Optogenetics seminars

Nearly all motivated behaviors require the ability to associate outcomes with specific actions and make adaptive decisions about future behavior. The nucleus accumbens (NAc) is integrally involved in these processes. The NAc is a heterogeneous population primarily composed of D1 and D2 medium spiny projection (MSN) neurons that are thought to have opposed roles in behavior, with D1 MSNs promoting reward and D2 MSNs promoting aversion. Here we examined what types of information are encoded by the D1 and D2 MSNs using optogenetics, fiber photometry, and cellular resolution calcium imaging. First, we showed that mice responded for optical self-stimulation of both cell types, suggesting D2-MSN activation is not inherently aversive. Next, we recorded population and single cell activity patterns of D1 and D2 MSNs during reinforcement as well as Pavlovian learning paradigms that allow dissociation of stimulus value, outcome, cue learning, and action. We demonstrated that D1 MSNs respond to the presence and intensity of unconditioned stimuli – regardless of value. Conversely, D2 MSNs responded to the prediction of these outcomes during specific cues. Overall, these results provide foundational evidence for the discrete aspects of information that are encoded within the NAc D1 and D2 MSN populations. These results will significantly enhance our understanding of the involvement of the NAc MSNs in learning and memory as well as how these neurons contribute to the development and maintenance of substance use disorders.

The hippocampus is crucial for spatial navigation and episodic memory formation. Hippocampal place cells exhibit spatially selective activity within an environment and have been proposed to form the neural basis of a cognitive map of space that supports these mnemonic functions. However, the direct influence of place cell activity on spatial navigation behaviour has not yet been demonstrated. Using an ‘all-optical’ combination of simultaneous two-photon calcium imaging and two-photon holographically targeted optogenetics, we identified and selectively activated place cells that encoded behaviourally relevant locations in a virtual reality environment. Targeted stimulation of a small number of place cells was sufficient to bias the behaviour of animals during a spatial memory task, providing causal evidence that hippocampal place cells actively support spatial navigation and memory. Time permitting, I will also describe new experiments aimed at understanding the fundamental encoding mechanism that supports episodic memory, focussing on the role of hippocampal sequences across multiple timescales and behaviours.

Inter-areal coherence between cortical field-potentials is a widespread phenomenon and depends on numerous behavioral and cognitive factors. It has been hypothesized that inter-areal coherence reflects phase-synchronization between local oscillations and flexibly gates communication. We reveal an alternative mechanism, where coherence results from and is not the cause of communication, and naturally emerges as a consequence of the fact that spiking activity in a sending area causes post-synaptic inputs both in the same area and in other areas. Consequently, coherence depends in a lawful manner on oscillatory power and phase-locking in a sending area and inter-areal connectivity. We show that changes in oscillatory power explain prominent changes in fronto-parietal beta-coherence with movement and memory, and LGN-V1 gamma-coherence with arousal and visual stimulation. Optogenetic silencing of a receiving area and E/I network simulations demonstrate that afferent synaptic inputs rather than spiking entrainment are the main determinant of inter-areal coherence. These findings suggest that the unique spectral profiles of different brain areas automatically give rise to large-scale inter-areal coherence patterns that follow anatomical connectivity and continuously reconfigure as a function of behavior and cognition.

The ability to reach, grasp, and manipulate objects is a remarkable expression of motor skill, and the loss of this ability in injury, stroke, or disease can be devastating. These behaviors are controlled by the coordinated activity of tens of millions of neurons distributed across many CNS regions, including the primary motor cortex. While many studies have characterized the activity of single cortical neurons during reaching, the principles governing the dynamics of large, distributed neural populations remain largely unknown. Recent work in primates has suggested that during the execution of reaching, motor cortex may autonomously generate the neural pattern controlling the movement, much like the spinal central pattern generator for locomotion. In this seminar, I will describe recent work that tests this hypothesis using large-scale neural recording, high-resolution behavioral measurements, dynamical systems approaches to data analysis, and optogenetic perturbations in mice. We find, by contrast, that motor cortex requires strong, continuous, and time-varying thalamic input to generate the neural pattern driving reaching. In a second line of work, we demonstrate that the cortico-cerebellar loop is not critical for driving the arm towards the target, but instead fine-tunes movement parameters to enable precise and accurate behavior. Finally, I will describe my future plans to apply these experimental and analytical approaches to the adaptive control of locomotion in complex environments.

