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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