Fusing left and right eye images into a single view is dependent on precise ocular alignment, which relies on coordinated eye movements. During movements of the head this alignment is maintained by numerous reflexes. Although rodents share with other mammals the key components of eye movement control, the coordination of eye movements in freely moving rodents is unknown. Here we show that movements of the two eyes in freely moving rats differ fundamentally from the precisely controlled eye movements used by other mammals to maintain continuous binocular fusion. The observed eye movements serve to keep the visual fields of the two eyes continuously overlapping above the animal during free movement, but not continuously aligned. Overhead visual stimuli presented to rats freely exploring an open arena evoke an immediate shelter-seeking behaviour, but are ineffective when presented beside the arena. We suggest that continuously overlapping visual fields overhead would be of evolutionary benefit for predator detection by minimizing blind spots.

We describe a miniaturized head-mounted multiphoton microscope and its use for recording Ca 2؉ transients from the somata of layer 2/3 neurons in the visual cortex of awake, freely moving rats. Images contained up to 20 neurons and were stable enough to record continuously for >5 min per trial and 20 trials per imaging session, even as the animal was running at velocities of up to 0.6 m/s. Neuronal Ca 2؉ transients were readily detected, and responses to various static visual stimuli were observed during free movement on a running track. Neuronal activity was sparse and increased when the animal swept its gaze across a visual stimulus. Neurons showing preferential activation by specific stimuli were observed in freely moving animals. These results demonstrate that the multiphoton fiberscope is suitable for functional imaging in awake and freely moving animals.calcium imaging ͉ head-mounted microscope ͉ neuronal activity ͉ two-photon ͉ visual cortex T he observation of neural activity has been central to the vast majority of efforts to understand information processing in the mammalian brain. Although some aspects of sensory processing can be studied in animals that are anesthetized or awake but head-fixed, to fully understand awake information processing, animals must be able to interact fully with their environment. Previously, this approach, which includes both allothetic and idiothetic cues, lead to the discovery of place cells (1), head-direction cells (2), and grid cells (3). Multiphoton (MP) imaging (4) of neurons bulk-loaded with calcium indicators (5-7) allows not only the unambiguous identification and precise anatomical localization of active neurons but also the simultaneous recording of activity in multiple neurons even at very low firing rates (8-10). Although one major limitation of conventional MP imaging has been the need to firmly hold the skull in position to prevent the brain from moving relative to the microscope objective, some aspects of free movement can be simulated in head-fixed animals by placing them in a virtual reality situation (11). For measurements in truly free-moving animals, the recording apparatus must be miniaturized and attached to the skull, similar to the approach used for the recording of extracellular (1, 12) and intracellular (13) electrical signals in freely moving rodents. It is possible to use optical fibers to deliver short-pulse light suitable for two-photon excitation, scan the excitation focus, and collect the emitted fluorescence (14). There have been a number of recent advances in scanning technology (15, 16) that have been applied to anesthetized animals by using two-photon excitation (17) or freely moving animals by using one-photon wide-field imaging (18). Here we show that it is possible to use two-photon microscopy to record activity from neuronal populations with cellular resolution in freely moving animals. Results and DiscussionWe developed a fiberscope (Fig. 1 A) that employs a customdesigned water-immersion lens and a leveraged, nonresonant fiber scan...

Multiphoton imaging is widely used for recording activity simultaneously from many neurons in superficial cortical layers in vivo. Here we combine regenerative amplification multiphoton microscopy (RAMM) with genetically encoded calcium indicators to extend multiphoton imaging of neuronal population activity into layer 5 of adult mouse somatosensory cortex. We show that this approach can be used to record and quantify spontaneous and sensory-evoked activity in populations of layer 5 neuronal somata located as much as 800µm below the pia. In addition, we show that RAMM can be used to simultaneously image activity from large (~80) populations of apical dendrites and follow these dendrites down to their somata of origin.3

