New biological tools for genetic manipulation of the mouse brain

The site-specific gene expression in the mouse brain is the most crucial issue for genetic studies of brain networks. In our studies, we used different fluorescent proteins (FPs) for monitoring brain anatomy, while for the functional analysis, proteins such as Cre recombinase and the tetracycline-controlled transactivator (tTA) were investigated. From the technical point of view, we attempted both mouse transgenic technology and recombinant adeno-associated virus (rAAV) gene delivery in vivo for transferring functional proteins into specific cell types of the mouse brain. First we analyzed the cell-type specificity of a bacterial artificial chromosome (BAC) transgene. We selected an enhanced green fluorescent protein (EGFP) recombined BAC (from Genesat project) which was supposed to have mitral/tufted cell specific expression in the olfactory bulb. By pronuclear injection of the BAC, different founders were obtained. Out of 41 potential founders, one was mitral cell specific, and two were specific for mitral and tufted cells. Regarding the limitations of cost and time using the mouse transgenic technology, we switched as an alternative approach to the rAAV gene delivery system. For visualizing cells that express the virus-delivered proteins, we applied two strategies: one with tTA/rtTA and its bi-directional responder Ptetbi to express Cre recombinase together with an FP in the specific brain regions. This provided a strong expression level of both Cre and FPs in cultured neurons and in neurons in the brain. For the second strategy, we applied a slightly modified T2A self-cleaving peptide bridge for the quantitative expression of Cre recombinase or tTA/rtTA together with FPs, respectively. By applying the T2A peptide approach, two proteins can be almost equally expressed with one rAAV construct. This opens a new avenue for gene function analysis in the central nervous system. Next we analyzed whether rAAV can be used for the cell-type-specific expression. We selected glia cells, since in the transgenic field, specific promoters are described for proteolipidprotein (PLP) and glial fibrillary acidic protein (GFAP). As second cell population we analyzed promoters for local interneurons, the glutamate decarboxylase 67 (GAD67) and the cholecystokinin (CCK). We investigated a complementation approach which splits Cre recombinase into two parts (N-Cre and C-Cre), each driven by a different promoter. The Cre recombinase is active when N-Cre and C-Cre are expressed in the same cell. With this genetic complementation approach, the infected Cre positive cells could be defined as PLP and GFAP or GAD67 and CCK double positive cells, respectively. Thus, the cell-type-specific expression is achieved via rAAV delivery, and the double positive cells for two different promoters are illustrated with the Cre complementation approach. BAC transgenic technology, rAAV gene in vivo delivery, tTA/rtTA inducible gene activation, 2A peptide cleavage approach and the Cre complementation system, all these newly developed biological tools can be taken advantage of different aspects for different experimental purposes. Moreover, they open a convenient avenue for site-specific and cell-type-specific gene expression in manipulating brain circuits

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

Inherited and de novo SHANK2 variants associated with autism spectrum disorder impair neuronal morphogenesis and physiology

Mutations in the postsynaptic scaffolding gene SHANK2 have recently been identified in individuals with autism spectrum disorder (ASD) and intellectual disability. However, the cellular and physiological consequences of these mutations in neurons remain unknown. We have analyzed the functional impact caused by two inherited and one de novo SHANK2 mutations from ASD individuals (L1008_P1009dup, T1127M, R462X). Although all three variants affect spine volume and have smaller SHANK2 cluster sizes, T1127M additionally fails to rescue spine volume in Shank2 knock-down neurons. R462X is not able to rescue spine volume and dendritic branching and lacks postsynaptic clustering, indicating the most severe dysfunction. To demonstrate that R462X when expressed in mouse can be linked to physiological effects, we analyzed synaptic transmission and behavior. Principal neurons of mice expressing rAAV-transduced SHANK2-R462X present a specific, long-lasting reduction in miniature postsynaptic AMPA receptor currents. This dominant negative effect translates into dose-dependent altered cognitive behavior of SHANK2-R462X-expressing mice, with an impact on the penetrance of ASD

Split-Cre Complementation Indicates Coincident Activity of Different Genes In Vivo

Cre/LoxP recombination is the gold standard for conditional gene regulation in mice in vivo. However, promoters driving the expression of Cre recombinase are often active in a wide range of cell types and therefore unsuited to target more specific subsets of cells. To overcome this limitation, we designed inactive “split-Cre” fragments that regain Cre activity when overlapping co-expression is controlled by two different promoters. Using transgenic mice and virus-mediated expression of split-Cre, we show that efficient reporter gene activation is achieved in vivo. In the brain of transgenic mice, we genetically defined a subgroup of glial progenitor cells in which the Plp1- and the Gfap-promoter are simultaneously active, giving rise to both astrocytes and NG2-positive glia. Similarly, a subset of interneurons was labelled after viral transfection using Gad67- and Cck1 promoters to express split-Cre. Thus, split-Cre mediated genomic recombination constitutes a powerful spatial and temporal coincidence detector for in vivo targeting

