A role for the ubiquitin-proteasome system in activity-dependent presynaptic silencing.

Chronic changes in electrical excitability profoundly affect synaptic transmission throughout the lifetime of a neuron. We have previously explored persistent presynaptic silencing, a form of synaptic depression at glutamate synapses produced by ongoing neuronal activity and by strong depolarization. Here we investigate the involvement of the ubiquitin-proteasome system (UPS) in the modulation of presynaptic function. We found that proteasome inhibition prevented the induction of persistent presynaptic silencing. Specifically, application of the proteasome inhibitor MG-132 (carbobenzoxy-L-leucyl-L-leucyl-L-leucinal) prevented decreases in the size of the readily releasable pool of vesicles and in the percentage of active synapses. Presynaptic silencing was accompanied by decreases in levels of the priming proteins Munc13-1 and Rim1. Importantly, overexpression of Rim1alpha prevented the induction of persistent presynaptic silencing. Furthermore, strong depolarization itself increased proteasome enzymatic activity measured in cell lysates. These results suggest that modulation of the UPS by electrical activity contributes to persistent presynaptic silencing by promoting the degradation of key presynaptic proteins.

A specific role for Ca2+-dependent adenylyl cyclases in recovery from adaptive presynaptic silencing.

Glutamate generates fast postsynaptic depolarization throughout the CNS. The positive-feedback nature of glutamate signaling likely necessitates flexible adaptive mechanisms that help prevent runaway excitation. We have previously explored presynaptic adaptive silencing, a form of synaptic plasticity produced by ongoing neuronal activity and by strong depolarization. Unsilencing mechanisms that maintain active synapses and restore normal function after adaptation are also important, but mechanisms underlying such presynaptic reactivation remain unexplored. Here we investigate the involvement of the cAMP pathway in the basal balance between silenced and active synapses, as well as the recovery of baseline function after depolarization-induced presynaptic silencing. Activation of the cAMP pathway activates synapses that are silent at rest, and pharmacological inhibition of cAMP signaling silences basally active synapses. Adenylyl cyclase (AC) 1 and AC8, the major Ca2+-sensitive AC isoforms, are not crucial for the baseline balance between silent and active synapses. In cells from mice doubly deficient in AC1 and AC8, the baseline percentage of active synapses was only modestly reduced compared with wild-type synapses, and forskolin unsilencing was similar in the two genotypes. Nevertheless, after strong presynaptic silencing, recovery of normal function was strongly inhibited in AC1/AC8-deficient synapses. The entire recovery phenotype of the double null was reproduced in AC8-deficient but not AC1-deficient cells. We conclude that, under normal conditions, redundant cyclase activity maintains the balance between presynaptically silent and active synapses, but AC8 plays a particularly important role in rapidly resetting the balance of active to silent synapses after adaptation to strong activity.

Vesicle pool heterogeneity at hippocampal glutamate and GABA synapses.

Glutamate and GABA are the major fast excitatory and inhibitory neurotransmitters, respectively, in the CNS. Although glutamate and GABA have clearly distinct postsynaptic actions, we are just beginning to appreciate that presynaptic differences between glutamatergic and GABAergic neurons may contribute to distinct functions of these transmitter systems. We therefore probed possible differences between the functional synaptic vesicle populations of glutamatergic and GABAergic neurons. We examined superecliptic synaptopHluorin (SpH) fluorescence during 20 Hz electrical stimulation in transfected hippocampal neurons and identified the phenotype of SpH-fluorescent synapses with post hoc immunostaining. With 200 stimuli (10 s), individual glutamate synapses displayed considerably more variability in peak SpH fluorescence than GABA synapses, without a strong difference in the mean SpH fluorescence increase. This spatial heterogeneity could not be accounted for by differences in endocytosis, which was nearly constant over these short time periods across glutamate and GABA synapses. Instead, variability in vesicle exocytosis correlated with variability in total vesicle staining and in measures of the total recycling pool size. Differences were also evident using FM1-43 [N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide] uptake. These data support the idea that the population of glutamate synapses exhibits more heterogeneity in release properties than the population of GABA synapses, possibly correlated with glutamatergic synaptic malleability.