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Mice have a large visual field that is constantly stabilized by vestibular ocular reflex (VOR) driven eye rotations that counter head-rotations. While maintaining their extensive visual coverage is advantageous for predator detection, mice also track and capture prey using vision. However, in the freely moving animal quantifying object location in the field of view is challenging. Here, we developed a method to digitally reconstruct and quantify the visual scene of freely moving mice performing a visually based prey capture task. By isolating the visual sense and combining a mouse eye optic model with the head and eye rotations, the detailed reconstruction of the digital environment and retinal features were projected onto the corneal surface for comparison, and updated throughout the behavior. By quantifying the spatial location of objects in the visual scene and their motion throughout the behavior, we show that the prey image consistently falls within a small area of the VOR-stabilized visual field. This functional focus coincides with the region of minimal optic flow within the visual field and consequently area of minimal motion-induced image-blur, as during pursuit mice ran directly toward the prey. The functional focus lies in the upper-temporal part of the retina and coincides with the reported high density-region of Alpha-ON sustained retinal ganglion cells.

The current study has investigated the electrophysiological responses evoked by histamine in bovine adrenal chromaffin cells using perforated‐patch techniques. Histamine caused a transient hyperpolarization followed by a sustained depolarization of 7.2 ± 1.4 mV associated with an increase in spontaneous action potential frequency. The hyperpolarization was abolished after depleting intracellular Ca2+ stores with thapsigargin (100 nm), and was reduced by 40 % with apamin (100 nm). Membrane resistance increased by about 60 % during the histamine‐induced depolarization suggesting inhibition of a K+ channel. An inward current relaxation, typical of an M‐current, was observed in response to negative voltage steps from a holding potential of −30 mV. This current reversed at −81.6 ± 1.8 mV and was abolished by the M‐channel inhibitor linopirdine (100 μm). During application of histamine, the amplitude of M‐currents recorded at a time corresponding with the sustained depolarization was reduced by 40 %. No inward current rectification was observed in the range −150 to −70 mV, and glibenclamide (10 μm) had no effect on either resting membrane potential or the response to histamine. The results show that an M‐current is present in bovine chromaffin cells and that this current is inhibited during sustained application of histamine, resulting in membrane depolarization and increased discharge of action potentials. These results demonstrate for the first time a possible mechanism coupling histamine receptors to activation of voltage‐operated Ca2+ channels in these cells.

Multiphoton imaging of genetically encoded calcium indicators is routinely used to report activity from populations of spatially resolved neurons in vivo. However, since the relationship between fluorescence and action potentials (APs) is nonlinear and varies over neurons, quantitatively inferring AP discharge is problematic. To address this we developed a biophysical model of calcium binding kinetics for the indicator GCaMP6s that accurately describes AP-evoked fluorescence changes in vivo. The model's physical interpretation allowed the same parameters to describe GCaMP6s binding kinetics for both in vitro binding assays and in vivo imaging. Using this model, we developed an algorithm to infer APs from fluorescence and measured its accuracy with cell-attached electrical recordings. This approach consistently inferred more accurate AP counts and times than alternative methods for firing rates from 0 to >20 Hz, while requiring less training data. These results demonstrate the utility of quantitative, biophysically grounded models for complex biological data. Greenberg et al. | 2018 | bioRχiv 2/84 Greenberg et al. | 2018 | bioRχiv 5/84

We investigated the effect of selective whisker trimming on the development of the cortical representation of a whisker deflection in layer 2/3 of rat somatosensory cortex using in vivo voltage-sensitive dye (vsd) imaging. Responses to deflection of D-row whiskers were recorded after trimming of A-row, B-row, and C-row whiskers, referred to as DE pairing, during postnatal development. Animals DE paired from postnatal day (p) 7 to p17 had a significant bias in the spread of the vsd signal, favoring spread toward the concomitantly nondeprived E-row columns. This resulted primarily from a strong decrease in signal spreading into the deprived C-row columns. In contrast, signal spread in control littermates was approximately symmetrical. DE pairing failed to elicit significant changes when begun after p14, thus defining a critical period for this phenomenon. The results suggest that sensory deprivation in this model results in lower connectivity being established between nondeprived columns and adjacent deprived ones.