Alexander A. Chubykin

Activity-dependent validation of excitatory versus inhibitory synapses by neuroligin-1 versus neuroligin-2

Neuroligins enhance synapse formation in vitro, but surprisingly are not required for the generation of synapses in vivo. We now show that in cultured neurons, neuroligin-1 overexpression increases excitatory, but not inhibitory, synaptic responses, and potentiates synaptic NMDAR/AMPAR ratios. In contrast, neuroligin-2 overexpression increases inhibitory, but not excitatory, synaptic responses. Accordingly, deletion of neuroligin-1 in knockout mice selectively decreases the NMDAR/AMPAR ratio, whereas deletion of neuroligin-2 selectively decreases inhibitory synaptic responses. Strikingly, chronic inhibition of NMDARs or CaM-Kinase II, which signals downstream of NMDARs, suppresses the synapse-boosting activity of neuroligin-1, whereas chronic inhibition of general synaptic activity suppresses the synapse-boosting activity of neuroligin-2. Taken together, these data indicate that neuroligins do not establish, but specify and validate, synapses via an activity-dependent mechanism, with different neuroligins acting on distinct types of synapses. This hypothesis reconciles the overexpression and knockout phenotypes and suggests that neuroligins contribute to the use-dependent formation of neural circuits.

A Splice Code for trans-Synaptic Cell Adhesion Mediated by Binding of Neuroligin 1 to α- and β-Neurexins

Previous studies suggested that postsynaptic neuroligins form a trans-synaptic complex with presynaptic β-neurexins, but not with presynaptic α-neurexins. Unexpectedly, we now find that neuroligins also bind α-neurexins and that α- and β-neurexin binding by neuroligin 1 is regulated by alternative splicing of neuroligin 1 (at splice site B) and of neurexins (at splice site 4). In neuroligin 1, splice site B is a master switch that determines α-neurexin binding but leaves β-neurexin binding largely unaffected, whereas alternative splicing of neurexins modulates neuroligin binding. Moreover, neuroligin 1 splice variants with distinct neurexin binding properties differentially regulate synaptogenesis: neuroligin 1 that binds only β-neurexins potently stimulates synapse formation, whereas neuroligin 1 that binds to both α- and β-neurexins more effectively promotes synapse expansion. These findings suggest that neuroligin binding to α- and β-neurexins mediates trans-synaptic cell adhesion but has distinct effects on synapse formation, indicating that expression of different neuroligin and neurexin isoforms specifies a trans-synaptic signaling code.

Selective capability of SynCAM and neuroligin for functional synapse assembly

Synaptic cell adhesion is central for synapse formation and function. Recently, the synaptic cell adhesion molecules neuroligin 1 (NL1) and SynCAM were shown to induce presynaptic differentiation in cocultured neurons when expressed in a non-neuronal cell. However, it is uncertain how similar the resulting artificial synapses are to regular synapses. Are these molecules isofunctional, or do all neuronal cell adhesion molecules nonspecifically activate synapse formation? To address these questions, we analyzed the properties of artificial synapses induced by NL1 and SynCAM, compared the actions of these molecules with those of other neuronal cell adhesion molecules, and examined the functional effects of NL1 and SynCAM overexpression in neurons. We found that only NL1 and SynCAM specifically induced presynaptic differentiation in cocultured neurons. The induced nerve terminals were capable of both spontaneous and evoked neurotransmitter release, suggesting that a full secretory apparatus was assembled. By all measures, SynCAM- and NL1-induced artificial synapses were identical. Overexpression in neurons demonstrated that only SynCAM, but not NL1, increased synaptic function in immature developing excitatory neurons after 8 d in vitro. Tests of chimeric molecules revealed that the dominant-positive effect of SynCAM on synaptic function in developing neurons was mediated by its intracellular cytoplasmic tail. Interestingly, morphological analysis of neurons overexpressing SynCAM or NL1 showed the opposite of the predictions from electrophysiological results. In this case, only NL1 increased the synapse number, suggesting a role for NL1 in morphological synapse induction. These results suggest that both NL1 and SynCAM act similarly and specifically in artificial synapse induction but that this process does not reflect a shared physiological function of these molecules.

Lovastatin Corrects Excess Protein Synthesis and Prevents Epileptogenesis in a Mouse Model of Fragile X Syndrome

Many neuropsychiatric symptoms of fragile X syndrome (FXS) are believed to be a consequence of altered regulation of protein synthesis at synapses. We discovered that lovastatin, a drug that is widely prescribed for the treatment of high cholesterol, can correct excess hippocampal protein synthesis in the mouse model of FXS and can prevent one of the robust functional consequences of increased protein synthesis in FXS, epileptogenesis. These data suggest that lovastatin is potentially disease modifying and could be a viable prophylactic treatment for epileptogenesis in FXS.

Characterization of the Interaction of a Recombinant Soluble Neuroligin-1 with Neurexin-1β

Neuroligins, proteins of the α/β-hydrolase fold family, are found as postsynaptic transmembrane proteins whose extracellular domain associates with presynaptic partners, proteins of the neurexin family. To characterize the molecular basis of neuroligin interaction with neurexin-β, we expressed five soluble and exportable forms of neuroligin-1 from recombinant DNA sources, by truncating the protein before the transmembrane span near its carboxyl terminus. The extracellular domain of functional neuroligin-1 associates as a dimer when analyzed by sedimentation equilibrium. By surface plasmon resonance, we established that soluble neuroligins-1 bind neurexin-1β, but the homologous α/β-hydrolase fold protein, acetylcholinesterase, failed to associate with the neurexins. Neuroligin-1 has a unique N-linked glycosylation pattern in the neuroligin family, and glycosylation and its processing modify neuroligin activity. Incomplete processing of the protein and enzymatic removal of the oligosaccharides chain or the terminal sialic acids from neuroligin-1 enhance its activity, whereas deglycosylation of neurexin-1β did not alter its association capacity. In particular, the N-linked glycosylation at position 303 appears to be a major determinant in modifying the association with neurexin-1β. We show here that glycosylation processing of neuroligin, in addition to mRNA splicing and gene selection, contributes to the specificity of the neurexin-β/neuroligin-1 association.

