Final Report Summary - HIPPOPROJECTION (Role of descending hippocampal outputs in anxiety studied using a novel pharmaco-genetic efferent inhibition tool)
We initially proposed to use a novel pharmaco-genetic tool based on the serotonin 1B receptor (Htr1b) to allow the inhibition of selected neuronal projections in behaving mice. This receptor is exclusively axonally located and its activation mediates inhibition of neurotransmitter release in various cell-types in the brain. For versatility, the system would depend on the expression of Htr1b via adeno-associated virus (AAV) delivery (in collaboration with Dr Valery Grinevich, MPI, Heidelberg, Germany) in mice lacking the endogenous receptor (Saudou et al., 1994). Rapid inhibition of anatomically distinct hippocampal efferent projections would be achieved by the tissue-specific injection (via chronically implanted cannulae) of the selective Htr1b receptor agonist, CP-94253. Selective inhibition of hippocampal projections to frontal cortex would be achieved by the bilateral delivery of AAV-Htr1b to different ventral or dorsal CA1 (and later also subiculum) subregions followed by injections of CP-94253 into the ventral medial frontal cortex. Changes in anxiety behavior in the presence or absence of the benzodiazepine, diazepam, would be assessed in the open field and elevated plus maze tests as well as in the dimensionality emergence assay (Fonio et al., 2009). In collaboration with Dr Josh Gordon (Columbia University, New York, United States (US)), we would also carry out in vivo electrophysiological recordings to assess associated changes in hippocampus-frontal cortex functional connectivity (Adhikari et al., 2010). We hypothesised that the selective inhibition of hippocampus-frontal cortex projections would be anxiolytic and would suppress the anxiolytic action of benzodiazepines.
Based on this, the three specific aims in which the project has been divided are the following:
Specific aim 1 - Development of novel tool for the rapid inhibition of genetically defined neural projections
A pharmaco-genetic tool for the rapid inhibition of selected neural projections has been developed in collaboration with Bianca Silva (PhD student, European Molecular Biology Laboratory (EMBL) Monterotondo). However, the system is based on the human M4 DREADD (hM4D DREADD) receptor instead of the serotonin Htr1b receptor originally proposed. The advantage of the new system is that it allows us to carry out our studies in wild type animals instead of the knock-out for the Htr1b receptor previously required.
Specific aim 2 - Validation of hM4D DREADD/CNO system for inhibition of hippocampal efferents
An adeno-associated virus (AAV) expressing the hM4D DREADD receptor has been produced in collaboration with Dr Valery Grinevich (MPI - Medical Research, Heidelberg, Germany). A group of mice was bilaterally injected in the ventral hippocampus and several parameters (titre, volume, route and location of injection) have been optimised in order to express hM4D DREADD in a significant fraction of ventral CA1 projection neurons.
Specific aim 3 - Role of hippocampus-frontal cortex outputs in anxiety
Besides the hippocampus-frontal cortex output, the hippocampus-septal projection is also being characterised. For this, recording electrodes have been chronically implanted in the ventral hippocampus (vHIP), prelimbic area of the medial prefrontal cortex (mPFC), and rostral lateral septum (rLS). Implanted animals have been subjected to the open field and elevated plus maze and prior to these tests in a familiar arena. Local field potentials recordings corresponding to the vHIP, mPFC and rLS have been analysed.
Although some behavioural experiments need to be completed, connectivity analysis of the local field potentials corresponding to the vHIP, mPFC and rLS indicate that, in a familiar environment, both vHIP-mPFC and vHIP-rLS projections are equally synchronised. Importantly, connectivity in both projections changes during the exposure to anxionegic environments (especially during exposure to the elevated plus maze) suggesting that both projections are similarly recruited during anxiety.