Physiological activity depresses synaptic function through an effect on vesicle priming.

Neurons engage compensatory, homeostatic synaptic changes to maintain their overall firing rate. We examined the induction and expression of a persistent presynaptic adaptation. We explored the effect of mild extracellular potassium elevation to increase hippocampal pyramidal neuron spiking over a physiological range. With several days of mild depolarization, glutamate release adapted, as revealed by an increased mismatch between the number of active, FM1-43-positive, glutamatergic synapses and the total number of synapses defined by vesicular glutamate transporter-1 antibody staining. Surprisingly, the adaptation of glutamate terminals was all-or-none; recycling vesicle pool size at remaining active synapses was not significantly altered by the adaptation. Tetrodotoxin (TTX), but not postsynaptic receptor blockade, reversed depolarization-induced adaptation, and TTX added to normal incubation medium increased the number of active synapses, suggesting that normal spiking activity sustains a steady-state percentage of inactive terminals. Chronic mild depolarization depressed EPSCs and decreased the size of the readily releasable pool of vesicles (RRP). Several hours of 10 Hz electrical stimulation also depressed the RRP size, confirming that spiking alone induces adaptation and that strong stimulation induces more rapid presynaptic adaptation. Despite the importance of RRP alteration to the adaptation, ultrastructural experiments revealed no changes in docked or total synaptic vesicle numbers. Furthermore, alpha-latrotoxin induced vesicle release at adapted synapses, consistent with the idea that adaptation resulted from changes in vesicle priming. These results show that glutamatergic neurotransmission persistently adapts to changes in electrical activity over a wide physiological range.

A clickable neurosteroid photolabel reveals selective Golgi compartmentalization with preferential impact on proximal inhibition.

Anesthetic, GABA-active neurosteroids potently augment GABAA receptor function, leading to important behavioral consequences. Neurosteroids and their synthetic analogues are also models for a wide variety of cell-permeant neuroactive compounds. Cell permeation and compartmentalization raise the possibility that these compounds' actions are influenced by their cellular partitioning, but these contributions are not typically considered experimentally or therapeutically. To examine the interplay between cellular accumulation and pharmacodynamics of neurosteroids, we synthesized a novel chemical biology analogue (bio-active, clickable photolabel) of GABA-active neurosteroids. We discovered that the analogue selectively photo-labels neuronal Golgi in rat hippocampal neurons. The active analogue's selective distribution was distinct from endogenous cholesterol and not completely shared by some non-GABA active, neurosteroid-like analogues. On the other hand, the distribution was not enantioselective and did not require energy, in contrast to other recent precedents from the literature. We demonstrate that the soma-selective accumulation can act as a sink or source for steroid actions at plasma-membrane GABA receptors, altering steady-state and time course of effects at somatic GABAA receptors relative to dendritic receptors. Our results suggest a novel mechanism for compartment-selective drug actions at plasma-membrane receptors.

Interaction between positive allosteric modulators and trapping blockers of the NMDA receptor channel.