Microchip amplifier for in vitro, in vivo, and automated whole cell patch-clamp recording.

Patch clamping is a gold-standard electrophysiology technique that has the temporal resolution and signal-to-noise ratio capable of reporting single ion channel currents, as well as electrical activity of excitable single cells. Despite its usefulness and decades of development, the amplifiers required for patch clamping are expensive and bulky. This has limited the scalability and throughput of patch clamping for single-ion channel and single-cell analyses. In this work, we have developed a custom patch-clamp amplifier microchip that can be fabricated using standard commercial silicon processes capable of performing both voltage- and current-clamp measurements. A key innovation is the use of nonlinear feedback elements in the voltage-clamp amplifier circuit to convert measured currents into logarithmically encoded voltages, thereby eliminating the need for large high-valued resistors, a factor that has limited previous attempts at integration. Benchtop characterization of the chip shows low levels of current noise [1.1 pA root mean square (rms) over 5 kHz] during voltage-clamp measurements and low levels of voltage noise (8.2 μV rms over 10 kHz) during current-clamp measurements. We demonstrate the ability of the chip to perform both current- and voltage-clamp measurement in vitro in HEK293FT cells and cultured neurons. We also demonstrate its ability to perform in vivo recordings as part of a robotic patch-clamping system. The performance of the patch-clamp amplifier microchip compares favorably with much larger commercial instrumentation, enabling benchtop commoditization, miniaturization, and scalable patch-clamp instrumentation.

Contrasting roles for parvalbumin-expressing inhibitory neurons in two forms of adult visual cortical plasticity.

The roles played by cortical inhibitory neurons in experience-dependent plasticity are not well understood. Here we evaluate the participation of parvalbumin-expressing (PV+) GABAergic neurons in two forms of experience-dependent modification of primary visual cortex (V1) in adult mice: ocular dominance (OD) plasticity resulting from monocular deprivation and stimulus-selective response potentiation (SRP) resulting from enriched visual experience. These two forms of plasticity are triggered by different events but lead to a similar increase in visual cortical response. Both also require the NMDA class of glutamate receptor (NMDAR). However, we find that PV+ inhibitory neurons in V1 play a critical role in the expression of SRP and its behavioral correlate of familiarity recognition, but not in the expression of OD plasticity. Furthermore, NMDARs expressed within PV+ cells, reversibly inhibited by the psychotomimetic drug ketamine, play a critical role in SRP, but not in the induction or expression of adult OD plasticity. DOI: http://dx.doi.org/10.7554/eLife.11450.001

A single-chip wireless microelectrode array for neural recording and stimulation

A single-chip ultra-low power wireless microelectrode array (MEA) for neural recording and stimulation is implemented. The device is fabricated in GlobalFoundries 45 nm CMOS SOI technology and post processed without using additional lithography to achieve a thin fully functional and flexible system. The design occupies a volume of 0.008 mm3 and integrates a 2D MEA with 9 active neural field potential channels and a miniaturized antenna for wireless powering and communication. The system consumes 15 μW of power per channel at a sensing rate of 26 kS/s. System measurement and in-vitro tests with live mouse brain cells have been conducted.

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a challenging and low-throughput technique due to its complexity and high reliance on user operation and control. This manuscript demonstrates an image-guided automatic patch clamp system for in vitro whole-cell patch clamp experiments in acute brain slices. Our system implements a computer vision-based algorithm to detect fluorescently labeled cells and to target them for fully automatic patching using a micromanipulator and internal pipette pressure control. The entire process is highly automated, with minimal requirements for human intervention. Real-time experimental information, including electrical resistance and internal pipette pressure, are documented electronically for future analysis and for optimization to different cell types. Although our system is described in the context of acute brain slice recordings, it can also be applied to the automated image-guided patch clamp of dissociated neurons, organotypic slice cultures, and other non-neuronal cell types.

Neurexins and Neuroligins: A Synaptic Code for Neuronal Wiring That Is Implicated in Autism

Neurexins and neuroligins are neuronal cell adhesion molecules (CAMs), which are specifically expressed in the nervous system and particularly in the brain. Presynaptic neurexins interact with postsynaptic neuroligins in a Ca2+-dependent manner and form trans-synaptic adhesion complexes, which are implicated in synaptogenesis. Differential splicing of neurexin and neuroligin transcripts results in a large variety of isoforms. These isoforms differ in their binding affinities and ligand specificities and are differentially distributed in the central nervous system. This may reflect a synaptic code, which along with other neuronal adhesion molecules determines the wiring diagram of neuronal connections in the brain. Mouse genetic studies suggest that neurexins and neuroligins are involved in the stabilization of transient, rather than the formation of de novo synapses. Mutations in neurexin and neuroligin genes, as well as their downstream signaling molecules, have been identified in patients with mental retardation and autism spectrum disorders. Mice with corresponding mouse mutations represent new promising experimental models, which display the typical phenotypes associated with these diseases. The first results obtained from these models suggest that a disruption of the balance between excitatory and inhibitory neurotransmission (E/I) is one of the potential pathophysiological mechanisms for autism and mental retardation.