The Miniscope Project -- an open-source collaborative effort—was created to accelerate innovation of miniature microscope technology and to increase global access to this technology. Currently, we are working on advancements ranging from optogenetic stimulation and wire-free operation to simultaneous optical and electrophysiological recording. Using these systems, we have uncovered mechanisms underlying temporal memory linking and investigated causes of cognitive deficits in temporal lobe epilepsy. Through innovation and optimization, this work aims to extend the reach of neuroscience research and create new avenues of scientific inquiry.

Join us for a comprehensive introduction to multielectrode recording technologies for in vivo neurophysiology. Whether you are new to the field or have experience with one type of technology, this webinar will provide you with information about a variety of technologies, with a main focus on Neuropixels probes. Dr Kris Schoepfer, US Product Specialist at Scientifica, will provide an overview of multielectrode technologies available to record from one or more brain areas simultaneously, including: DIY multielectrode probes; Tetrodes / Hyperdrives; Silicon probes; Neuropixels. Dr Sylvia Schröder, University of Sussex, will delve deeper into the advantages of Neuropixels, highlighting the value of channel depth and the types of new biological insights that can be explored thanks to the advancements this technology brings. Presenting exciting data from the optic tract and superior colliculus, Sylvia will also discuss how Neuropixels recordings can be combined with optogenetics, and how histology can be used to identify the location of probes.

Motivational drives are internal states that can be different even in similar interactions with external stimuli. Curiosity as the motivational drive for novelty-seeking and investigating the surrounding environment is for survival as essential and intrinsic as hunger. Curiosity, hunger, and appetitive aggression drive three different goal-directed behaviors—novelty seeking, food eating, and hunting— but these behaviors are composed of similar actions in animals. This similarity of actions has made it challenging to study novelty seeking and distinguish it from eating and hunting in nonarticulating animals. The brain mechanisms underlying this basic survival drive, curiosity, and novelty-seeking behavior have remained unclear. In spite of having well-developed techniques to study mouse brain circuits, there are many controversial and different results in the field of motivational behavior. This has left the functions of motivational brain regions such as the zona incerta (ZI) still uncertain. Not having a transparent, nonreinforced, and easily replicable paradigm is one of the main causes of this uncertainty. Therefore, we chose a simple solution to conduct our research: giving the mouse freedom to choose what it wants—double freeaccess choice. By examining mice in an experimental battery of object free-access double-choice (FADC) and social interaction tests—using optogenetics, chemogenetics, calcium fiber photometry, multichannel recording electrophysiology, and multicolor mRNA in situ hybridization—we uncovered a cell type–specific cortico-subcortical brain circuit of the curiosity and novelty-seeking behavior. We found in mice that inhibitory neurons in the medial ZI (ZIm) are essential for the decision to investigate an object or a conspecific. These neurons receive excitatory input from the prelimbic cortex to signal the initiation of exploration. This signal is modulated in the ZIm by the level of investigatory motivation. Increased activity in the ZIm instigates deep investigative action by inhibiting the periaqueductal gray region. A subpopulation of inhibitory ZIm neurons expressing tachykinin 1 (TAC1) modulates the investigatory behavior.

Coping with threatening situations requires both identifying stimuli predicting danger and selecting adaptive behavioral responses in order to survive. The dorso medial prefrontal cortex (dmPFC) is a critical structure involved in the regulation of threat-related behaviour, yet it is still largely unclear how threat-predicting stimuli and defensive behaviours are associated within prefrontal networks in order to successfully drive adaptive responses. To address these questions, we used a combination of extracellular recordings, neuronal decoding approaches, and optogenetic manipulations to show that threat representations and the initiation of avoidance behaviour are dynamically encoded in the overall population activity of dmPFC neurons. These data indicate that although dmPFC population activity at stimulus onset encodes sustained threat representations and discriminates threat- from non-threat cues, it does not predict action outcome. In contrast, transient dmPFC population activity prior to action initiation reliably predicts avoided from non-avoided trials. Accordingly, optogenetic inhibition of prefrontal activity critically constrained the selection of adaptive defensive responses in a time-dependent manner. These results reveal that the adaptive selection of active fear responses relies on a dynamic process of information linking threats with defensive actions unfolding within prefrontal networks.

