Extended field-of-view ultrathin microendoscopes for high-resolution two-photon imaging with minimal invasiveness.

Imaging neuronal activity with high and homogeneous spatial resolution across the field-of-view (FOV) and limited invasiveness in deep brain regions is fundamental for the progress of neuroscience, yet is a major technical challenge. We achieved this goal by correcting optical aberrations in gradient index lens-based ultrathin (≤500 µm) microendoscopes using aspheric microlenses generated through 3D-microprinting. Corrected microendoscopes had extended FOV (eFOV) with homogeneous spatial resolution for two-photon fluorescence imaging and required no modification of the optical set-up. Synthetic calcium imaging data showed that, compared to uncorrected endoscopes, eFOV-microendoscopes led to improved signal-to-noise ratio and more precise evaluation of correlated neuronal activity. We experimentally validated these predictions in awake head-fixed mice. Moreover, using eFOV-microendoscopes we demonstrated cell-specific encoding of behavioral state-dependent information in distributed functional subnetworks in a primary somatosensory thalamic nucleus. eFOV-microendoscopes are, therefore, small-cross-section ready-to-use tools for deep two-photon functional imaging with unprecedentedly high and homogeneous spatial resolution.

Two-Photon Bidirectional Control and Imaging of Neuronal Excitability with High Spatial Resolution In Vivo.

Sensory information is encoded within the brain in distributed spatiotemporal patterns of neuronal activity. Understanding how these patterns influence behavior requires a method to measure and to bidirectionally perturb with high spatial resolution the activity of the multiple neuronal cell types engaged in sensory processing. Here, we combined two-photon holography to stimulate neurons expressing blue light-sensitive opsins (ChR2 and GtACR2) with two-photon imaging of the red-shifted indicator jRCaMP1a in the mouse neocortex in vivo. We demonstrate efficient control of neural excitability across cell types and layers with holographic stimulation and improved spatial resolution by opsin somatic targeting. Moreover, we performed simultaneous two-photon imaging of jRCaMP1a and bidirectional two-photon manipulation of cellular activity with negligible effect of the imaging beam on opsin excitation. This all-optical approach represents a powerful tool to causally dissect how activity patterns in specified ensembles of neurons determine brain function and animal behavior.

Benzodiazepine treatment induces subtype-specific changes in GABA(A) receptor trafficking and decreases synaptic inhibition.

Benzodiazepines potentiate γ-aminobutyric acid type A receptor (GABA(A)R) activity and are widely prescribed to treat anxiety, insomnia, and seizure disorders. Unfortunately, clinical use of benzodiazepines (BZs) is severely limited by tolerance. The mechanisms leading to BZ tolerance are unknown. BZs bind at the interface between an α and γ subunit of GABA(A)Rs, preferentially enhancing synaptic receptors largely composed of α(1-3, 5), ß3, and γ2 subunits. Using confocal imaging and patch-clamp approaches, we show that treatment with the BZ flurazepam decreases GABA(A)R surface levels and the efficacy of neuronal inhibition in hippocampal neurons. A dramatic decrease in surface and total levels of α2 subunit-containing GABA(A)Rs occurred within 24 h of flurazepam treatment, whereas GABA(A)Rs incorporating α1 subunits showed little alteration. The GABA(A)R surface depletion could be reversed by treatment with the BZ antagonist Ro 15-1788. Coincident with decreased GABA(A)R surface levels, flurazepam treatment reduced miniature inhibitory postsynaptic current amplitude, which returned to control levels with acute Ro 15-1788 treatment. GABA(A)R endocytosis and insertion rates were unchanged by flurazepam treatment. Treatment with leupeptin restored flurazepam lowered receptor surface levels, strongly suggesting that flurazepam increases lysosomal degradation of GABA(A)Rs. Together, these data suggest that flurazepam exposure enhances degradation of α2 subunit-containing GABA(A)Rs after their removal from the plasma membrane, leading to a reduction in inhibitory synapse size and number along with a decrease in the efficacy of synaptic inhibition. These reported subtype-specific changes in GABA(A)R trafficking provide significant mechanistic insight into the initial neuroadaptive responses occurring with BZ treatment.

Intracellular chloride concentration influences the GABAA receptor subunit composition.

GABA(A) receptors (GABA(A)Rs) exist as different subtype variants showing unique functional properties and defined spatio-temporal expression pattern. The molecular mechanisms underlying the developmental expression of different GABA(A)R are largely unknown. The intracellular concentration of chloride ([Cl(-)](i)), the main ion permeating through GABA(A)Rs, also undergoes considerable changes during maturation, being higher at early neuronal stages with respect to adult neurons. Here we investigate the possibility that [Cl(-)](i) could modulate the sequential expression of specific GABA(A)Rs subtypes in primary cerebellar neurons. We show that [Cl(-)](i) regulates the expression of α3-1 and δ-containing GABA(A) receptors, responsible for phasic and tonic inhibition, respectively. Our findings highlight the role of [Cl(-)](i) in tuning the strength of GABAergic responses by acting as an intracellular messenger.

