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Optical Interrogation of Hippocampal Dentate Granule Circuit Dynamics in Health and Disease

Periodic Reporting for period 1 - Circdyn (Optical Interrogation of Hippocampal Dentate Granule Circuit Dynamics in Health and Disease)

Reporting period: 2017-01-01 to 2018-12-31

The specific objectives of my H2020 MSCA proposal was to establish a decision-making task amenable to all-optical recording, and apply large-scale, chronic calcium imaging in awake behaving mice to measure neural activity to determine their causal role in such behavior and to test for a potential contribution of neural circuit alterations in animal models of neurodevelopmental disorders such as Rett syndrome (RTT). This work is extended to study decision-making in behavioural flexibility that we are about to submit as a manuscript. The work on behavioural flexibility is now extended to RTT animals that show deficiency of cognitive flexibility.

Understanding neural correlates of brain dynamics in health and brain disorders is a fundamental yet long-standing challenge of neuroscience. With the advancement of several ground-breaking neurotechnological tools for studying neural circuits in an unprecedented spatio-temporal resolution, we have started to understand mechanisms underlying behaviour-dependent circuit dynamics and their alterations in brain disorders. Rodent models of neurodevelopmental disorders have provided an important tool to study dysfunctions at cellular, molecular and circuit levels as well (Banerjee et al. 2019 BRAIN).

Our overall objective is to study neural correlates of complex cognitive behaviour and identify mechanisms that would allow us to study cognitive dysfunctions seen in neurodevelopmental disorders in the autism spectrum.
"Probing Mesoscale Neural Circuits underlying Flexible Decision-Making.

One of the key aspects of decision-making is behavioural flexibility i.e. switching between appropriate goal-directed strategies based on stimulus/action-outcome evaluation. The orbitofrontal cortex (OFC), play an important role in invoking rule-based strategies to regulate adaptive decision-making. Flexible decision-making is severely compromised in neurological disorders such as autism including RTT. Behavioural assay probing flexible decision-making in cortical circuits: To study neural circuits underlying cognitive flexibility and its dysfunctions in RTT, I designed a novel reversal learning task in awake and head-fixed mice. This allowed monitoring sensory learning and reversal learning induced changes in goal-directed behaviour. Wild-type (WT) mice implanted with custom-designed head plates are placed on a water restriction regime. Once habituated for 3-4 days, mice proceed to a Go/No-go tactile-discrimination task, learning to lick for water reward in response to detecting a coarse sandpaper texture (P100), while withhold licking in response to a fine sandpaper (P1200). When learning performance is proficient, the reward contingencies are switched (P1200 becomes the new target texture for reward). Mice performing reversal learning tasks exhibited high performance scores for learning and reversal upon alterations in reward contingency (Fig. 4A-C, see preliminary data proposed project), [discrimination sensitivity (d’) > 1.5 for all mice, one session of 300 trials per day, n = 8 WT mice). To further study the causal link between S1, OFC and reversal learning, inhibitory DREADD constructs (hM4Di) were injected in either S1 or OFC. Virus injected mice were trained on the learning-phase of the discrimination task, CNO was injected intraperitoneally (i.p.) to silence neuronal activity in the OFC during rule-switching. OFC silenced animals showed impaired reversal learning with intact acquisition of the task, whereas, i.p. administration of CNO in animals injected with hM4Di in S1 showed learning deficits (Fig. 4D, see preliminary data proposed project). Deep imaging of OFC neurons with a gradient-index (GRIN) lens and a novel optical preparation. To study neural correlates of reversal learning, genetically-encoded fluorescent calcium indicator (AAV2.5-hSyn-GCaMP6f) is injected in specific cortical structures to enable simultaneous imaging of learning and reversal-induced changes in specific neural circuits. To directly probe how altered reward contingency is encoded and retrieved in neuronal populations in the mouse OFC, I have developed a novel optical preparation. OFC neurons expressing GCaMP6 were imaged using a metal cannula (diameter 1.2 mm) with a glass coverslip at the base, through which a gradient-index (GRIN) lens (diameter 1.0 mm) was inserted and imaged using a custom-built two-photon microscope. By longitudinal imaging of trial-by-trial Ca2+ responses from OFC neurons (first of its kind in mice), we revealed that neurons in the OFC are a key predictive substrate assigning value to sensory context upon reinforcer devaluation. Silencing OFC during reversal impairs prediction-update signals from OFC to alter selective of S1 ‘choice-selective’ neurons. Taken together, our experiments shed considerable light on the circuit mechanisms of tactile decision-making and the role of mouse orbital areas in behavioural flexibility.

Dissemination of the research results: Detailed methodologies, such as the memory-guided decision task, volume scanning of neural populations, was presented and openly discussed (with the goal of promoting the use of these methods in other laboratories), annually with the wider neuroscience community at smaller national and international conferences (e.g. EMBO meeting 2019, ZNZ symposium, Zurich 2018, Giessbach meeting 2019). An abstract is currently being prepared to be submitted to Society for Neurosciences meeting in Chicago (October 2019). A mini-symposium is being organised on ‘Learning dynamics’ to be submitted for the FENS meeting in Glasgow 2020. I have also dissipated knowledge through EMBO ""Two-photon imaging"" course that Dr. Helmchen organises (EMBO meeting in Zurich 2017). Finally, working with Prof. Benjamin Grewe at ETH-Zurich, I organised INI-HiFO symposium 2017, 2018, 2019, to dissipate acquired knowledge and skills to undergraduates and researchers to further encourage collaborations.

Progress beyond the state of the art: Finding causes, cures, and effective treatments for neurobiological disorders: While the ongoing work is promising, our next aims involves training several mice on both linear treadmill and behavioural flexibility task, causally manipulate circuit elements and record neuronal population activity in DG, CA1, and OFC to understand their functional role in such behaviour, and finally, use MeCP2 Het female or global Mecp2 knock out male animals to study dysfunctions in Rett syndrome (RTT). This approach will open-up new avenues to elucidate granule cell function within the complex hippocampal and deeper cortical network during distinct stages of memory processing and behavioural flexibility and their functional alterations in RTT.