Two-photon calcium imaging provides an optical readout of neuronal activity in populations of neurons with subcellular resolution. However, conventional two-photon imaging systems are limited in their field of view to ~1 mm2, precluding the visualization of multiple cortical areas simultaneously. Here, we demonstrate a two-photon microscope with an expanded field of view (>9.5 mm2) for rapidly reconfigurable simultaneous scanning of widely separated populations of neurons. We custom designed and assembled an optimized scan engine, objective, and two independently positionable, temporally multiplexed excitation pathways. We used this new microscope to measure activity correlations between two cortical visual areas in mice during visual processing.

Understanding how the brain operates requires understanding how large sets of neurons function together. Modern recording technology makes it possible to simultaneously record the activity of hundreds of neurons, and technological developments will soon allow recording of thousands or tens of thousands. As with all experimental techniques, these methods are subject to confounds that complicate the interpretation of such recordings, and could lead to erroneous scientific conclusions. Here, we discuss methods for assessing and improving the quality of data from these techniques, and outline likely future directions in this field.

Visual cortex exhibits smooth retinotopic organization on the macroscopic scale, but it is unknown how receptive fields are organized at the level of neighboring neurons. This information is crucial for discriminating among models of visual cortex. We used in vivo two-photon calcium imaging to independently map ON and OFF receptive field subregions of local populations of layer 2/3 neurons in mouse visual cortex. We found that receptive field subregions are often precisely shared among multiple neighboring neurons. Furthermore, large subregions appear to be assembled from multiple smaller, non-overlapping subregions of other neurons in the same local population. These experiments provide the first characterization of the diversity of receptive fields in a dense local network of visual cortex, and reveal elementary units of receptive field organization. Our results suggest that a limited pool of afferent receptive fields is available to a local population of neurons, and reveal new organizational principles for the neural circuitry of the mouse visual cortex.

Neural circuitry has evolved to form distributed networks that act dynamically across large volumes. Collecting data from individual planes, conventional microscopy cannot sample circuitry across large volumes at the temporal resolution relevant to neural circuit function and behaviors. Here, we review emerging technologies for rapid volume imaging of neural circuitry. We focus on two critical challenges: the inertia of optical systems, which limits image speed, and aberrations, which restrict the image volume. Optical sampling time must be long enough to ensure high-fidelity measurements, but optimized sampling strategies and point spread function engineering can facilitate rapid volume imaging of neural activity within this constraint. We also discuss new computational strategies for the processing and analysis of volume imaging data of increasing size and complexity. Together, optical and computational advances are providing a broader view of neural circuit dynamics, and help elucidate how brain regions work in concert to support behavior.

Currently, therapeutic platelet concentrates can be stored for only 5 days. We have developed a procedure that permits long-term storage of fixed and lyophilized platelets that retain hemostatic properties after rehydration. These rehydrated lyophilized platelets (RL platelets) restore hemostasis in thrombocytopenic rats and become incorporated in the hemostatic plug of bleeding time wounds of normal dogs as well as von Willebrand disease dogs with partially replenished plasma von Willebrand factor. Ultrastructurally, these platelets are well preserved and are comparable to control normal washed platelets. Flow cytometry analysis shows that RL platelets react with antibodies to the major surface receptors, glycoprotein (GP)Ib and GPIIb/IIIa. These receptors are involved in platelet agglutination, aggregation, and adhesion. In vitro functional tests document the ability of RL platelets to adhere to denuded subendothelium and to spread on a foreign surface. Circulating RL platelets participated in carotid arterial thrombus formation induced in normal canine subjects. The participation of RL platelets in these vital hemostatic properties suggests that with further development they could become a stable platelet product for transfusion.To promote effective hemostasis, platelets must respond quickly to changes in normal blood flow or vessel injury (1, 2). After vascular injury, platelets adhere to exposed subendothelium, aggregate, and form a primary platelet plug. Platelet activation and initiation of coagulation follow with stabilization of the platelet plug by the formation of fibrin. The initiation of a thrombus at a site of vascular injury is mediated through platelet membrane glycoprotein (GP) receptors (3, 4). Platelet adhesion to a damaged vessel wall and its extracellular matrix at high shear is primarily mediated through the specific interaction of the platelet membrane GPIb-IX complex and bound von Willebrand factor (vWF) (5-7), which is synthesized and released into plasma and the vessel wall by endothelial cells (1). Platelet adhesion at low shear rates is mediated by several interactions, including collagen with the a2131 integrin (7). Platelet adhesion stimulates a spreading of the platelet (8).Although the mechanism of platelet spreading has not been completely characterized, recent in vitro studies have shown that platelets will spread on surfaces coated with fibrinogen (9) or polymerized fibrin (10). The activation of the GPIIb/IIIa receptor by agents such as ADP results in a conformational change in the receptor (11-13). The activated receptor binds fibrinogen, which forms a "bridge" between the platelets, and causes aggregation (1,14,15). Activated platelets provide the phospholipid surface for the assembly of blood clotting enzyme complexes, and the concentration and localization of activated coagulant proteins at sites of vessel wall injury may be facilitated by adherent platelets (16). Internal storage granules in platelets release clot-promoting contents in response to activation of bi...

