Holographic stimulation of opposing amygdala ensembles bidirectionally modulates valence-specific behavior via mutual inhibition.

The basolateral amygdala (BLA) is an evolutionarily conserved brain region, well known for valence processing. Despite this central role, the relationship between activity of BLA neuronal ensembles in response to appetitive and aversive stimuli and the subsequent expression of valence-specific behavior has remained elusive. Here, we leverage two-photon calcium imaging combined with single-cell holographic photostimulation through an endoscopic lens to demonstrate a direct causal role for opposing ensembles of BLA neurons in the control of oppositely valenced behavior in mice. We report that targeted photostimulation of either appetitive or aversive BLA ensembles results in mutual inhibition and shifts behavioral responses to promote consumption of an aversive tastant or reduce consumption of an appetitive tastant, respectively. Here, we identify that neuronal encoding of valence in the BLA is graded and relies on the relative proportion of individual BLA neurons recruited in a stable appetitive or quinine ensemble.

Deep-brain optical recording of neural dynamics during behavior.

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.

Stimulus-specific regulation of visual oddball differentiation in posterior parietal cortex.

The frequency at which a stimulus is presented determines how it is interpreted. For example, a repeated image may be of less interest than an image that violates the prior sequence. This process involves integration of sensory information and internal representations of stimulus history, functions carried out in higher-order sensory areas such as the posterior parietal cortex (PPC). Thus far, there are few detailed reports investigating the single-neuron mechanisms for processing of stimulus presentation frequency in PPC. To address this gap in knowledge, we recorded PPC activity using 2-photon calcium imaging and electrophysiology during a visual oddball paradigm. Calcium imaging results reveal differentiation at the level of single neurons for frequent versus rare conditions which varied depending on whether the stimulus was preferred or non-preferred by the recorded neural population. Such differentiation of oddball conditions was mediated primarily by stimulus-independent adaptation in the frequent condition.

Arousal dependent modulation of thalamo-cortical functional interaction.

Ongoing changes in arousal influence sensory processing and behavioral performance. Yet the circuit-level correlates for this influence remain poorly understood. Here, we investigate how functional interaction between posterior parietal cortex (PPC) and lateral posterior (LP)/Pulvinar is influenced by ongoing fluctuations in pupil-linked arousal, which is a non-invasive measure of neuromodulatory tone in the brain. We find that fluctuations in pupil-linked arousal correlate with changes to PPC to LP/Pulvinar oscillatory interaction, with cortical alpha oscillations driving activity during low arousal states, and LP/Pulvinar driving PPC in the theta frequency band during higher arousal states. Active visual exploration by saccadic eye movements elicits similar transitions in thalamo-cortical interaction. Furthermore, the presentation of naturalistic video stimuli induces thalamo-cortical network states closely resembling epochs of high arousal in the absence of visual input. Thus, neuromodulators may play a role in dynamically sculpting the patterns of thalamo-cortical functional interaction that underlie visual processing.

MIN1PIPE: A Miniscope 1-Photon-Based Calcium Imaging Signal Extraction Pipeline.

In vivo calcium imaging using a 1-photon-based miniscope and a microendoscopic lens enables studies of neural activities in freely behaving animals. However, the high and fluctuating background, the inevitable movements and distortions of imaging field, and the extensive spatial overlaps of fluorescent signals emitted from imaged neurons inherent in this 1-photon imaging method present major challenges for extracting neuronal signals reliably and automatically from the raw imaging data. Here, we develop a unifying algorithm called the miniscope 1-photon imaging pipeline (MIN1PIPE), which contains several stand-alone modules and can handle a wide range of imaging conditions and qualities with minimal parameter tuning and automatically and accurately isolate spatially localized neural signals. We have quantitatively compared MIN1PIPE with other existing partial methods using both synthetic and real datasets obtained from different animal models and show that MIN1PIPE has superior efficiency and precision in analyzing noisy miniscope calcium imaging data.

Theta Oscillations Organize Spiking Activity in Higher-Order Visual Thalamus during Sustained Attention.

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.

Early Development of Network Oscillations in the Ferret Visual Cortex.

Although oscillations during development have been characterized in a wide range of neural systems, little is known about the interaction between these network oscillations and neuronal spiking, and the interactions among different oscillation frequencies. Here we recorded the spontaneous and visual-elicited local field potential (LFP) and multi-unit activity (MUA) in the visual cortex of freely-moving juvenile ferrets before and after eye-opening. We found that both the spontaneous and visually-elicited LFP power was increased after eye-opening, especially in higher frequency bands (>30 Hz). Spike LFP phase coupling was decreased for lower frequency bands (theta and alpha) but slightly increased for higher frequencies (high-gamma band). A similar shift towards faster frequencies also occurred for phase-amplitude coupling; with maturation, the coupling of the theta/alpha/beta band amplitude to the delta phase was decreased and the high-gamma amplitude coupling to theta/alpha phase was increased. This shift towards higher frequencies was also reflected in the visual responses; the LFP oscillation became more entrained by visual stimulation with higher frequencies (>10 Hz). Taken together, these results suggest gamma oscillation as a signature of the maturation of cortical circuitry.

Resting state network topology of the ferret brain.

Resting state functional magnetic resonance imaging (rsfMRI) has emerged as a versatile tool for non-invasive measurement of functional connectivity patterns in the brain. RsfMRI brain dynamics in rodents, non-human primates, and humans share similar properties; however, little is known about the resting state functional connectivity patterns in the ferret, an animal model with high potential for developmental and cognitive translational study. To address this knowledge-gap, we performed rsfMRI on anesthetized ferrets using a 9.4T MRI scanner, and subsequently performed group-level independent component analysis (gICA) to identify functionally connected brain networks. Group-level ICA analysis revealed distributed sensory, motor, and higher-order networks in the ferret brain. Subsequent connectivity analysis showed interconnected higher-order networks that constituted a putative default mode network (DMN), a network that exhibits altered connectivity in neuropsychiatric disorders. Finally, we assessed ferret brain topological efficiency using graph theory analysis and found that the ferret brain exhibits small-world properties. Overall, these results provide additional evidence for pan-species resting-state networks, further supporting ferret-based studies of sensory and cognitive function.

Oscillatory Dynamics in the Frontoparietal Attention Network during Sustained Attention in the Ferret.

Sustained attention requires the coordination of neural activity across multiple cortical areas in the frontoparietal network, in particular the prefrontal cortex (PFC) and posterior parietal cortex (PPC). Previous work has demonstrated that activity in these brain regions is coordinated by neuronal oscillations of the local field potential (LFP). However, the underlying coordination of activity in terms of organization of single unit (SU) spiking activity has remained poorly understood, particularly in the freely moving animal. We found that long-range functional connectivity between anatomically connected PFC and PPC was mediated by oscillations in the theta frequency band. SU activity in PFC was phase locked to theta oscillations in PPC, and spiking activity in PFC and PPC was locked to local high-gamma activity. Together, our results support a model in which frequency-specific synchronization mediates functional connectivity between and within PFC and PPC of the frontoparietal attention network in the freely moving animal.

Dorso-Lateral Frontal Cortex of the Ferret Encodes Perceptual Difficulty during Visual Discrimination.

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.

Visualization of cortical, subcortical and deep brain neural circuit dynamics during naturalistic mammalian behavior with head-mounted microscopes and chronically implanted lenses.

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.