via the powerful climbing fiber (CF) synapse. CF activity is an error signal and the association of CF and granule cell parallel fiber (PF) activity results in LTD at coactivated PF synapses. Here we examine the timing dependence of LTD by using an induction protocol consisting of a single CF activation paired with a PF burst, with the relative timing of CF and PF activation systematically varied. LTD was most prominent when PF activation occurred before CF activation. A plot of LTD magnitude as a function of PF and CF timing was well approximated by a fit in which LTD peaked for PF activity approximately 80 ms before CF activation and the half width was approximately 300 ms. This indicates that the timing dependence of LTD is well suited to allow a CF to depress preceding PF inputs that generated inappropriate motor outputs. We also find that LTD induction and endocannabinoid release have a similar dependence on PF and CF timing. This suggests that the properties of endocannabinoid release may underlie the timing dependence of some forms of motor learning.

SummaryThe long-term depression (LTD) of parallel fiber (PF) synapses onto Purkinje cells plays a central role in motor learning. Endocannabinoid release and LTD induction both depend upon activation of the metabotropic glutamate receptor mGluR1, require postsynaptic calcium increases, are synapse specific, and have a similar dependence on the associative activation of PF and climbing fiber synapses. These similarities suggest that endocannabinoid release could account for many features of cerebellar LTD. Here we show that LTD induction is blocked by a cannabinoid receptor (CB1R) antagonist, by inhibiting the synthesis of the endocannabinoid 2-arachidonyl glycerol (2-AG), and is absent in mice lacking the CB1R. Although CB1Rs are prominently expressed presynaptically at PF synapses, LTD is expressed postsynaptically. In contrast, a previously described transient form of inhibition mediated by endocannabinoids is expressed presynaptically. This indicates that Purkinje cells release 2-AG that activates CB1Rs to both transiently inhibit release and induce a postsynaptic form of LTD.

Chronically ill rural women must manage complex illness without easy access to health care resources including support and health information. The Women to Women project is a technology-based program with an overarching aim to assist rural women in the day-to-day management of their illnesses. An important aspect of the Women to Women program is teaching the women how to use the Internet to meet their support and informational needs. The purposes of this article are to examine changes in 1) the level of computer skills, 2) degree of comfort in using the computer, and 3) knowledge of Internet functions for the participants in the Women to Women computer-based intervention. Results of the initial analysis of data from 63 women (intervention group n = 29, control group n = 34) indicate that women participating in the intervention reported greater computer skills and computer comfort and greater knowledge of specific aspects of Internet use than women in the control group. These findings were further strengthened considering that intervention and control group differentials were sustained 8 months after the end of the women’s participation in the computer intervention. With the attainment of computer and Internet skills, it is expected that these rural women will have a sustained ability to access quality Internet information that will allow them to better manage and adapt to their chronic illnesses.

SummaryLocal interneurons provide feed-forward inhibition from retinal ganglion cells (RGCs) to thalamocortical (TC) neurons, but questions remain regarding the timing, magnitude, and functions of this inhibition. Here, we identify two types of inhibition that are suited to play distinctive roles. We recorded excitatory and inhibitory postsynaptic currents (EPSCs/IPSCs) in TC neurons in mouse brain slices and activated individual RGC inputs. In 34% of TC neurons, we identified EPSCs and IPSCs with identical thresholds that were tightly correlated, indicating activation by the same RGC. Such “locked” IPSCs occurred 1 ms after EPSC onset. The remaining neurons had only “nonlocked” inhibition, in which EPSCs and IPSCs had different thresholds, indicating activation by different RGCs. Nonlocked inhibition may refine receptive fields within the LGN by providing surround inhibition. In contrast, dynamic-clamp recordings suggest that locked inhibition improves the precision of synaptically evoked responses in individual TC neurons by eliminating secondary spikes.

The larynx is one of the most important structures in the upper aerodigestive tract. Functional impairment of the larynx is a bur-den to patients who suffer from laryngeal carcinoma, the second most common cancer of the head and neck region after the oral cavity.

Presynaptic calcium influx is mediated by a variety of different calcium channel subtypes with distinct pharmacological and biophysical properties. In this issue of Neuron , Dietrich et al. show that although Ca V 2.3 calcium channels do not contribute to fast transmitter release at hippocampal mossy fiber synapses, they play a specialized role in induction of multiple presynaptic forms of synaptic plasticity.

Synapses that reliably activate their postsynaptic targets typically release neurotransmitter with high probability, are not very sensitive to changes in calcium entry, and depress. We have determined the mechanisms that give rise to these characteristic features at the climbing fiber to Purkinje cell synapse. We find that saturation of presynaptic calcium entry, of presynaptic release, and of postsynaptic receptors combine to produce a postsynaptic response that is near maximal. Postsynaptic receptor saturation also accelerates recovery from depression, in part by accentuating a rapid calcium-dependent recovery phase. Thus, postsynaptic receptor saturation interacts with presynaptic mechanisms to produce highly reliable synapses that can effectively drive their targets even during sustained activation.

Recent studies suggest that endocannabinoids act as retrograde messengers at many synapses in the central nervous system. Activation of phospholipases, either through calcium-mediated or receptor-mediated signaling, leads to the formation and release of endocannabinoids. These lipophilic signaling molecules diffuse to nearby presynaptic terminals where they bind to specific G-protein-coupled receptors and inhibit neurotransmitter release for tens of seconds. Thus, an important physiological role of endocannabinoids may be to provide a mechanism by which neurons can rapidly regulate the strength of their synaptic inputs.

Fluorometric calcium measurements have revealed presynaptic residual calcium (Ca res ) to be an important regulator of synaptic strength. However, in the mammalian brain, it has not been possible to monitor Ca res in fibers that project from one brain region to another. Here, we label neuronal projections by injecting dextran-conjugated calcium indicators into brain nuclei in vivo. Currently available dextran conjugates distort Ca res due to their high affinity for calcium. Therefore, we synthesized a low-affinity indicator, fluo-4 dextran, that can more accurately measure the amplitude and time course of Ca res . We then demonstrate the utility of fluo-4 dextran by measuring Ca res at climbing fiber presynaptic terminals. This method promises to facilitate the study of many synapses in the mammalian CNS, both in brain slices and in vivo.