Mazahir T. Hasan

Two-photon imaging to a depth of 1000 µm microm in living brains by use of a Ti:Al2O3 regenerative amplifier

It is shown that two-photon fluorescence images can be obtained throughout almost the entire gray matter of the mouse neocortex by using optically amplified femtosecond pulses. The achieved imaging depth approaches the theoretical limit set by excitation of out-of-focus fluorescence.

Functional fluorescent Ca2+ indicator proteins in transgenic mice under TET control.

Genetically encoded fluorescent calcium indicator proteins (FCIPs) are promising tools to study calcium dynamics in many activity-dependent molecular and cellular processes. Great hopes—for the measurement of population activity, in particular—have therefore been placed on calcium indicators derived from the green fluorescent protein and their expression in (selected) neuronal populations. Calcium transients can rise within milliseconds, making them suitable as reporters of fast neuronal activity. We here report the production of stable transgenic mouse lines with two different functional calcium indicators, inverse pericam and camgaroo-2, under the control of the tetracycline-inducible promoter. Using a variety of in vitro and in vivo assays, we find that stimuli known to increase intracellular calcium concentration (somatically triggered action potentials (APs) and synaptic and sensory stimulation) can cause substantial and rapid changes in FCIP fluorescence of inverse pericam and camgaroo-2.

IKK mediates ischemia-induced neuronal death

The IκB kinase complex IKK is a central component of the signaling cascade that controls NF-κB–dependent gene transcription. So far, its function in the brain is largely unknown. Here, we show that IKK is activated in a mouse model of stroke. To investigate the function of IKK in brain ischemia we generated mice that contain a targeted deletion of Ikbkb (which encodes IKK2) in mouse neurons and mice that express a dominant inhibitor of IKK in neurons. In both lines, inhibition of IKK activity markedly reduced infarct size. In contrast, constitutive activation of IKK2 enlarged the infarct size. A selective small-molecule inhibitor of IKK mimicked the effect of genetic IKK inhibition in neurons, reducing the infarct volume and cell death in a therapeutic time window of 4.5 h. These data indicate a key function of IKK in ischemic brain damage and suggest a potential role for IKK inhibitors in stroke therapy.

Single-spike detection in vitro and in vivo with a genetic Ca2+ sensor.

Measurement of population activity with single-action-potential, single-neuron resolution is pivotal for understanding information representation and processing in the brain and how the brains responses are altered by experience. Genetically encoded indicators of neuronal activity allow long-term, cell type–specific expression. Fluorescent Ca2+ indicator proteins (FCIPs), a main class of reporters of neural activity, initially suffered, in particular, from an inability to report single action potentials in vivo. Although suboptimal Ca2+-binding dynamics and Ca2+-induced fluorescence changes in FCIPs are important factors, low levels of expression also seem to play a role. Here we report that delivering D3cpv, an improved fluorescent resonance energy transfer–based FCIP, using a recombinant adeno-associated virus results in expression sufficient to detect the Ca2+ transients that accompany single action potentials. In upper-layer cortical neurons, we were able to detect transients associated with single action potentials firing at rates of <1 Hz, with high reliability, from in vivo recordings in living mice.

Optical recording of neuronal activity with a genetically−encoded calcium indicator in anesthetized and freely moving mice

Fluorescent calcium (Ca2+) indicator proteins (FCIPs) are promising tools for functional imaging of cellular activity in living animals. However, they have still not reached their full potential for in vivo imaging of neuronal activity due to limitations in expression levels, dynamic range, and sensitivity for reporting action potentials. Here, we report that viral expression of the ratiometric Ca2+ sensor yellow cameleon 3.60 (YC3.60) in pyramidal neurons of mouse barrel cortex enables in vivo measurement of neuronal activity with high dynamic range and sensitivity across multiple spatial scales. By combining juxtacellular recordings and two-photon imaging in vitro and in vivo, we demonstrate that YC3.60 can resolve single action potential (AP)-evoked Ca2+ transients and reliably reports bursts of APs with negligible saturation. Spontaneous and whisker-evoked Ca2+ transients were detected in individual apical dendrites and somata as well as in local neuronal populations. Moreover, bulk measurements using wide-field imaging or fiber-optics revealed sensory-evoked YC3.60 signals in large areas of the barrel field. Fiber-optic recordings in particular enabled measurements in awake, freely moving mice and revealed complex Ca2+ dynamics, possibly reflecting different behavior-related brain states. Viral expression of YC3.60 – in combination with various optical techniques – thus opens a multitude of opportunities for functional studies of the neural basis of animal behavior, from dendrites to the levels of local and large-scale neuronal populations.

