A positively tuned voltage indicator for extended electrical recordings in the brain.

Genetically encoded voltage indicators (GEVIs) enable optical recording of electrical signals in the brain, providing subthreshold sensitivity and temporal resolution not possible with calcium indicators. However, one- and two-photon voltage imaging over prolonged periods with the same GEVI has not yet been demonstrated. Here, we report engineering of ASAP family GEVIs to enhance photostability by inversion of the fluorescence-voltage relationship. Two of the resulting GEVIs, ASAP4b and ASAP4e, respond to 100-mV depolarizations with ≥180% fluorescence increases, compared with the 50% fluorescence decrease of the parental ASAP3. With standard microscopy equipment, ASAP4e enables single-trial detection of spikes in mice over the course of minutes. Unlike GEVIs previously used for one-photon voltage recordings, ASAP4b and ASAP4e also perform well under two-photon illumination. By imaging voltage and calcium simultaneously, we show that ASAP4b and ASAP4e can identify place cells and detect voltage spikes with better temporal resolution than commonly used calcium indicators. Thus, ASAP4b and ASAP4e extend the capabilities of voltage imaging to standard one- and two-photon microscopes while improving the duration of voltage recordings.

Kinase pathway inhibition restores PSD95 induction in neurons lacking fragile X mental retardation protein.

Fragile X syndrome (FXS) is the leading monogenic cause of autism and intellectual disability. FXS is caused by loss of expression of fragile X mental retardation protein (FMRP), an RNA-binding protein that regulates translation of numerous mRNA targets, some of which are present at synapses. While protein synthesis deficits have long been postulated as an etiology of FXS, how FMRP loss affects distributions of newly synthesized proteins is unknown. Here we investigated the role of FMRP in regulating expression of new copies of the synaptic protein PSD95 in an in vitro model of synaptic plasticity. We find that local BDNF application promotes persistent accumulation of new PSD95 at stimulated synapses and dendrites of cultured neurons, and that this accumulation is absent in FMRP-deficient mouse neurons. New PSD95 accumulation at sites of BDNF stimulation does not require known mechanisms regulating FMRP-mRNA interactions but instead requires the PI3K-mTORC1-S6K1 pathway. Surprisingly, in FMRP-deficient neurons, BDNF induction of new PSD95 accumulation can be restored by mTORC1-S6K1 blockade, suggesting that constitutively high mTORC1-S6K1 activity occludes PSD95 regulation by BDNF and that alternative pathways exist to mediate induction when mTORC1-S6K1 is inhibited. This study provides direct evidence for deficits in local protein synthesis and accumulation of newly synthesized protein in response to local stimulation in FXS, and supports mTORC1-S6K1 pathway inhibition as a potential therapeutic approach for FXS.

Homeostatic competition between phasic and tonic inhibition.

The GABAA receptors are the major inhibitory receptors in the brain and are localized at both synaptic and extrasynaptic membranes. Synaptic GABAA receptors mediate phasic inhibition, whereas extrasynaptic GABAA receptors mediate tonic inhibition. Both phasic and tonic inhibitions regulate neuronal activity, but whether they regulate each other is not very clear. Here, we investigated the functional interaction between synaptic and extrasynaptic GABAA receptors through various molecular manipulations. Overexpression of extrasynaptic α6ß3δ-GABAA receptors in mouse hippocampal pyramidal neurons significantly increased tonic currents. Surprisingly, the increase of tonic inhibition was accompanied by a dramatic reduction of the phasic inhibition, suggesting a possible homeostatic regulation of the total inhibition. Overexpressing the α6 subunit alone induced an up-regulation of δ subunit expression and suppressed phasic inhibition similar to overexpressing the α6ß3δ subunits. Interestingly, blocking all GABAA receptors after overexpressing α6ß3δ receptors could not restore the synaptic GABAergic transmission, suggesting that receptor activation is not required for the homeostatic interplay. Furthermore, insertion of a gephyrin-binding-site (GBS) into the α6 and δ subunits recruited α6(GBS)ß3δ(GBS) receptors to postsynaptic sites but failed to rescue synaptic GABAergic transmission. Thus, it is not the positional effect of extrasynaptic α6ß3δ receptors that causes the down-regulation of phasic inhibition. Overexpressing α5ß3γ2 subunits similarly reduced synaptic GABAergic transmission. We propose a working model that both synaptic and extrasynaptic GABAA receptors may compete for limited receptor slots on the plasma membrane to maintain a homeostatic range of the total inhibition.

A fast and responsive voltage indicator with enhanced sensitivity for unitary synaptic events.

A remaining challenge for genetically encoded voltage indicators (GEVIs) is the reliable detection of excitatory postsynaptic potentials (EPSPs). Here, we developed ASAP5 as a GEVI with enhanced activation kinetics and responsivity near resting membrane potentials for improved detection of both spiking and subthreshold activity. ASAP5 reported action potentials (APs) in vivo with higher signal-to-noise ratios than previous GEVIs and successfully detected graded and subthreshold responses to sensory stimuli in single two-photon trials. In cultured rat or human neurons, somatic ASAP5 reported synaptic events propagating centripetally and could detect ∼1-mV EPSPs. By imaging spontaneous EPSPs throughout dendrites, we found that EPSP amplitudes decay exponentially during propagation and that amplitude at the initiation site generally increases with distance from the soma. These results extend the applications of voltage imaging to the quantal response domain, including in human neurons, opening up the possibility of high-throughput, high-content characterization of neuronal dysfunction in disease.

Aberrant mPFC GABAergic synaptic transmission and fear behavior in neuroligin-2 R215H knock-in mice.

