Pierre F. Apostolides

Rapid, activity-independent turnover of vesicular transmitter content at a mixed glycine/GABA synapse.

The release of neurotransmitter via the fusion of transmitter-filled, presynaptic vesicles is the primary means by which neurons relay information. However, little is known regarding the molecular mechanisms that supply neurotransmitter destined for vesicle filling, the endogenous transmitter concentrations inside presynaptic nerve terminals, or the dynamics of vesicle refilling after exocytosis. We addressed these issues by recording from synaptically coupled pairs of glycine/GABA coreleasing interneurons (cartwheel cells) of the mouse dorsal cochlear nucleus. We find that the plasma membrane transporter GlyT2 and the intracellular enzyme glutamate decarboxylase supply the majority of glycine and GABA, respectively. Pharmacological block of GlyT2 or glutamate decarboxylase led to rapid and complete rundown of transmission, whereas increasing GABA synthesis via intracellular glutamate uncaging dramatically potentiated GABA release within 1 min. These effects were surprisingly independent of exocytosis, indicating that prefilled vesicles re-equilibrated upon acute changes in cytosolic transmitter. Titration of cytosolic transmitter with postsynaptic responses indicated that endogenous, nonvesicular glycine/GABA levels in nerve terminals are 5–7 mm, and that vesicular transport mechanisms are not saturated under basal conditions. Thus, cytosolic transmitter levels dynamically set the strength of inhibitory synapses in a release-independent manner.

Regulation of interneuron excitability by gap junction coupling with principal cells

Electrical coupling of inhibitory interneurons can synchronize activity across multiple neurons, thereby enhancing the reliability of inhibition onto principal cell targets. It is unclear whether downstream activity in principal cells controls the excitability of such inhibitory networks. Using paired patch-clamp recordings, we show that excitatory projection neurons (fusiform cells) and inhibitory stellate interneurons of the dorsal cochlear nucleus form an electrically coupled network through gap junctions containing connexin36 (Cxc36, also called Gjd2). Remarkably, stellate cells were more strongly coupled to fusiform cells than to other stellate cells. This heterologous coupling was functionally asymmetric, biasing electrical transmission from the principal cell to the interneuron. Optogenetically activated populations of fusiform cells reliably enhanced interneuron excitability and generated GABAergic inhibition onto the postsynaptic targets of stellate cells, whereas deep afterhyperpolarizations following fusiform cell spike trains potently inhibited stellate cells over several hundred milliseconds. Thus, the excitability of an interneuron network is bidirectionally controlled by distinct epochs of activity in principal cells.