Influence of GABAAR monoliganded states on GABAergic responses.

To reach the open state, the GABA(A) receptor (GABA(A)R) is assumed to bind two agonist molecules. Although it is currently believed that GABA(A)R could also operate in the monoliganded state, the gating properties of singly bound GABA(A)R are poorly understood and their physiological role is still obscure. In the present study, we characterize for the first time the gating properties of singly bound GABA(A)Rs by using a mutagenesis approach and we propose that monoliganded GABA(A)R contribute in shaping synaptic responses. At saturating GABA concentrations, currents mediated by recombinant GABA(A)Rs with a single functional binding site display slow onset, fast deactivation kinetics, and slow rate of desensitization-resensitization. GABA(A)Rs with two binding sites activated by brief pulses of subsaturating GABA concentrations (in the range of the GABA concentration profile in the synaptic cleft) could also mediate fast deactivating currents, displaying deactivation kinetics similar to those mediated by GABA(A)Rs with a single functional binding site. Model simulations of receptors activated by realistic synaptic GABA waves revealed that a considerable proportion of GABA(A) receptors open in the monoliganded state during synaptic transmission, therefore contributing in shaping IPSCs.

Endogenous nonneuronal modulators of synaptic transmission control cortical slow oscillations in vivo.

Gliotransmission, the release of molecules from astrocytes, regulates neuronal excitability and synaptic transmission in situ. Whether this process affects neuronal network activity in vivo is not known. Using a combination of astrocyte-specific molecular genetics, with in vivo electrophysiology and pharmacology, we determined that gliotransmission modulates cortical slow oscillations, a rhythm characterizing nonrapid eye movement sleep. Inhibition of gliotransmission by the expression of a dominant negative SNARE domain in astrocytes affected cortical slow oscillations, reducing the duration of neuronal depolarizations and causing prolonged hyperpolarizations. These network effects result from the astrocytic modulation of intracortical synaptic transmission at two sites: a hypofunction of postsynaptic NMDA receptors, and by reducing extracellular adenosine, a loss of tonic A1 receptor-mediated inhibition. These results demonstrate that rhythmic brain activity is generated by the coordinated action of the neuronal and glial networks.

GABA(A) receptor membrane trafficking regulates spine maturity.

GABA(A) receptors (GABA(A)Rs), the principal sites of synaptic inhibition in the brain, are dynamic entities on the neuronal cell surface, but the role their membrane trafficking plays in shaping neuronal activity remains obscure. Here, we examined this by using mutant receptor beta3 subunits (beta3S408/9A), which have reduced binding to the clathrin adaptor protein-2, a critical regulator of GABA(A)R endocytosis. Neurons expressing beta3S408/9A subunits exhibited increases in the number and size of inhibitory synapses, together with enhanced inhibitory synaptic transmission due to reduced GABA(A)R endocytosis. Furthermore, neurons expressing beta3S408/9A subunits had deficits in the number of mature spines and reduced accumulation of postsynaptic density protein-95 at excitatory synapses. This deficit in spine maturity was reversed by pharmacological blockade of GABA(A)Rs. Therefore, regulating the efficacy of synaptic inhibition by modulating GABA(A)R membrane trafficking may play a critical role in regulating spine maturity with significant implications for synaptic plasticity together with behavior.

A role for 12/15 lipoxygenase in the amyloid beta precursor protein metabolism.

12/15 Lipoxygenase (12/15LO) protein levels and activity are increased in pathologically affected regions of Alzheimer's disease (AD) brains, compared with controls. Its metabolic products are elevated in cerebrospinal fluid of patients with AD and individuals with mild cognitive impairment, suggesting that this enzyme may be involved early in AD pathogenesis. Herein, we investigate the effect of pharmacologic inhibition of 12/15LO on the amyloid beta precursor protein (APP) metabolism. To this end, we used CHO and N2A cells stably expressing human APP with the Swedish mutant, and two structurally distinct and selective 12/15LO inhibitors, PD146176 and CDC. Our results demonstrated that both drugs dose-dependently reduced Abeta formation without affecting total APP levels. Interestingly, in the same cells we observed a significant reduction in secreted (s)APPbeta and beta-secretase (BACE), but not sAPPalpha and ADAM10 protein levels. Together, these data show for the first time that this enzymatic pathway influences Abeta formation whereby modulating the BACE proteolytic cascade. We conclude that specific pharmacologic inhibition of 12/15LO could represent a novel therapeutic target for treating or preventing AD pathology in humans.