Dysregulation of emotional processing and its integration with cognitive functions are central features of many mental/emotional disorders associated both with externalizing problems (aggressive, antisocial behaviors) and internalizing problems (anxiety, depression). As Dr. Joseph LeDoux, our invited speaker of this program, wrote in his famous book “Synaptic self: How Our Brains Become Who We Are”—the brain’s synapses—are the channels through which we think, act, imagine, feel, and remember. Synapses encode the essence of personality, enabling each of us to function as a distinctive, integrated individual from moment to moment. Thus, exploring the functioning of synapses leads to the understanding of the mechanism of (patho)physiological function of our brain. In this context, we have investigated the pathophysiology of psychiatric disorders, with particular emphasis on the synaptic function of model mice of various psychiatric disorders such as schizophrenia, autism, depression, and PTSD. Our current interest is how synaptic inputs are integrated to generate the action potential. Because the spatiotemporal organization of neuronal firing is crucial for information processing, but how thousands of inputs to the dendritic spines drive the firing remains a central question in neuroscience. We identified a distinct pattern of synaptic integration in the disease-related models, in which extra-large (XL) spines generate NMDA spikes within these spines, which was sufficient to drive neuronal firing. We experimentally and theoretically observed that XL spines negatively correlated with working memory. Our work offers a whole new concept for dendritic computation and network dynamics, and the understanding of psychiatric research will be greatly reconsidered. The second half of my talk is the development of a novel synaptic tool. Because, no matter how beautifully we can illuminate the spine morphology and how accurately we can quantify the synaptic integration, the links between synapse and brain function remain correlational. In order to challenge the causal relationship between synapse and brain function, we established AS-PaRac1, which is unique not only because it can specifically label and manipulate the recently potentiated dendritic spine (Hayashi-Takagi et al, 2015, Nature). With use of AS-PaRac1, we developed an activity-dependent simultaneous labeling of the presynaptic bouton and the potentiated spines to establish “functional connectomics” in a synaptic resolution. When we apply this new imaging method for PTSD model mice, we identified a completely new functional neural circuit of brain region A→B→C with a very strong S/N in the PTSD model mice. This novel tool of “functional connectomics” and its photo-manipulation could open up new areas of emotional/psychiatric research, and by extension, shed light on the neural networks that determine who we are.

Transient changes in dopamine activity in response to reward and punishment have been known to regulate reward-related learning. However, the cellular basis that detects the transient dopamine signaling has long been unclear. Using two-photon microscopy and optogenetics, I have shown that transient increases and decreases of dopamine modulate plasticity of dopamine D1 and D2 receptor-expressing cells in the nucleus accumbens, respectively. At the behavioral level, I characterized that these D1 and D2 cells cooperatively tune learning by generalization and discrimination learning. Interestingly, disturbance of the dopamine signaling impaired D2 cell plasticity and discrimination learning, which was analogous to salience misattribution seen in subjects with schizophrenia.

During sexual behavior, copulation related sensory information and modulatory signals from the brain must be integrated and converted into the motor and secretory outputs that characterize ejaculation (Lenschow and Lima, Current Opinion in Neurobiology, 2020). Studies in humans and rats suggest the existence of interneurons in the lumbar spinal cord that mediates that step: the spinal ejaculation generator (SEG). My work aimed at gaining mechanistic insights about the neuronal circuits controlling ejaculation thereby applying cutting-edge techniques. More specifically, we mapped anatomically and functionally the spinal circuit for ejaculation starting from the main muscle being involved in sperm expulsion: the bulbospongiosus muscle (BSM). Combining viral tracing strategies with electrophysiology, we specifically show that the BSM motoneurons receive direct synaptic input from a group of interneurons located in between lumbar segment 2 and 3 and expressing the peptide galanin. Electrically and optogenetically activating the galanin positive cells (the SEG) lead to the activation of the motoneurons innervating the BSM and the muscle itself. Finally, inhibition of SEG cells using DREADDs (Designer Receptors Exclusively Activated by Designer Drugs) in sexual behaving animals is currently conducted to reveal whether ejaculation can be prevented.

