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

Summary: 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. : Forli et al. developed an all-optical method to image and bidirectionally manipulate brain networks with high spatial resolution and minimal crosstalk in the intact mammalian brain. They validate the method across cell types and layers in the mouse neocortex. Keywords: optogenetics, two-photon excitation, digital holography, patterned illumination, two-photon imagin

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

Benzodiazepines potentiate gamma-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 a and gamma subunit of GABA(A)Rs, preferentially enhancing synaptic receptors largely composed of alpha(1-3, 5), beta 3, and gamma 2 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 alpha 2 subunit-containing GABA(A)Rs occurred within 24 h of flurazepam treatment, whereas GABA(A)Rs incorporating alpha 1 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, flurazepamtreatment 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 alpha 2 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

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

Pharmacologic Blockade of 5-Lipoxygenase Improves the Amyloidotic Phenotype of an Alzheimer's Disease Transgenic Mouse Model: Involvement of γ-Secretase

The 5-lipoxygenase (5-LO) enzyme is widely distributed within the central nervous system. Previous works showed that this protein is up-regulated in Alzheimer's disease (AD) and that its genetic absence results in a reduction of amyloid β (Aβ) levels in Tg2576 mice. In the present study, we examined the effect of 5-LO pharmacological inhibition on the amyloidotic phenotype of these mice. Aβ deposition in the brains of mice receiving zileuton, a selective and specific 5-LO inhibitor, was significantly reduced when compared with control Tg2576 mice receiving vehicle. This reduction was associated with a similar decrease in brain Aβ peptides levels. Zileuton treatment did not induce any change in the steady state levels of amyloid-β precursor protein (APP), BACE1 or ADAM10. By contrast, it resulted in a significant reduction of presenilin 1 (PSEN1, alias PS1), nicastrin (NCSTN) , presenilin enhancer 2 homolog (PSNEN, alias, Pen-2), and anterior pharynx defective 1 (APH-1), the four components of the γ-secretase complex—at the protein and message level. Furthermore, in vitro studies confirmed that zileuton prevents Aβ formation by modulating γ-secretase complex levels without affecting Notch signaling. These data establish a functional role for 5-LO in the pathogenesis of AD-like amyloidosis, whereby it modulates the γ-secretase pathway. They suggest that pharmacological inhibition of 5-LO could provide a novel therapeutic opportunity for AD