Understanding the neuronal circuitry underlying Memory and Anxiety: the roles of GABA and CCK

Lead Research Organisation: Kiel University
Department Name: Institute of Physiology

Abstract

Our brain processes information in a network of nerve cells. The communication between these nerve cells (neurons) occurs via specialized structures called synapses. When an electrical signal (action potential) travels along the neuron and reaches the synapse then a messenger substance is released from the synapse. This messenger substance is received by a receptor protein in the membrane of the adjacent nerve cell. In general nerve cells can be grouped into ?Yes-neurons?, which activate other neurons via their messenger substance and communicate information between different brain regions, and ?No-neurons?, which inactivate the ?Yes-cells? and thus regulate the flow of information. Dysfunction of subclasses of ?No-neurons? leads to pathological information-processing in psychiatric disorders. Recent studies have indicated that one particular sub-type of the ?No-neurons?, the so called CCK+ interneurons might be very important for controlling the flow of information related to the formation of memories and the regulation of mood and anxiety. We want to directly test the function of these particular ?No-neurons? in memory formation and fear/anxiety. To this end we are developing new methods that allow us to functionally remove specific nerve cells and messenger substances in model systems to test their behavioural relevance - similar to an electrician who goes through the components of a circuit board to identify a problem. Understanding how ?Yes neurons? and ?No-neurons? interact to generate memories and emotions will help to understand what is going wrong in memory and anxiety disorders and may lead to the development of new therapeutic strategies to treat such diseases.

Technical Summary

Memory deficits as well as anxiety/mood disorders have a very high prevalence, cause substantial personal distress and represent a significant economic burden for society. Understanding the neuronal circuits underlying these pathologies is a prerequisite for new therapeutic strategies. However, progress in the field of circuit analysis has been slow due to the lack of methods with sufficient resolution. Using newly developed molecular tools we want to directly test the involvement of cholecystokinin (CCK)-expressing GABAergic interneurons in learning and memory and mood regulation. In the hippocampus, a structure critical for learning, recent studies indicate that CCK+ interneurons play a key role in orchestrating the encoding of spatial information by individual principal cells. Apart from GABA CCK+ interneurons also release the neuropeptide CCK, which strongly enhances memory performance after intra-hippocampal application, whereas CCK antagonists or knockout of CCK (or its receptor) impair memory performance. In addition, CCK-interneurons in the hippocampus and basolateral amygdala have been implicated in the regulation of emotion: They form a hub for inputs from neurotransmitters such as serotonin and neuromodulatory lipids such as endocannabinoids that control mood. Furthermore, CCK+ interneurons act through GABAA receptor types, which form the target of the most commonly used anxiolytics. Whereas injections of CCK, evoke fear and anxiety, CCK antagonists are strongly anxiolytic and reduce panic attacks in patients.
The molecular techniques for high resolution in vivo circuit dissection that we have developed and will develop for this proposal put us into a unique position to directly test the hypothesis that CCK-interneurons play key roles in memory and emotion. To analyse their role in memory we will 1, functionally remove them from the hippocampus by cell-type-selective block of synaptic output with tetanus toxin and 2, independently perturb their GABAergic and CCKergic out-put by cell-type-selective knock down to dissect the neurotransmitter functions. We will investigate CCK-interneurons in anxiety by 3, targeting these cells in the basolateral amygdala. These perturbations will be analysed at the cellular (in vitro electrophysiology), network (local field potential recordings) and behavioural (memory and anxiety tests) level.

Publications

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Description Our goal is to investigate the behavioural relevance of CCK-positive interneurons. To do this we require to selectively target these neurons, which is complicated by the fact that principal cells also express CCK. We have used various approaches to target CCK-positive interneurons.
We have tried published short sequence promoters in AAV viruses, which however showed non-specific expression. We have tried an intersectional approach based on CCK-Cre and interneuron Flp mice. Here we found that previously published floxed stop sequences did not inhibit transgene expression, which was unexpected. We developped a new expression control system based on the use of heterotypic sets of antiparallel FRT sites, which shows no leakiness and finally allowed to selectively target expression to neurons that express both Cre- and Flp-recombinase. Using this system in compound CCK-Cre/ interneuron Flp mice we found that only about 50% of interneurons really expressed Flp in interneurons. Accordingly we have now changed the strategy to express Flp from an AAV, which only expresses in priincipal cells. In this scenario Flp expression prohibits transgene expression from a second AAV. We are hopeful that this approach will finally permitt cell-type-selective targeting of these interneurons. First results are promising. We are currently working on a confirmation of these results and will then proceed with the proposed program of work.
Exploitation Route We have spent much time on the development of molecular/viral tools and have not produced much data regarding the original question yet. The tools will be useful for the scientific community. We hope to soon produce data on the behavioural function of CCK-positive neurons, which may be relevant to psychiatry.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Title AAV that expresses Flp recombinase under the CamKII promoter 
Description The CamKII promoter should drive expression of Flp-recombinase mainly in principal cells. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact This virus should allow removal or activation of genes in principal cells. 
 
Title Conditional AAV, where gene expression is activated by Cre recombinase and deactivated by Flp expression 
Description The transgene to be expressed by an adeno-associated virus is inserted between two sets of heterologous antiparallel sets of loxP sites (FLEX cassette), which is then flaked by two parallel Frt sites. This should allow gene expression in a poulation of neurons, which expresses Cre recombinase but not Flp recombinase in an intersectional gene targeting approach. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact This should allow gene expression in a poulation of neurons, which expresses Cre recombinase but not Flp recombinase in an intersectional gene targeting approach. 
 
Title Conditional AAV, where gene expression is only activated in the presence of Cre and Flp recombinase 
Description We built a modular construct, which permits expüression of a gene of interest under the condition that the 2 recombinases Cre and Flp are present. This vector can be used to enhance the specificity of viral gene transfer e.g. in the mouse brain. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? Yes  
Impact We expect, that this vector will allow us to persue our research goals. 
 
Title Flex cassette adapted to FRT sites 
Description In analogy of the FLEX cassette which permits 100% tight Cre dependent transgene expression from e.g. AAV vectors, we have generated a FRT site-based cassette which works in vitro and vivo. 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? No  
Impact Combination of Cre and Flp based cassettes will permit leak-free intersectional targeting of transgene expression. 
 
Title cell-specific rAAV mediated knock down 
Description we have generated cre recombinase-dependent rAAVs for microRNA-based shRNA knock down as proposed in the application. First Experiments in vitro suggest very good knock down efficiency. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact This tool wil permitt the cell-type- and region-specific manipulation of defined molecules. We will use this tool to dissect the role of different but co-released transmitters in the brain.