The hippocampal formation has been implicated in both cognitive functions as well as the sensing and control of endocrine states. To identify a candidate activity pattern which may link such disparate functions, we simultaneously measured electrophysiological activity from the hippocampus and interstitial glucose concentrations in the body of freely behaving rats. We found that clusters of sharp wave-ripples (SPW-Rs) recorded from both dorsal and ventral hippocampus reliably predicted a decrease in peripheral glucose concentrations within ~10 minutes. This correlation was less dependent on circadian, ultradian, and meal-triggered fluctuations, it could be mimicked with optogenetically induced ripples, and was attenuated by pharmacogenetically suppressing activity of the lateral septum, the major conduit between the hippocampus and subcortical structures. Our findings demonstrate that a novel function of the SPW-R is to modulate peripheral glucose homeostasis and offer a mechanism for the link between sleep disruption and blood glucose dysregulation seen in type 2 diabetes and obesity.

Animals constantly modify their behavior through experience. Flexible behavior is key to our ability to adapt to the ever-changing environment. My laboratory is interested in studying the activity of neuronal ensembles in behaving animals, and how it changes with learning. We have recently set up a paradigm where mice learn to associate sensory information (two different odors) to motor outputs (lick vs no-lick) under head-fixation. We combined this with two-photon calcium imaging, which can monitor the activity of a microcircuit of many tens of neurons simultaneously from a small area of the brain. Imaging the motor cortex during the learning of this task revealed neurons with diverse task-related response types. Intriguingly, different response types were spatially intermingled; even immediately adjacent neurons often had very different response types. As the mouse learned the task under the microscope, the activity coupling of neurons with similar response types specifically increased, even though they are intermingled with neurons with dissimilar response types. This suggests that intermingled subnetworks of functionally-related neurons form in a learning-related way, an observation that became possible with our cutting-edge technique combining imaging and behavior. We are working to extend this study. How plastic are neuronal microcircuits during other forms of learning? How plastic are they in other parts of the brain? What are the cellular and molecular mechanisms of the microcircuit plasticity? Are the observed activity and plasticity required for learning? How does the activity of identified individual neurons change over days to weeks? We are asking these questions, combining a variety of techniques including in vivo two-photon imaging, optogenetics, electrophysiology, genetics and behavior.

Over 60 years of research has established that medial temporal lobe structures, including the hippocampus and entorhinal cortex, are necessary for the formation of episodic memories (i.e. memories of specific personal events that occur in spatial and temporal context). While prior work to establish the neural mechanisms underlying episodic memory has largely focused on questions related spatial context, recently we have begun to investigate how these brain structures could be involved in encoding aspects of temporal context. In particular, we have focused on how medial entorhinal cortex, a structure well known for its role in spatial memory, may also be involved in encoding interval time. To answer this question we have developed an instrumental paradigm for head-fixed mice that requires both immobile interval timing and locomotion-dependent navigation behavior. By combining this behavioral paradigm with large-scale cellular resolution functional imaging and optogenetic-mediated inactivation, our results suggest that MEC is required for learning of interval timing behavior and that interval timing could be mediated through regular, sequential neural activity of a distinct subpopulation of neurons in MEC that encode elapsed time during periods of immobility (Heys and Dombeck, 2018; Heys et al, 2020; Issa et al., 2020). In this talk, I will discuss these findings and discuss our on-going work to investigate the principles underlying the role of medial temporal lobe structures in timing behavior and episodic memory.

