Convergent, functionally independent signaling by mu and delta opioid receptors in hippocampal parvalbumin interneurons

Functional interactions between G protein-coupled receptors are poised to enhance neuronal sensitivity to neuromodulators and therapeutic drugs. Mu and Delta opioid receptors (MORs and DORs) can interact when overexpressed in the same cells, but whether co-expression of endogenous MORs and DORs in neurons leads to functional interactions is unclear. Here, in mice, we show that both MORs and DORs inhibit parvalbumin-expressing basket cells (PV-BCs) in hippocampal CA1 through partially occlusive signaling pathways that terminate on somato-dendritic potassium channels and presynaptic calcium channels. Using photoactivatable opioid neuropeptides, we find that DORs dominate the response to enkephalin in terms of both ligand-sensitivity and kinetics, which may be due to relatively low expression levels of MOR. Opioid-activated potassium channels do not show heterologous desensitization, indicating that MORs and DORs signal independently. In a direct test for heteromeric functional interactions, the DOR antagonist TIPP-Psi does not alter the kinetics or potency of either the potassium channel or synaptic responses to photorelease of the MOR agonist DAMGO. Thus, aside from largely redundant and convergent signaling, MORs and DORs do not functionally interact in PV-BCs in a way that impacts somato-dendritic potassium currents or synaptic transmission. These findings imply that crosstalk between MORs and DORs, either in the form of physical interactions or synergistic intracellular signaling, is not a preordained outcome of co-expression in neurons.

Using ephaptic coupling to estimate the synaptic cleft resistivity of the calyx of Held synapse

At synapses, the pre- and postsynaptic cells get so close that currents entering the cleft do not flow exclusively along its conductance, gcl. A prominent example is found in the calyx of Held synapse in the medial nucleus of the trapezoid body (MNTB), where the presynaptic action potential can be recorded in the postsynaptic cell in the form of a prespike. Here, we developed a theoretical framework for ephaptic coupling via the synaptic cleft, and we tested its predictions using the MNTB prespike recorded in voltage-clamp. The shape of the prespike is predicted to resemble either the first or the second derivative of the inverted presynaptic action potential if cleft currents dissipate either mostly capacitively or resistively, respectively. We found that the resistive dissipation scenario provided a better description of the prespike shape. Its size is predicted to scale with the fourth power of the radius of the synapse, explaining why intracellularly recorded prespikes are uncommon in the central nervous system. We show that presynaptic calcium currents also contribute to the prespike shape. This calcium prespike resembled the first derivative of the inverted calcium current, again as predicted by the resistive dissipation scenario. Using this calcium prespike, we obtained an estimate for gcl of ~1 μS. We demonstrate that, for a circular synapse geometry, such as in conventional boutons or the immature calyx of Held, gcl is scale-invariant and only defined by extracellular resistivity, which was ~75 Ωcm, and by cleft height. During development the calyx of Held develops fenestrations. We show that these fenestrations effectively minimize the cleft potentials generated by the adult action potential, which might otherwise interfere with calcium channel opening. We thus provide a quantitative account of the dissipation of currents by the synaptic cleft, which can be readily extrapolated to conventional, bouton-like synapses.

Activation of Neuronal Nicotinic Receptors Inhibits Acetylcholine Release in the Neuromuscular Junction by Increasing Ca2+ Flux through Cav1 Channels

Cholinergic neurotransmission is a key signal pathway in the peripheral nervous system and in several branches of the central nervous system. Despite the fact that it has been studied extensively for a long period of time, some aspects of its regulation still have not yet been established. One is the relationship between the nicotine-induced autoregulation of acetylcholine (ACh) release with changes in the concentration of presynaptic calcium levels. The mouse neuromuscular junction of m. Levator Auris Longus was chosen as the model of the cholinergic synapse. ACh release was assessed by electrophysiological methods. Changes in calcium transients were recorded using a calcium-sensitive dye. Nicotine hydrogen tartrate salt application (10 μM) decreased the amount of evoked ACh release, while the calcium transient increased in the motor nerve terminal. Both of these effects of nicotine were abolished by the neuronal ACh receptor antagonist dihydro-beta-erythroidine and Cav1 blockers, verapamil, and nitrendipine. These data allow us to suggest that neuronal nicotinic ACh receptor activation decreases the number of ACh quanta released by boosting calcium influx through Cav1 channels.