Chłop i praca w przemyśle amerykańskim. Pole semantyczne "robota" w listach emigrantów polskich ze Stanów Zjednoczonych z lat 1890-1891 / ETNOGRAFIA POLSKA 1990 t.34

ETNOGRAFIA POLSKA 1990 t.34, s.107-11

Aquaporin-4-independent volume dynamics of astroglial endfeet during cortical spreading depression

Cortical spreading depression (CSD) is a slowly propagating wave of depolarization of gray matter. This phenomenon is believed to underlie the migraine aura and similar waves of depolarization may exacerbate injury in a number of neurological disease states. CSD is characterized by massive ion dyshomeostasis, cell swelling, and multiphasic blood flow changes. Recently, it was shown that CSD is associated with a closure of the paravascular space (PVS), a proposed exit route for brain interstitial fluid and solutes, including excitatory and inflammatory substances that increase in the wake of CSD. The PVS closure was hypothesized to rely on swelling of astrocytic endfeet due to their high expression of aquaporin‐4 (AQP4) water channels. We investigated whether CSD is associated with swelling of endfeet around penetrating arterioles in the cortex of living mice. Endfoot cross‐sectional area was assessed by two‐photon microscopy of mice expressing enhanced green fluorescent protein in astrocytes and related to the degree of arteriolar constriction. In anesthetized mice CSD triggered pronounced endfoot swelling that was short‐lasting and coincided with the initial arteriolar constriction. Mice lacking AQP4 displayed volume changes of similar magnitude. CSD‐induced endfoot swelling and arteriolar constriction also occurred in awake mice, albeit with faster kinetics than in anesthetized mice. We conclude that swelling of astrocytic endfeet is a robust event in CSD. The early onset and magnitude of the endfoot swelling is such that it may significantly delay perivascular drainage of interstitial solutes in neurological conditions where CSD plays a pathophysiological role

Astroglial endfeet exhibit distinct Ca2+ signals during hypoosmotic conditions

Astrocytic endfeet cover the brain surface and form a sheath around the cerebral vasculature. An emerging concept is that endfeet control blood–brain water transport and drainage of interstitial fluid and waste along paravascular pathways. Little is known about the signaling mechanisms that regulate endfoot volume and hence the width of these drainage pathways. Here, we used the genetically encoded fluorescent Ca2+ indicator GCaMP6f to study Ca2+ signaling within astrocytic somata, processes, and endfeet in response to an osmotic challenge known to induce cell swelling. Acute cortical slices were subjected to artificial cerebrospinal fluid with 20% reduction in osmolarity while GCaMP6f fluorescence was imaged with two‐photon microscopy. Ca2+ signals induced by hypoosmotic conditions were observed in all astrocytic compartments except the soma. The Ca2+ response was most prominent in subpial and perivascular endfeet and included spikes with single peaks, plateau‐type elevations, and rapid oscillations, the latter restricted to subpial endfeet. Genetic removal of the type 2 inositol 1,4,5‐triphosphate receptor (IP3R2) severely suppressed the Ca2+ responses in endfeet but failed to affect brain water accumulation in vivo after water intoxication. Furthermore, the increase in endfoot Ca2+ spike rate during hypoosmotic conditions was attenuated in mutant mice lacking the aquaporin‐4 anchoring molecule dystrophin and after blockage of transient receptor potential vanilloid 4 channels. We conclude that the characteristics and underpinning of Ca2+ responses to hypoosmotic stress differ within the astrocytic territory and that IP3R2 is essential for the Ca2+ signals only in subpial and perivascular endfeet

Faithful expression of multiple proteins via 2A-peptide self-processing: a versatile and reliable method for manipulating brain circuits

Editor’s Note: Toolboxes are intended to briefly highlight a new method or a resource of general use in neuroscience or to criticall

Augmentation of Ca2+ signaling in astrocytic endfeet in the latent phase of temporal lobe epilepsy

Astrocytic endfeet are specialized cell compartments whose important homeostatic roles depend on their enrichment of water and ion channels anchored by the dystrophin associated protein complex (DAPC). This protein complex is known to disassemble in patients with mesial temporal lobe epilepsy and in the latent phase of experimental epilepsies. The mechanistic underpinning of this disassembly is an obvious target of future therapies, but remains unresolved. Here we show in a kainate model of temporal lobe epilepsy that astrocytic endfeet display an enhanced stimulation-evoked Ca(2+) signal that outlast the Ca(2+) signal in the cell bodies. While the amplitude of this Ca(2+) signal is reduced following group I/II metabotropic receptor (mGluR) blockade, the duration is sustained. Based on previous studies it has been hypothesized that the molecular disassembly in astrocytic endfeet is caused by dystrophin cleavage mediated by Ca(2+) dependent proteases. Using a newly developed genetically encoded Ca(2+) sensor, the present study bolsters this hypothesis by demonstrating long-lasting, enhanced stimulation-evoked Ca(2+) signals in astrocytic endfeet