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Developmental principles for the functional specialisation of inhibitory circuits in neocortical areas

Periodic Reporting for period 2 - DEVINCI (Developmental principles for the functional specialisation of inhibitory circuits in neocortical areas)

Reporting period: 2020-01-01 to 2021-06-30

The mammalian neocortex consists of discrete, but highly interconnected, functional areas that collectively encode features of the environment, form associations between stimuli and drive behaviour by transforming sensory input into motor output. All neocortical areas are organised into six layers containing two major classes of neurons, excitatory glutamatergic pyramidal cells and inhibitory GABAergic interneurons. However, each area has distinctive cytoarchitectonical features and inputs that largely determine its computational capabilities.

We hypothesised that distinct patterns of inhibitory connectivity influence the functional specialisation of neocortical areas. To test this idea, our research programme has three specific objectives:

1. Characterise the developmental emergence of interneuron diversity across neocortical areas

2. Determine how the normal process of interneuron cell death impacts the distribution of interneurons in different neocortical areas.

3. Identify causal links between interneuron distributions and the function of cortical areas.
1. Interneuron diversity across neocortical areas

We are generating quantitative four-dimensional maps of the distribution of cortical interneurons across cortical areas. To this end, we are using relatively straightforward mouse genetic approaches (Cre-based reporters) to identify the distribution of large subclasses of interneuron (PV, SST and 5HTR). In addition, we are using more sophisticated mouse genetics (Cre- and Flp-based reporters) to identify specific subtypes of cortical interneuron (e.g. VIP/CR bipolar cells). We have made good progress in the generation of the relevant mouse colonies, but some of them involve up to three different alleles and took over 9 months to produce. In parallel, we are developing methods to quantify the density different subtypes of interneuron throughout the entire cerebral cortex and across different layers, along with the appropriate statistical approaches to identify significant differences in distribution patterns. The results of the first round of analysis involving the three main subclasses of interneuron are being analysed at the moment.

2. Impact of cell death in interneuron distribution

We are generating mice in which cell death is genetically prevented by deletion of Bax and Bak in all GABAergic interneurons using Vgat-Cre line. We will generate our-dimensional maps of the distribution of cortical interneurons across cortical areas in the absence of programme cell death, which will be compared to those generated under aim 1. The analysis of these experiments will reveal in which cortical areas and layers the final distribution of specific subtypes of interneuron is more strongly modulated by developmental cell death. In addition, we are expanding our understanding of interneuron programmed cell death by investigating the mechanisms mediating this process in different types of interneuron within the 5HTR subclass. Interneurons also undergo programme cell death in the striatum, and we are currently investigating whether their survival is dependent on local signals or, as our data so far seem to suggest, on inputs from cortical pyramidal cells during the normal period of programmed cell death.

3. Interneuron distributions and the function of cortical areas

We are beginning to investigate how disruption of the normal process of programmed cell death (in pyramidal cells and interneurons or only in interneurons) impact early network activity in different cortical areas. To this end, we have generated mice in which we can both prevent the normal process of programmed cell death and monitor the activity of pyramidal cells and interneurons using calcium imaging. We will investigate network dynamics in these mice starting from postnatal day 6. We are also exploring the impact of perturbing the final number of cortical interneurons in the function of different cortical areas in adult mice. We have established a visual discrimination learning paradigm in head-fixed mice in which we assess the ability of mice to distinguish between two different gratings. We are planning to set up a similar paradigm based on somatosensory discrimination mediated by the whiskers. Once this second paradigm is in place, we will investigate how modulation of the final number of interneurons in the visual and somatosensory cortices impact performance.
In the next few months we will perform the following experiments:

1. We will finalise the quantification of the distribution of interneuron subclasses and subtypes throughout the entire cerebral cortex. We will also have more granular data (relative densities, laminar distributions, etc.) for a selective number of cortical areas, including all three main primary sensory areas (somatosensory, visual and auditory), motor cortex, and representatives of the prefrontal cortex such as the prelimbic and infralimbic areas.

2. We will perform single-cell RNA sequencing (scRNA-seq) experiments to obtain a much more granular characterisation of interneuron diversity in selected cortical areas. These experiments will be validated through multiplex fluorescence in situ hybridisation approaches (with up to 12 probes).

3. We will quantify the distribution of interneuron subclasses and subtypes throughout the entire cerebral cortex in mice in which programmed cell death is prevented in all interneurons. The maps obtained from these experiments will be compared to control distributions.

4. We will carry out further experiments to determine the mechanisms controlling the survival of bipolar interneurons and striatal interneurons.

5. We will perform calcium imaging experiments in vivo to monitor network activity during early postnatal development in mice with abnormal distributions of cortical interneurons.

6. We will establish a tactile (whisker-mediated) discrimination task to evaluate somatosensory perception in mice with abnormal distributions of cortical interneurons.
Regional distribution of different interneuron subclasses