Short-term dynamics of long-range corticocortical synapses revealed by selective optical stimulation

Synapses are continually regulated by their own activity. In the neocortex, direct interactions between cortical areas play a central role in cognitive function, but the dynamic regulation of these long-range corticocortical synapses by activity and their impact on a postsynaptic target neuron is unclear. Here, we use an optogenetic strategy to study the connections between mouse somatosensory and motor cortex. We found that short-term synaptic facilitation was strong in both corticocortical synapses, resulting in far more sustained responses than local intra-cortical and thalamocortical connections. This facilitation was dependent on the presynaptic calcium sensor synaptotagmin-7 and altered by several optogenetic approaches. Recordings revealed that during repetitive activation, the short-term dynamics of corticocortical synapses enhanced the excitability of layer 2/3 pyramidal neurons, increasing the probability of spiking with activity. Furthermore, the properties of the connections linking primary with secondary somatosensory cortex resemble those between somatosensory-motor areas. These results reveal a synaptic mechanism by which corticocortical projections may mediate specific changes in cellular excitability over relatively extended periods.

Activation of neuronal nicotinic receptors inhibits acetylcholine release in the neuromuscular junction by increasing Ca2+ flux through Cav1 channels

Background and Purpose: Cholinergic neurotransmission is a key signal pathway in the peripheral nervous system (PNS) and in several branches of the central nervous system (CNS). Despite the fact that it has been studied extensively for a long period of time, some aspects of its regulation still have not yet been established. One is relationship between nicotine-induced autoregulation of acetylcholine (ACh) release with changes in the concentration of presynaptic calcium levels. Experimental Approach: The mouse neuromuscular junction of m. Levator Auris Longus was chosen as the model of the cholinergic synapse. ACh release was assessed by electrophysiological methods. Changes in the calcium transients were recorded using a calcium-sensitive dye. Functional interaction between nicotinic ACh receptors and calcium channels was investigated pharmacologically using specific agonists and antagonists. Key Results: Nicotine hydrogen tartrate salt (considered as a stable form for potential therapeutic delivery of nicotine) effects on the parameters of ACh release from the nerve ending were analyzed. Nicotine application (10 μM) decrease the amount of evoked ACh release, while calcium transient increase in the motor nerve terminal. Both of these effects of nicotine were abolished by the neuronal ACh receptor antagonist dihydro-beta-erythroidine and Cav1 blockers, verapamil and nitrendipine. Conclusion and Implications: Neuronal nicotinic ACh receptors activation decreases the number of ACh quanta released by boosting calcium influx through Cav1 channels. Understanding of mechanisms of autoregulation of ACh release is important for the searching new approaches treat diseases associated with cholinergic dysfunction.

Presynaptic NMDA receptors facilitate short-term plasticity and BDNF release at hippocampal mossy fiber synapses

Neurotransmitter release is a highly controlled process by which synapses can critically regulate information transfer within neural circuits. While presynaptic receptors –typically activated by neurotransmitters and modulated by neuromodulators– provide a powerful way of fine-tuning synaptic function, their contribution to activity-dependent changes in transmitter release remains poorly understood. Here, we report that presynaptic NMDA receptors (preNMDARs) at mossy fiber boutons in the rodent hippocampus can be activated by physiologically relevant patterns of activity and selectively enhance short-term synaptic plasticity at mossy fiber inputs onto CA3 pyramidal cells and mossy cells, but not onto inhibitory interneurons. Moreover, preNMDARs facilitate brain-derived neurotrophic factor (BDNF) release and contribute to presynaptic calcium rise. Taken together, our results indicate that by increasing presynaptic calcium, preNMDARs fine tune mossy fiber neurotransmission and can control information transfer during dentate granule cell burst activity that normally occur in vivo.

Using ephaptic coupling to estimate the synaptic cleft resistivity of the calyx of Held synapse.

At synapses, the pre- and postsynaptic cell get so close that currents entering the cleft do not flow exclusively along its conductance, gcl. A prominent example is found in the calyx of Held synapse in the medial nucleus of the trapezoid body, where the presynaptic action potential can be recorded in the postsynaptic cell in the form of a prespike. Here, we developed a theoretical framework for ephaptic coupling via the synaptic cleft. We found that the capacitive component of the prespike recorded in voltage clamp is closely approximated by the second time derivative of the presynaptic action potential. Its size scales with the fourth power of the radius of the synapse, explaining why intracellularly recorded prespikes are uncommon in the CNS. We show that presynaptic calcium currents can contribute to the prespike and that their contribution is closely approximated by the scaled first derivative of these currents. We confirmed these predictions in juvenile rat brainstem slices, and used the presynaptic calcium currents to obtain an estimate for gcl of ~1 μS. We demonstrate that for a typical synapse geometry, gcl is scale-invariant and only defined by extracellular resistivity, which was ~75 Ωcm, and by cleft height. During development the calyx of Held develops fenestrations. These fenestrations effectively minimize the cleft potentials generated by the adult action potential, which would otherwise interfere with calcium channel opening. We thus provide a quantitative account of the dissipation of currents by the synaptic cleft, which can be readily extrapolated to conventional, bouton-like synapses.