Imaging the activity of neurons that are widely distributed across brain regions deep in scattering tissue at high speed remains challenging. Here, we introduce an open-source system with Dual Independent Enhanced Scan Engines for Large field-of-view Two-Photon imaging (Diesel2p). Combining optical design, adaptive optics, and temporal multiplexing, the system offers subcellular resolution over a large field-of-view of ~25 mm2, encompassing distances up to 7 mm, with independent scan engines. We demonstrate the flexibility and various use cases of this system for calcium imaging of neurons in the living brain.

Global motion perception is a function of higher, or extrastriate, visual system circuitry. These circuits can be engaged in visually driven navigation, a behavior at which mice are adept. However, the properties of global motion perception in mice are unclear. Therefore, we developed a touchscreen-based, two-alternative forced choice (2AFC) task to explore global motion detection in mice using random dot kinematograms (RDK). Performance data was used to compute coherence thresholds for global motion perception. The touchscreen-based task allowed for parallel training and testing with multiple chambers and minimal experimenter intervention with mice performing hundreds of trials per session. Parameters of the random dot kinematograms, including dot size, lifetime, and speed, were tested. Mice learned to discriminate kinematograms whose median motion direction differed by 90 degrees in 7-24 days after a 10-14 day pre-training period. The average coherence threshold (measured at 70% correct) in mice for this task was 22 ± 5%, with a dot diameter of 3.88 mm and speed of 58.2 mm/second. Our results confirm the ability of mice to perform global motion discriminations, and the touchscreen assay provides a flexible, automated, and relatively high throughput method with which to probe complex visual function in mice.

Apoptosis plays an essential role during brain development, yet the precise mechanism by which this pathway is regulated in the brain remains unknown. In particular, mammalian cells are known to express multiple anti-apoptotic Bcl-2 family proteins. However, the cells of the developing brain could also exist in a primed state in which the loss of a single anti-apoptotic Bcl-2 family protein is sufficient to trigger apoptosis. Here, we examined the critical role of Bcl-xL, an anti-apoptotic protein, during brain development. Using conditional knock-out mice in which Bcl-xL is deleted in neural progenitor cells (Bcl-xLEmx1-Cre ), we show that the loss of Bcl-xL is not sufficient to trigger apoptosis in these proliferating progenitors. In contrast, specific populations of postmitotic neurons derived from these progenitors, including upper layer cortical neurons and the CA1-CA3 regions of the hippocampus, were acutely dependent on Bcl-xL. Consistent with this finding, deletion of Bcl-xL selectively in the postmitotic neurons in the brain (Bcl-xLNex-Cre ) also resulted in similar patterns of apoptosis. This Bcl-xL deficiency-induced neuronal death was a consequence of activation of the apoptotic pathway, because the cell death was rescued with codeletion of the proapoptotic proteins Bax and Bak. Importantly, the loss of these Bcl-xL-dependent neurons led to severe neurobehavioral abnormalities, including deficits in motor learning, hyperactivity, and increased risk-taking and self-injurious behaviors. Together, our results identify a population of neurons in the developing brain that are acutely dependent on Bcl-xL during the peak period of synaptic connectivity that are important for the establishment of higher-order complex behaviors.