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In vivo fluorescence recording techniques have produced landmark discoveries in neuroscience, providing insight into how single cell and circuit-level computations mediate sensory processing and generate complex behaviors. While much attention has been given to recording from cortical brain regions, deep-brain fluorescence recording is more complex because it requires additional measures to gain optical access to harder to reach brain nuclei. Here we discuss detailed considerations and tradeoffs regarding deep-brain fluorescence recording techniques and provide a comprehensive guide for all major steps involved, from project planning to data analysis. The goal is to impart guidance for new and experienced investigators seeking to use in vivo deep fluorescence optical recordings in awake, behaving rodent models., Competing Interests: Declaration of interests M.R.B. is a co-founder and scientific advisory board member of Neurolux, Inc, a Neurotechnology company. None of the research noted here is related to those efforts., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Higher-order visual thalamus plays a fundamental but poorly understood role in attention-demanding tasks. To investigate how neuronal dynamics in higher-order visual thalamus are modulated by sustained attention, we performed multichannel electrophysiological recordings in the lateral posterior-pulvinar complex (LP/pulvinar) in the ferret ( Mustela putorius furo ). We recorded single unit activity and local field potential (LFP) during the performance of the five-choice serial reaction time task (5-CSRTT), which is used in both humans and animals as an assay of sustained attention. We found that half of the units exhibited an increasing firing rate during the delay period before stimulus onset (attention-modulated units). In contrast, the non-attention-modulated units responded to the stimulus, but not during the delay period. Spike-field coherence (SFC) of only the attention-modulated neurons significantly increased from the start of the delay period until screen touch, predominantly in the θ frequency band. In addition, θ power and θ/γ phase amplitude coupling (PAC) were elevated throughout the delay period. Our findings suggest that the θ oscillation plays a central role in orchestrating thalamic signaling during sustained attention.

Visual discrimination requires sensory processing followed by a perceptual decision. Despite a growing understanding of visual areas in this behavior, it is unclear what role top-down signals from prefrontal cortex play, in particular as a function of perceptual difficulty. To address this gap, we investigated how neurons in dorso-lateral frontal cortex (dl-FC) of freely-moving ferrets encode task variables in a two-alternative forced choice visual discrimination task with high- and low-contrast visual input. About two-thirds of all recorded neurons in dl-FC were modulated by at least one of the two task variables, task difficulty and target location. More neurons in dl-FC preferred the hard trials; no such preference bias was found for target location. In individual neurons, this preference for specific task types was limited to brief epochs. Finally, optogenetic stimulation confirmed the functional role of the activity in dl-FC before target touch; suppression of activity in pyramidal neurons with the ArchT silencing opsin resulted in a decrease in reaction time to touch the target but not to retrieve reward. In conclusion, dl-FC activity is differentially recruited for high perceptual difficulty in the freely-moving ferret and the resulting signal may provide top-down behavioral inhibition.

Genetically encoded calcium indicators for visualizing dynamic cellular activity have greatly expanded our understanding of the brain. However, owing to the light-scattering properties of the brain, as well as the size and rigidity of traditional imaging technology, in vivo calcium imaging has been limited to superficial brain structures during head-fixed behavioral tasks. These limitations can now be circumvented by using miniature, integrated microscopes in conjunction with an implantable microendoscopic lens to guide light into and out of the brain, thus permitting optical access to deep brain (or superficial) neural ensembles during naturalistic behaviors. Here we describe steps to conduct such imaging studies using mice. However, we anticipate that the protocol can be easily adapted for use in other small vertebrates. Successful completion of this protocol will permit cellular imaging of neuronal activity and the generation of data sets with sufficient statistical power to correlate neural activity with stimulus presentation, physiological state and other aspects of complex behavioral tasks. This protocol takes 6-11 weeks to complete., Competing Interests: Dr. G.D. Stuber is the recipient of a DECODE grant from Inscopix, Palo Alto, CA.