Convergent, functionally independent signaling by mu and delta opioid receptors in hippocampal parvalbumin interneurons

Functional interactions between G protein-coupled receptors are poised to enhance neuronal sensitivity to neuromodulators and therapeutic drugs. Mu and Delta opioid receptors (MORs and DORs) can interact when overexpressed in the same cells, but whether co-expression of endogenous MORs and DORs in neurons leads to functional interactions is unclear. Here, we show that both MORs and DORs inhibit parvalbumin-expressing basket cells (PV-BCs) in hippocampal CA1 through partially occlusive signaling pathways that terminate on somato-dendritic potassium channels and presynaptic calcium channels. Using photoactivatable opioid neuropeptides, we find that DORs dominate the response to enkephalin in terms of both ligand-sensitivity and kinetics, which may be due to relatively low expression levels of MOR. Opioid-activated potassium channels do not show heterologous desensitization, indicating that MORs and DORs signal independently. In a direct test for heteromeric functional interactions, the DOR antagonist TIPP-Psi does not alter the kinetics or potency of either the potassium channel or synaptic responses to photorelease of the MOR agonist DAMGO. Thus, despite largely redundant and convergent signaling, MORs and DORs do not functionally interact in PV-BCs. These findings imply that crosstalk between MORs and DORs, either in the form of physical interactions or synergistic intracellular signaling, is not a preordained outcome of co-expression in neurons.

Presynaptic α2δ subunits are key organizers of glutamatergic synapses

In nerve cells the genes encoding for α2δ subunits of voltage-gated calcium channels have been linked to synaptic functions and neurological disease. Here we show that α2δ subunits are essential for the formation and organization of glutamatergic synapses. Using a cellular α2δ subunit triple-knockout/knockdown model, we demonstrate a failure in presynaptic differentiation evidenced by defective presynaptic calcium channel clustering and calcium influx, smaller presynaptic active zones, and a strongly reduced accumulation of presynaptic vesicle-associated proteins (synapsin and vGLUT). The presynaptic defect is associated with the downscaling of postsynaptic AMPA receptors and the postsynaptic density. The role of α2δ isoforms as synaptic organizers is highly redundant, as each individual α2δ isoform can rescue presynaptic calcium channel trafficking and expression of synaptic proteins. Moreover, α2δ-2 and α2δ-3 with mutated metal ion-dependent adhesion sites can fully rescue presynaptic synapsin expression but only partially calcium channel trafficking, suggesting that the regulatory role of α2δ subunits is independent from its role as a calcium channel subunit. Our findings influence the current view on excitatory synapse formation. First, our study suggests that postsynaptic differentiation is secondary to presynaptic differentiation. Second, the dependence of presynaptic differentiation on α2δ implicates α2δ subunits as potential nucleation points for the organization of synapses. Finally, our results suggest that α2δ subunits act as transsynaptic organizers of glutamatergic synapses, thereby aligning the synaptic active zone with the postsynaptic density.

Single calcium channel nanodomains drive presynaptic calcium entry at lamprey reticulospinal presynaptic terminals

Synchronous neurotransmission is central to efficient information transfer in neural circuits, requiring precise coupling between action potentials, Ca2+ entry and neurotransmitter release. However, determinations of Ca2+ requirements for release, which may originate from entry through single voltage-gated Ca2+ channels, remain largely unexplored in simple active zone synapses common in the nervous system. Understanding these requirements is key to addressing Ca2+ channel and synaptic dysfunction underlying numerous neurological and neuropsychiatric disorders. Here, we present single channel analysis of evoked active zone Ca2+ entry, using cell-attached patch clamp and lattice light sheet microscopy over active zones at single central lamprey reticulospinal presynaptic terminals. Our findings show a small pool (mean of 23) of Ca2+ channels at each terminal, comprising subtypes N-type (CaV2.2), P/Q-type (CaV2.1) and R-type (CaV2.3), available to gate neurotransmitter release. Significantly, of this pool only 1-6 (mean of 4) channels open upon depolarization. High temporal fidelity lattice light sheet imaging reveals AP-evoked Ca2+ transients exhibiting quantal amplitude variations between action potentials and stochastic variation of precise locations of Ca2+ entry within the active zone. Further, Ca2+ channel numbers at each active zone correlate to the number of presynaptic primed synaptic vesicles. Together, our findings indicate 1:1 association of Ca2+ channels with primed vesicles, suggesting Ca2+ entry via as few as one channel may trigger neurotransmitter release.