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 Rim1␣ 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.

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, ␣-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.

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 Ca 2ϩ -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.

Oxysterols have emerged as important biomarkers in disease and as signaling molecules. We recently showed that the oxysterol 24(S)-hydroxycholesterol, the major brain cholesterol metabolite, potently and selectively enhances NMDA receptor function at a site distinct from other modulators. Here we further characterize the pharmacological mechanisms of 24(S)-hydroxycholesterol and its synthetic analogue SGE201. We describe an oxysterol antagonist of this positive allosteric modulation, 25-hydroxycholesterol. We found that 24(S)-hydroxycholesterol and SGE201 primarily increased the efficacy of NMDAR agonists but did not directly gate the channel or increase functional receptor number. Rather than binding to a direct aqueous-accessible site, oxysterols may partition into the plasma membrane to access the NMDAR, likely explaining slow onset and offset kinetics of modulation. Interestingly, oxysterols were ineffective when applied to the cytosolic face of inside-out membrane patches or through a whole-cell pipette solution, suggesting a non-intracellular site. We also found that another natural oxysterol, 25-hydroxycholesterol, although exhibiting slight potentiation on its own, non-competitively and enantioselectively antagonized the effects of 24(S)-hydroxycholesterol analogues. In summary, we suggest two novel allosteric sites on NMDARs that separately modulate channel gating, but together oppose each other.

Voltage-sensitive dyes are important tools for assessing network and single-cell excitability, but an untested premise in most cases is that the dyes do not interfere with the parameters (membrane potential, excitability) that they are designed to measure. We found that popular members of several different families of voltage-sensitive dyes modulate GABAAreceptor with maximum efficacy and potency similar to clinically used GABAAreceptor modulators. Di-4-ANEPPS and DiBAC4(3) potentiated GABA function with micromolar and high nanomolar potency, respectively, and yielded strong maximum effects similar to barbiturates and neurosteroids. Newer blue oxonols had biphasic effects on GABAAreceptor function at nanomolar and micromolar concentrations, with maximum potentiation comparable to that of saturating benzodiazepine effects. ANNINE-6 and ANNINE-6plus had no detectable effect on GABAAreceptor function. Even dyes with no activity on GABAAreceptors at baseline induced photodynamic enhancement of GABAAreceptors. The basal effects of dyes were sufficient to prolong IPSCs and to dampen network activity in multielectrode array recordings. Therefore, the dual effects of voltage-sensitive dyes on GABAergic inhibition require caution in dye use for studies of excitability and network activity.