In addition, the researcher has been working on another line of research previously established in the host laboratory and to which she contributed significantly. We have examined the contribution of DG to hippocampal learning and plasticity combining a pharmacogenetic tool for the rapid and transient suppression of neural activity with in vivo electrophysiology during trace eye-blink conditioning. Our studies confirm that activity in DG granule cells is not necessary for the retrieval of hippocampal memory. However, suppression of DG during learning was associated with rapid and persistent memory loss, as revealed by long-term suppression of both conditioned responding and learning-associated plasticity. Pharmacological studies demonstrated a role for adenosine A1 receptor activation in this synaptic depotentiation, suggesting that direct entorhinal-CA1 inputs promote memory loss during learning. Similar memory impairment could be induced by DG suppression during contextual fear conditioning, demonstrating its generalization across hippocampus-dependent memory tasks. Finally, we show that activation of an endogenous inhibitory receptor selectively expressed in DG granule cells can induce rapid and persistent memory loss, opening the possibility of the targeted erasure of hippocampal memories.
Based on this, the three specific aims in which the project has been divided are the following:
Specific aim 1 - Development of novel tool for the rapid inhibition of genetically defined neural projections
A pharmaco-genetic tool for the rapid inhibition of selected neural projections has been developed in collaboration with Bianca Silva (PhD student, European Molecular Biology Laboratory (EMBL) Monterotondo). However, the system is based on the human M4 DREADD (hM4D DREADD) receptor instead of the serotonin Htr1b receptor originally proposed. The advantage of the new system is that it allows us to carry out our studies in wild type animals instead of the knock-out for the Htr1b receptor previously required.
Specific aim 2 - Validation of hM4D DREADD/CNO system for inhibition of hippocampal efferents
An adeno-associated virus (AAV) expressing the hM4D DREADD receptor has been produced in collaboration with Dr Valery Grinevich (MPI - Medical Research, Heidelberg, Germany). A group of mice was bilaterally injected in the ventral hippocampus and several parameters (titre, volume, route and location of injection) have been optimised in order to express hM4D DREADD in a significant fraction of ventral CA1 projection neurons.
Specific aim 3 - Role of hippocampus-frontal cortex outputs in anxiety
Besides the hippocampus-frontal cortex output, the hippocampus-septal projection is also being characterised. For this, recording electrodes have been chronically implanted in the ventral hippocampus (vHIP), prelimbic area of the medial prefrontal cortex (mPFC), and rostral lateral septum (rLS). Implanted animals have been subjected to the open field and elevated plus maze and prior to these tests in a familiar arena. Local field potentials recordings corresponding to the vHIP, mPFC and rLS have been analysed.
Although some behavioural experiments need to be completed, connectivity analysis of the local field potentials corresponding to the vHIP, mPFC and rLS indicate that, in a familiar environment, both vHIP-mPFC and vHIP-rLS projections are equally synchronised. Importantly, connectivity in both projections changes during the exposure to anxionegic environments (especially during exposure to the elevated plus maze) suggesting that both projections are similarly recruited during anxiety.
In addition, the researcher has been working on another line of research previously established in the host laboratory and to which she contributed significantly. We have examined the contribution of DG to hippocampal learning and plasticity combining a pharmacogenetic tool for the rapid and transient suppression of neural activity with in vivo electrophysiology during trace eye-blink conditioning. Our studies confirm that activity in DG granule cells is not necessary for the retrieval of hippocampal memory. However, suppression of DG during learning was associated with rapid and persistent memory loss, as revealed by long-term suppression of both conditioned responding and learning-associated plasticity. Pharmacological studies demonstrated a role for adenosine A1 receptor activation in this synaptic depotentiation, suggesting that direct entorhinal-CA1 inputs promote memory loss during learning. Similar memory impairment could be induced by DG suppression during contextual fear conditioning, demonstrating its generalization across hippocampus-dependent memory tasks. Finally, we show that activation of an endogenous inhibitory receptor selectively expressed in DG granule cells can induce rapid and persistent memory loss, opening the possibility of the targeted erasure of hippocampal memories.