Aberrant medial prefrontal cortex (mPFC) activity is associated with neuropsychiatric disorders such as schizophrenia, but the precise role of mPFC GABAergic neurotransmission in the pathogenesis of schizophrenia remains not well understood. Neuroligin-2 (Nlgn 2) is a postsynaptic cell-adhesion protein playing an important role in inhibitory synapse formation and function. Mutations of Nlgn 2 have been reported to be associated with schizophrenia. Using a Nlgn 2 Arg215 â†’ His215 mutation knock-in (NL2 R215H KI) mouse model of schizophrenia, we show here that inhibitory synaptic transmission, such as miniature and evoked inhibitory postsynaptic currents (mIPSCs, eIPSCs), is significantly reduced in the mPFC of NL2 R215H KI mice. The levels of inhibition-related proteins, including parvalbumin (PV), the γ2 subunit of the GABAA receptor, and a vesicular GABA transporter vGAT, are also reduced significantly in NL2 R215H KI mPFC. The reduction of GABAergic inhibition disrupts the excitation/inhibition (E/I) ratio in mPFC, and results in the subsequent abnormal gamma oscillation in the mPFC of NL2 R215H KI mice. Behavioral evaluation suggests that GABAergic deficits contribute, at least in part, to alterations in fear response, which requires balanced E/I ratio of mPFC neurons. These results suggest a pivotal role of Nlgn 2 in maintaining E/I balance in the mPFC and in the maintenance of normal behaviors governed by the mPFC.

Spectroscopic analysis of tylosin adsorption on extracellular DNA reveals its interaction mechanism.

Extracellular DNA (eDNA), which is commonly detected in aquatic and terrestrial environments, may be involved in gene transfer, increases in genetic diversity, and evolution. However, it has been reported that some small organic molecules or heavy metal ions can influence the transformation of DNA and even destroy its structure. We previously found that tylosin (TYL, a kind of antibiotic) is adsorbed onto salmon sperm DNA in a mixed solution. However, it is not clear whether this antibiotic affects the structure of DNA, and the mechanism of their interaction needs to be clarified. Therefore, we investigated the adsorption of TYL on different concentrations of salmon sperm DNA using agarose gel electrophoresis, ultraviolet-visible (UV-vis) spectroscopy, fluorescence spectroscopy, and surface enhanced Raman spectroscopy (SERS) to elucidate the interaction mechanism between TYL and DNA. The results showed that the adsorption of TYL decreased with increased concentrations of DNA. The electrophoresis band of pristine DNA was at 5000 bps. The brightness of the DNA band decreased with the TYL concentration and their incubation time. As the concentration of TYL increased, the fluorescence absorption intensity of DNA decreased significantly. Redshift and hyperchromicity were observed in the UV-vis adsorption spectrum with the presence of TYL in DNA solution, and they weakened as the DNA concentration increased. The Raman spectrum intensities of characteristic peaks in the mixed solution were weaker than that of pure TYL solution, and the peak intensity increased with increasing DNA concentration. Even a part of TYL characteristic peaks disappeared in the mixed solution. These results indicated that the pyran and macrolide of TYL might intercalate into the base pair plane of DNA. In addition, electrostatic attraction between TYL and DNA and interactions among TYL molecules may also play a role in the interaction mechanism. However, the double helix structure of DNA was not subject to the interaction of TYL.

GABAergic deficits and schizophrenia-like behaviors in a mouse model carrying patient-derived neuroligin-2 R215H mutation.

Schizophrenia (SCZ) is a severe mental disorder characterized by delusion, hallucination, and cognitive deficits. We have previously identified from schizophrenia patients a loss-of-function mutation Arg215→His215 (R215H) of neuroligin 2 (NLGN2) gene, which encodes a cell adhesion molecule critical for GABAergic synapse formation and function. Here, we generated a novel transgenic mouse line with neuroligin-2 (NL2) R215H mutation. The single point mutation caused a significant loss of NL2 protein in vivo, reduced GABAergic transmission, and impaired hippocampal activation. Importantly, R215H KI mice displayed anxiety-like behavior, impaired pre-pulse inhibition (PPI), cognition deficits and abnormal stress responses, recapitulating several key aspects of schizophrenia-like behaviors. Our results demonstrate a significant impact of a single point mutation NL2 R215H on brain functions, providing a novel animal model for the study of schizophrenia and neuropsychiatric disorders.

Regulation of epileptiform activity by two distinct subtypes of extrasynaptic GABAA receptors.

BACKGROUND: GABAergic deficit is one of the major mechanisms underlying epileptic seizures. Previous studies have mainly focused on alterations of synaptic GABAergic inhibition during epileptogenesis. Recent work suggested that tonic inhibition may also play a role in regulating epileptogenesis, but the underlying mechanism is not well understood. RESULTS: We employed molecular and pharmacological tools to investigate the role of tonic inhibition during epileptogenesis both in vitro and in vivo. We overexpressed two distinct subtypes of extrasynaptic GABAA receptors, α5ß3γ2 and α6ß3δ receptors, in cultured hippocampal neurons. We demonstrated that overexpression of both α5ß3γ2 and α6ß3δ receptors enhanced tonic inhibition and reduced epileptiform activity in vitro. We then showed that injection of THIP (5 µM), a selective agonist for extrasynaptic GABAA receptors at low concentration, into rat brain also suppressed epileptiform burst activity and behavioral seizures in vivo. Mechanistically, we discovered that low concentration of THIP had no effect on GABAergic synaptic transmission and did not affect the basal level of action potentials, but significantly inhibited high frequency neuronal activity induced by epileptogenic agents. CONCLUSIONS: Our studies suggest that extrasynaptic GABAA receptors play an important role in controlling hyperexcitatory activity, such as that during epileptogenesis, but a less prominent role in modulating a low level of basal activity. We propose that tonic inhibition may play a greater role under pathological conditions than in physiological conditions in terms of modulating neural network activity.