Deconstructing behavioral neuropharmacology with cellular specificity

A tailored look at behavioral pharmacology It is important to understand how animal behavior is mediated by molecular, cellular, and circuit components of the brain. However, it has been difficult to link the activity of specific molecules in defined cells to behavioral roles. Shields et al. developed an approach to deconstruct behavioral neuropharmacology with cellular specificity. The technique, termed DART (drugs acutely restricted by tethering), uses enzymatic capture to restrict standard drugs to the surface of genetically specified cells without prior modification of the native pharmacological target. The method provides cell-type specificity, endogenous-protein specificity, acute onset, and utility in behaving animals. This enables the activity of specific molecules in defined circuit elements to be causally linked to behavior. Science, this issue p. eaaj2161 A technique, DART (drugs acutely restricted by tethering), that rapidly localizes drugs to the surface of defined neurons is developed. INTRODUCTION Animal behavior is mediated by molecular, cellular, and circuit components of the brain. However, because many proteins are broadly expressed, it has been difficult to link the activity of specific proteins in defined cells to behavioral roles. This challenge has particular relevance to neuropsychiatric disorders, which have largely been understood in relation to clinically effective drugs that act on known molecular targets. Such knowledge has been difficult to extend to circuit-level insight because cell type specificity has not been possible with traditional pharmacology. RATIONALE Here, we combined the speed and molecular specificity of pharmacology with the cell type specificity of genetic tools. DART (drugs acutely restricted by tethering) is a technique that uses a bacterial enzyme called HaloTag to capture and tether drugs to the surface of defined cells. A key feature is that the method does not require prior modification or overexpression of the native pharmacological target because HaloTag is expressed as a separate protein. Drug capture proceeds rapidly over seconds to minutes, producing a factor of ~100 enrichment of the drug at the surface of HaloTag-expressing cells. The method provides a unique feature set: (i) Cell type specificity arises from expression of HaloTag under control of a cell type–specific promoter; (ii) molecular specificity is inherited from the drug that is tethered; and (iii) acute onset upon delivery of the DART ligand is similar to that of traditional pharmacology, thus averting compensatory phenomena. RESULTS We first developed a DART that antagonizes the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR), a broadly expressed postsynaptic glutamate receptor. We validated the speed, cell type specificity, and molecular specificity of the method in cultured neuronal assays, in coronal slices of mouse dorsal striatum, and in behaving mice. We then applied the technique to a mouse model of Parkinson’s disease (PD). In PD, AMPARs are subject to dysregulated long-term potentiation (LTP) and long-term depression (LTD) in distinct cell types of the striatum, a brain region critical for movement. AMPAR antagonists have been studied extensively in animal models of PD and in human clinical trials for the disorder. However, it has been difficult to link AMPAR activity in defined cells to motor deficits. We found that motor deficits were causally attributed to AMPARs in indirect spiny projection neurons (iSPNs) and to excess phasic firing of tonically active interneurons (TANs) of the striatum. Together, iSPNs and TANs (i.e., D2 cells) drove akinesia, whereas movement execution deficits reflected the ratio of AMPARs in D2 versus D1 cells. Finally, we designed a muscarinic antagonist DART in one iteration, demonstrating applicability of the method to diverse targets. CONCLUSION Neuropsychiatric disorders have been examined with acute manipulations featuring either circuit or molecular specificity. DART combines these features, enabling interrogation of specific proteins in defined cells. The approach may provide a platform whereby the mechanism of action for widely prescribed drugs can be examined with cellular specificity in animal models of several disorders. Such studies could inform new translational strategies by advancing nonobvious drug combinations, or by providing a road map for the design of bivalent therapeutics based on the “message-address” concept of Schwyzer. Deconstructing behavioral neuropharmacology. Drugs that manipulate specific molecules in the brain (e.g., green but not purple postsynaptic receptor) have shaped our understanding of neuropathologies. The DART technique uses HaloTag (red) to capture and spatially restrict drugs to the surface of genetically defined cells. Behavioral effects of drugs can thus be deconstructed into the individual and combinatorial contributions produced by defined cell types. Illustration: Julia Kuhl Behavior has molecular, cellular, and circuit determinants. However, because many proteins are broadly expressed, their acute manipulation within defined cells has been difficult. Here, we combined the speed and molecular specificity of pharmacology with the cell type specificity of genetic tools. DART (drugs acutely restricted by tethering) is a technique that rapidly localizes drugs to the surface of defined cells, without prior modification of the native target. We first developed an AMPAR antagonist DART, with validation in cultured neuronal assays, in slices of mouse dorsal striatum, and in behaving mice. In parkinsonian animals, motor deficits were causally attributed to AMPARs in indirect spiny projection neurons (iSPNs) and to excess phasic firing of tonically active interneurons (TANs). Together, iSPNs and TANs (i.e., D2 cells) drove akinesia, whereas movement execution deficits reflected the ratio of AMPARs in D2 versus D1 cells. Finally, we designed a muscarinic antagonist DART in one iteration, demonstrating applicability of the method to diverse targets.

Inhibitory Gating of Input Comparison in the CA1 Microcircuit

Spatial and temporal features of synaptic inputs engage integration mechanisms on multiple scales, including presynaptic release sites, postsynaptic dendrites, and networks of inhibitory interneurons. Here we investigate how these mechanisms cooperate to filter synaptic input in hippocampal area CA1. Dendritic recordings from CA1 pyramidal neurons reveal that proximal inputs from CA3 as well as distal inputs from entorhinal cortex layer III (ECIII) sum sublinearly or linearly at low firing rates due to feedforward inhibition, but sum supralinearly at high firing rates due to synaptic facilitation, producing a high-pass filter. However, during ECIII and CA3 input comparison, supralinear dendritic integration is dynamically balanced by feedforward and feedback inhibition, resulting in suppression of dendritic complex spiking. We find that a particular subpopulation of CA1 interneurons expressing neuropeptide Y (NPY) contributes prominently to this dynamic filter by integrating both ECIII and CA3 input pathways and potently inhibiting CA1 pyramidal neuron dendrites.

Dendritic calcium channels and their activation by synaptic signals in auditory coincidence detector neurons.

The avian nucleus laminaris (NL) encodes the azimuthal location of low-frequency sound sources by detecting the coincidence of binaural signals. Accurate coincidence detection requires precise developmental regulation of the lengths of the fine, bitufted dendrites that characterize neurons in NL. Such regulation has been suggested to be driven by local, synaptically mediated, dendritic signals such as Ca(2+). We examined Ca(2+) signaling through patch clamp and ion imaging experiments in slices containing nucleus laminaris from embryonic chicks. Voltage-clamp recordings of neurons located in the NL showed the presence of large Ca(2+) currents of two types, a low voltage-activated, fast inactivating Ni(2+) sensitive channel resembling mammalian T-type channels, and a high voltage-activated, slowly inactivating Cd(2+) sensitive channel. Two-photon Ca(2+) imaging showed that both channel types were concentrated on dendrites, even at their distal tips. Single action potentials triggered synaptically or by somatic current injection immediately elevated Ca(2+) throughout the entire cell. Ca(2+) signals triggered by subthreshold synaptic activity were highly localized. Thus when electrical activity is suprathreshold, Ca(2+) channels ensure that Ca(2+) rises in all dendrites, even those that are synaptically inactive.