Broadly, the Mauro Costa-Mattioli laboratory (The MCM Lab) encompasses two complementary lines of research. The first one, more traditional but very important, aims at unraveling the molecular mechanisms underlying memory formation (e.g., using state-of-the-art molecular and cell-specific genetic approaches). Learning and memory disorders can strike the brain during development (e.g., Autism Spectrum Disorders and Down Syndrome), as well as during adulthood (e.g., Alzheimer’s disease). We are interested in understanding the specific circuits and molecular pathways that are primarily targeted in these disorders and how they can be restored. To tackle these questions, we use a multidisciplinary, convergent and cross-species approach that combines mouse and fly genetics, molecular biology, electrophysiology, stem cell biology, optogenetics and behavioral techniques. The second line of research, more recent and relatively unexplored, is focused on understanding how gut microbes control CNS driven-behavior and brain function. Our recent discoveries, that microbes in the gut could modulate brain function and behavior in a very powerful way, have added a whole new dimension to the classic view of how complex behaviors are controlled. The unexpected findings have opened new avenues of study for us and are currently driving my lab to answer a host of new and very interesting questions: - What are the gut microbes (and metabolites) that regulate CNS-driven behaviors? Would it be possible to develop an unbiased screening method to identify specific microbes that regulate different behaviors? - If this is the case, can we identify how members of the gut microbiome (and their metabolites) mechanistically influence brain function? - What is the communication channel between the gut microbiota and the brain? Do different gut microbes use different ways to interact with the brain? - Could disruption of the gut microbial ecology cause neurodevelopmental dysfunction? If so, what is the impact of disruption in young and adult animals? - More importantly, could specific restoration of selected bacterial strains (new generation probiotics) represent a novel therapeutic approach for the targeted treatment of neurodevelopmental disorders? - Finally, can we develop microbiota-directed therapeutic foods to repair brain dysfunction in a variety of neurological disorders?

The developmental journey of cortical interneurons encounters several activity-dependent milestones. During the early postnatal period in developing mice, GABAergic neurons are transient preferential recipients of thalamic inputs and undergo activity-dependent migration arrest, wiring and programmed cell-death. But cortical GABAergic neurons are also specified by very early developmental programs. For example, the earliest born GABAergic neurons develop into hub cells coordinating spontaneous activity in hippocampal slices. Despite their importance for the emergence of sensory experience, their role in coordinating network dynamics, and the role of activity in their integration into cortical networks, the collective in vivo dynamics of GABAergic neurons during the neonatal postnatal period remain unknown. Here, I will present data related to the coordinated activity between GABAergic cells of the mouse barrel cortex and hippocampus in non-anesthetized pups using the recent development of all optical methods to record and manipulate neuronal activity in vivo. I will show that the functional structure of developing GABAergic circuits is remarkably patterned, with segregated assemblies of prospective parvalbumin neurons and highly connected hub cells, both shaped by sensory-dependent processes.

Team work makes sensory streams work: our senses work together, learn from each other, and stand in for one another, the result of which is perception and understanding. Learned associations between stimuli in different sensory modalities can shape the way we perceive these stimuli (Mcgurk and Macdonald, 1976). During audio-visual associative learning, auditory cortex is thought to underlie multi-modal plasticity in visual cortex (McIntosh et al., 1998; Mishra et al., 2007; Zangenehpour and Zatorre, 2010). However, it is not well understood how processing in visual cortex is altered by an auditory stimulus that is predictive of a visual stimulus and what the mechanisms are that mediate such experience-dependent, audio-visual associations in sensory cortex. Here we describe a neural mechanism by which an auditory input can shape visual representations of behaviorally relevant stimuli through direct interactions between auditory and visual cortices. We show that the association of an auditory stimulus with a visual stimulus in a behaviorally relevant context leads to an experience-dependent suppression of visual responses in primary visual cortex (V1). Auditory cortex axons carry a mixture of auditory and retinotopically-matched visual input to V1, and optogenetic stimulation of these axons selectively suppresses V1 neurons responsive to the associated visual stimulus after, but not before, learning. Our results suggest that cross-modal associations can be stored in long-range cortical connections and that with learning these cross-modal connections function to suppress the responses to predictable input.