Faithful Expression of Multiple Proteins via 2A-Peptide Self-Processing: A Versatile and Reliable Method for Manipulating Brain Circuits

A method allowing for efficient and quantitative coexpression of multiple heterologous proteins in neurons in vivo would be highly valuable for many applications in neuroscience. To date, different approaches, such as internal ribosomal entry site (IRES) elements ([Douin et al., 2004][1]),

Tetracycline-controlled genetic switches.

Unlike recombinase-mediated gene manipulations, tetracycline (Tet)-controlled genetic switches permit reversible control of gene expression in the mouse. Trancriptional activation can be induced by activators termed tTA (Tet-Off) or rtTA (Tet-On) in the absence and presence of Tet, respectively. The Tet-Off and Tet-On systems are complementary, and the decision to choose one over the other depends on the particular experimental strategy. Both systems were optimized over the years and can now be used to develop mouse models.

Somatic cell genetic and biochemical characterization of cell lines resulting from human genomic DNA transfections of Chinese hamster ovary cell mutants defective in sterol−dependent activation of sterol synthesis and LDL receptor expression

We have isolated several non-leaky mutant Chinese hamster ovary (CHO) cell clones (M4, M19, and M21) requiring cholesterol and unsaturated fatty acid for growth. These mutants belong to the same complementation group as the mutant M1 cells previously reported from this laboratory. M19 cells reverted to lipid prototrophy at very low frequency and were chosen as recipients to perform DNA-mediated gene-transfer experiments using total human genomic DNAs. Biochemical characterization of these transfectant clones indicated that, unlike their parental M19 cells, they were able to exhibit activation of cholesterol biosynthesis and LDL receptor expression in response to sterol removal from the growth medium. RNA blotting analysis indicated that these transfectants were able to increase HMG-CoA synthase gene transcripts in response to sterol removal. From the genomic DNAs of a representative secondary transfectant cell, we cloned a unique human DNA fragment (designated as hλ2) and showed that hλ2 is closely linked with the presumptive human M1 gene.

Topological specificity and hierarchical network of the circadian calcium rhythm in the suprachiasmatic nucleus.

The circadian pacemaker in the hypothalamic suprachiasmatic nucleus (SCN) is a hierarchical multioscillator system in which neuronal networks play crucial roles in expressing coherent rhythms in physiology and behavior. However, our understanding of the neuronal network is still incomplete. Intracellular calcium mediates the input signals, such as phase-resetting stimuli, to the core molecular loop involving clock genes for circadian rhythm generation and the output signals from the loop to various cellular functions, including changes in neurotransmitter release. Using a unique large-scale calcium imaging method with genetically encoded calcium sensors, we visualized intracellular calcium from the entire surface of SCN slice in culture including the regions where autonomous clock gene expression was undetectable. We found circadian calcium rhythms at a single-cell level in the SCN, which were topologically specific with a larger amplitude and more delayed phase in the ventral region than the dorsal. The robustness of the rhythm was reduced but persisted even after blocking the neuronal firing with tetrodotoxin (TTX). Notably, TTX dissociated the circadian calcium rhythms between the dorsal and ventral SCN. In contrast, a blocker of gap junctions, carbenoxolone, had only a minor effect on the calcium rhythms at both the single-cell and network levels. These results reveal the topological specificity of the circadian calcium rhythm in the SCN and the presence of coupled regional pacemakers in the dorsal and ventral regions. Neuronal firings are not necessary for the persistence of the calcium rhythms but indispensable for the hierarchical organization of rhythmicity in the SCN.

Select Overexpression of Homer1a in Dorsal Hippocampus Impairs Spatial Working Memory

Long Homer proteins forge assemblies of signaling components involved in glutamate receptor signaling in postsynaptic excitatory neurons, including those underlying synaptic transmission and plasticity. The short immediate-early gene (IEG) Homer1a can dynamically uncouple these physical associations by functional competition with long Homer isoforms. To examine the consequences of Homer1a-mediated “uncoupling” for synaptic plasticity and behavior, we generated forebrain-specific tetracycline (tet) controlled expression of Venus-tagged Homer1a (H1aV) in mice. We report that sustained overexpression of H1aV impaired spatial working but not reference memory. Most notably, a similar impairment was observed when H1aV expression was restricted to the dorsal hippocampus (HP), which identifies this structure as the principal cortical area for spatial working memory. Interestingly, H1aV overexpression also abolished maintenance of CA3-CA1 long-term potentiation (LTP). These impairments, generated by sustained high Homer1a levels, identify a requirement for long Homer forms in synaptic plasticity and temporal encoding of spatial memory.