Axonal Filtering Allows Reliable Output during Dendritic Plateau-Driven Complex Spiking in CA1 Neurons.

In CA1 pyramidal neurons, correlated inputs trigger dendritic plateau potentials that drive neuronal plasticity and firing rate modulation. Given the strong electrotonic coupling between soma and axon, the >25 mV depolarization associated with the plateau could propagate through the axon to influence action potential initiation, propagation, and neurotransmitter release. We examined this issue in brain slices, awake mice, and a computational model. Despite profoundly inactivating somatic and proximal axon Na(+) channels, plateaus evoked action potentials that recovered to full amplitude in the distal axon (>150 μm) and triggered neurotransmitter release similar to regular spiking. This effect was due to strong attenuation of plateau depolarizations by axonal K(+) channels, allowing full axon repolarization and Na(+) channel deinactivation. High-pass filtering of dendritic plateaus by axonal K(+) channels should thus enable accurate transmission of gain-modulated firing rates, allowing neuronal firing to be efficiently read out by downstream regions as a simple rate code.

Control of Interneuron Firing by Subthreshold Synaptic Potentials in Principal Cells of the Dorsal Cochlear Nucleus

Voltage-gated ion channels amplify, compartmentalize, and normalize synaptic signals received by neurons. We show that voltage-gated channels activated during subthreshold glutamatergic synaptic potentials in a principal cell generate an excitatory→inhibitory synaptic sequence that excites electrically coupled interneurons. In fusiform cells of the dorsal cochlear nucleus, excitatory synapses activate a TTX-sensitive Na(+) conductance and deactivate a resting Ih conductance, leading to a striking reshaping of the synaptic potential. Subthreshold voltage changes resulting from activation/deactivation of these channels subsequently propagate through gap junctions, causing slow excitation followed by inhibition in GABAergic stellate interneurons. Gap-junction-mediated transmission of voltage-gated signals accounts for the majority of glutamatergic signaling to interneurons, such that subthreshold synaptic events from a single principal cell are sufficient to drive spikes in coupled interneurons. Thus, the interaction between a principal cells synaptic and voltage-gated channels may determine the spike activity of networks without firing a single action potential.

Chemical synaptic transmission onto superficial stellate cells of the mouse dorsal cochlear nucleus.

The dorsal cochlear nucleus (DCN) is a cerebellum-like auditory brain stem region whose functions include sound localization and multisensory integration. Although previous in vivo studies have shown that glycinergic and GABAergic inhibition regulate the activity of several DCN cell types in response to sensory stimuli, data regarding the synaptic inputs onto DCN inhibitory interneurons remain limited. Using acute DCN slices from mice, we examined the properties of excitatory and inhibitory synapses onto the superficial stellate cell, a poorly understood cell type that provides inhibition to DCN output neurons (fusiform cells) as well as to local inhibitory interneurons (cartwheel cells). Excitatory synapses onto stellate cells activated both NMDA receptors and fast-gating, Ca(2+)-permeable AMPA receptors. Inhibition onto superficial stellate cells was mediated by glycine and GABAA receptors with different temporal kinetics. Paired recordings revealed that superficial stellate cells make reciprocal synapses and autapses, with a connection probability of ∼ 18-20%. Unexpectedly, superficial stellate cells co-released both glycine and GABA, suggesting that co-transmission may play a role in fine-tuning the duration of inhibitory transmission.

Superficial stellate cells of the dorsal cochlear nucleus

The dorsal cochlear nucleus (DCN) integrates auditory and multisensory signals at the earliest levels of auditory processing. Proposed roles for this region include sound localization in the vertical plane, head orientation to sounds of interest, and suppression of sensitivity to expected sounds. Auditory and non-auditory information streams to the DCN are refined by a remarkably complex array of inhibitory and excitatory interneurons, and the role of each cell type is gaining increasing attention. One inhibitory neuron that has been poorly appreciated to date is the superficial stellate cell. Here we review previous studies and describe new results that reveal the surprisingly rich interactions that this tiny interneuron has with its neighbors, interactions which enable it to respond to both multisensory and auditory afferents.