Sequential temporal ordering and patterning are key features of natural signals, used by the brain to decode stimuli and perceive them as sensory objects. Touch is one sensory modality where temporal patterning carries key information, and the rodent whisker system is a prominent model for understanding neuronal coding and plasticity underlying touch sensation. Neurons in this system are precise encoders of fluctuations in whisker dynamics down to a timescale of milliseconds, but it is not clear whether they can refine their encoding abilities as a result of learning patterned stimuli. For example, can they enhance temporal integration to become better at distinguishing sequences? To explore how cortical coding plasticity underpins sequence discrimination, we developed a task in which mice distinguished between tactile ‘word’ sequences constructed from distinct vibrations delivered to the whiskers, assembled in different orders. Animals licked to report the presence of the target sequence. Optogenetic inactivation showed that the somatosensory cortex was necessary for sequence discrimination. Two-photon imaging in layer 2/3 of the primary somatosensory “barrel” cortex (S1bf) revealed that, in well-trained animals, neurons had heterogeneous selectivity to multiple task variables including not just sensory input but also the animal’s action decision and the trial outcome (presence or absence of the predicted reward). Many neurons were activated preceding goal-directed licking, thus reflecting the animal’s learnt action in response to the target sequence; these neurons were found as soon as mice learned to associate the rewarded sequence with licking. In contrast, learning evoked smaller changes in sensory response tuning: neurons responding to stimulus features were already found in naïve mice, and training did not generate neurons with enhanced temporal integration or categorical responses. Therefore, in S1bf sequence learning results in neurons whose activity reflects the learnt association between target sequence and licking, rather than a refined representation of sensory features. Taken together with results from other laboratories, our findings suggest that neurons in sensory cortex are involved in task-specific processing and that an animal does not sense the world independently of what it needs to feel in order to guide behaviour.

My lab seeks to understand how the mammalian brain performs the computations that underlie cognitive functions, including decision-making, short-term memory, and spatial navigation, at the level of the building blocks of the nervous system, cell types and neural populations organized into circuits. We have developed methods to measure, manipulate, and analyze neural circuits across various spatial and temporal scales, including technology for virtual reality, optical imaging, optogenetics, intracellular electrophysiology, molecular sensors, and computational modeling. I will present recent work that uses large scale calcium imaging to reveal the functional organization of the mouse posterior cortex for flexible decision-making during spatial navigation in virtual reality. I will also discuss work that uses optogenetics and calcium imaging during a variety of decision-making tasks to highlight how cognitive experience and context greatly alter the cortical circuits necessary for navigation decisions.

Neural codes, such as temporal codes (precisely timed spikes) and rate codes (instantaneous spike firing rates), are believed to be used in encoding sensory information into spike trains of cortical neurons. Temporal and rate codes co-exist in the spike train and such multiplexed neural code-carrying spike trains have been shown to be spatially synchronized in multiple neurons across different cortical layers during sensory information processing. Inhibition is suggested to promote such synchronization, but it is unclear whether distinct subtypes of interneurons make different contributions in the synchronization of multiplexed neural codes. To test this, in vivo single-unit recordings from barrel cortex were combined with optogenetic manipulations to determine the contributions of parvalbumin (PV)- and somatostatin (SST)-positive interneurons to synchronization of precisely timed spike sequences. We found that PV interneurons preferentially promote the synchronization of spike times when instantaneous firing rates are low (<12 Hz), whereas SST interneurons preferentially promote the synchronization of spike times when instantaneous firing rates are high (>12 Hz). Furthermore, using a computational model, we demonstrate that these effects can be explained by PV and SST interneurons having preferential contribution to feedforward and feedback inhibition, respectively. Overall, these results show that PV and SST interneurons have distinct frequency (rate code)-selective roles in dynamically gating the synchronization of spike times (temporal code) through preferentially recruiting feedforward and feedback inhibitory circuit motifs. The inhibitory neural circuit mechanisms we uncovered here his may have critical roles in regulating neural code-based somatosensory information processing in the neocortex.