BACKGROUND AND PURPOSE: Memantine and ketamine are clinically used, open-channel blockers of NMDA receptors exhibiting remarkable pharmacodynamic similarities despite strikingly different clinical profiles. Although NMDA channel gating constitutes an important difference between memantine and ketamine, it is unclear how positive allosteric modulators (PAMs) might affect the pharmacodynamics of these NMDA blockers. EXPERIMENTAL APPROACH: We used two different PAMs: SGE-201, an analogue of an endogenous oxysterol, 24S-hydroxycholesterol, along with pregnenolone sulphate (PS), to test on memantine and ketamine responses in single cells (oocytes and cultured neurons) and networks (hippocampal slices), using standard electrophysiological techniques. KEY RESULTS: SGE-201 and PS had no effect on steady-state block or voltage dependence of a channel blocker. However, both PAMs increased the actions of memantine and ketamine on phasic excitatory post-synaptic currents, but neither revealed underlying pharmacodynamic differences. SGE-201 accelerated the re-equilibration of blockers during voltage jumps. SGE-201 also unmasked differences among the blockers in neuronal networks - measured either by suppression of activity in multi-electrode arrays or by neuroprotection against a mild excitotoxic insult. Either potentiating NMDA receptors while maintaining the basal activity level or increasing activity/depolarization without potentiating NMDA receptor function is sufficient to expose pharmacodynamic blocker differences in suppressing network function and in neuroprotection. CONCLUSIONS AND IMPLICATIONS: Positive modulation revealed no pharmacodynamic differences between NMDA receptor blockers at a constant voltage, but did expose differences during spontaneous network activity. Endogenous modulator tone of NMDA receptors in different brain regions may underlie differences in the effects of NMDA receptor blockers on behaviour.

Phosphatidylinositol 4,5-bisphosphate depletion fails to affect neurosteroid modulation of GABAA receptor function.

RATIONALE: Neurosteroids and likely other lipid modulators access transmembrane sites on the GABAA receptor (GABAAR) by partitioning into and diffusing through the plasma membrane. Therefore, specific components of the plasma membrane may affect the potency or efficacy of neurosteroid-like modulators. Here, we tested a possible role for phosphatidylinositol 4,5-bisphosphate (PIP2), a phospholipid that governs activity of many channels and transporters, in modulation or function of GABAARs. OBJECTIVES: In these studies, we sought to deplete plasma-membrane PIP2 and probe for a change in the strength of potentiation by submaximal concentrations of the neurosteroid allopregnanolone (3α5αP) and other anesthetics, including propofol, pentobarbital, and ethanol. We also tested for a change in the behavior of negative allosteric modulators pregnenolone sulfate and dipicrylamine. METHODS: We used Xenopus oocytes expressing the ascidian voltage-sensitive phosphatase (Ci-VSP) to deplete PIP2. Voltage pulses to positive membrane potentials were used to deplete PIP2 in Ci-VSP-expressing cells. GABAARs composed of α1ß2γ2L and α4ß2δ subunits were challenged with GABA and 3α5αP or other modulators before and after PIP2 depletion. KV7.1 channels and NMDA receptors (NMDARs) were used as positive controls to verify PIP2 depletion. RESULTS: We found no evidence that PIP2 depletion affected modulation of GABAARs by positive or negative allosteric modulators. By contrast, Ci-VSP-induced PIP2 depletion depressed KV7.1 activation and NMDAR activity. CONCLUSIONS: We conclude that despite a role for PIP2 in modulation of a wide variety of ion channels, PIP2 does not affect modulation of GABAARs by neurosteroids or related compounds.

Presynaptically silent synapses studied with light microscopy.

Synaptic plasticity likely underlies the nervous system's ability to learn and remember and may also represent an adaptability that prevents otherwise damaging insults from becoming neurotoxic. We have been studying a form of presynaptic plasticity that is interesting in part because it is expressed as a digital switching on and off of a presynaptic terminal s ability to release vesicles containing the neurotransmitter glutamate. Here we demonstrate a protocol for visualizing the activity status of presynaptic terminals in dissociated cell cultures prepared from the rodent hippocampus. The method relies on detecting active synapses using staining with a fixable form of the styryl dye FM1-43, commonly used to label synaptic vesicles. This staining profile is compared with immunostaining of the same terminals with an antibody directed against the vesicular glutamate transporter 1 (vGluT-1), a stain designed to label all glutamate synapses regardless of activation status. We find that depolarizing stimuli induce presynaptic silencing. The population of synapses that is silent under baseline conditions can be activated by prolonged electrical silencing or by activation of cAMP signaling pathways.