1. Overview of Research Plans.
The successful treatment of memory impairments and neurodegenerative disorders critically depends on our understanding of the storage and recollection of memory episodes. Specifically, an understanding of the interaction between amnesic and neurodegenerative syndromes would aid the development of effective treatments. One of the key questions in neuroscience is: how do brain networks encode experience-dependent memory? Answering this question will give us universal tool to treat multiple brain disorders. The anatomical focus of my current research interests is the interaction between limbic, basal forebrain and nigro-striatal circuits as a possible candidate for interregional coordination of spatial navigation and context-dependent components of episodic memory.
I plan to examine if 1) neuromodulation from cholinergic neurons in forebrain and pontine tegmentum and 2) neuromodulation from dopaminergic and GABA-ergic neurons from ventral tegmental area (VTA) and substantia nigra can regulate the properties of spatial experience-dependent spatial navigation to guide behavior. Understanding the fundamental principles of this inter-regional signal processing will allow me to address my next goal: to apply pharmacological, electrophysiological and genetic tools to treat memory and movement disorders. The cellular response to cholinergic and dopaminergic neurotransmitters in hippocampal region is largely explored, but little is known about the optimizing effect of extrinsic manipulation of the neuromodulatory projections on the encoding properties of hippocampal neurons and mnemonic function of the limbic system. Therefore, the present proposal addresses two broad questions: 1) Can we enhance the hippocampal spatial representation by activation of the cholinergic/dopaminergic systems? 2) Should medial temporal lobe amnesia and depression be regarded as independent syndromes, or are they interlinked?
2. Current Research.
My concurrent investigation targets the septal area, which is interconnected with 1) the hippocampus which mediates spatial memory 2) ventral dopaminergic tegmentum, which mediates reward-motivated behavior, and 3) medial cholinergic septal inputs, which regulate of the limbic excitability and network rhythmicity, that are crucially involved in the neuropathology of the Alzheimer’s disease. There is, however, limited information about how hippocampal spatial and tegmental reward systems maintain feedback loop to synchronize their activity for place and reward. I propose that septal neurons integrate spatial and context reward value, enabling episodic memory for past experience to support future adaptive behavior. Using electrophysiological recordings from rats performing spatial and reinforcement tasks I am currently identifying the signal processing of the space and reward for each septal sub-region (manuscript in preparation). My optognetic stimulation design also explores the role of pathophysiological alteration of septal cholinergic and tegmental dopaminergic inputs on septo-hippocampal neuronal responses and adaptive behaviour.
3. Next Research Aims.
Hippocampus mediates the formation of adaptive memory for positive or negative experiences, but the neurophysiological mechanisms of this learning process however remain unknown (Shapiro et al., 2006). Hippocampus may encode locations independently from the stimuli and events, which are associated with these locations (Leutgeb et al., 2005). Recent findings deduced artificial association between place cells and place preference through use of optognetic (Ramirez et al., 2013; Redondo et al., 2014; Ryan et al., 2015) or electrical stimulation (de Lavilleon et al., 2015). These results provide key evidence linking place cell activity and context-dependent encoding of space (Eichenbaum, 2000).
3.1. Chronic Activation of Cholinergic Projections to the Episodic Memory Networks.
Using photostimulation protocols that mimic the spiking activity of septal neurons, I have shown that the modulatory role of septal cholinergic control over the hippcampal place field representation is mediated by the rhythmic entrainment of hippocampal neurons (Mamad et al., 2015). Recent work in my lab showed that the place cells are not simply coincidence detectors but they actively mediate the learning between context and location and this process is regulated by the dopaminergic neuromoudulator inputs (Mamad et al., 2017a). My next research aims target the investigation of cholinergic optogenetic and pharmacological control of neuromodulation in aged animals and Alzheimer model animals.
(i) Does selective cholinergic stimulation improves the hippocampal spatial representation in aged rats (RQ1)?
(ii) Does chronic cholinergic optogenetic stimulation improves the spatial memory performance in Alzheimer model rats (RQ2)?
(iii) Does chronic pharmacological agonist treatment improves the spatial representation and place field plasticity in aged rats (RQ3)?
(iv) Does chronic cholinergic optogenetic stimulation improves the hippocampal long-term synaptic plasticity (RQ4)?
(v) Is there a direct correlation between degree of optogenetic or pharmacological effect on the spatial memory performance and the hippocampal spatial representation or plasticity (RQ5)?
(vi) Are the electrophysiological, pharmacological and behavioural data related to decrease of amyloid plaques formation, neurofibrillary tangles alteration, restricted neuronal loss and neuroinflammation (RQ6)?
The optogenetic stimulation will target medial septum and pedunculopontine nucleus. To achieve these goals I plan to implement a methodological innovation: cross-bread of Alzheimer model of rat, strain: F344-Tg(Prp-APP,Prp-PS1) with transgenic model of rat that allows selective optogenetic activation of the septal cholinergic neurons, strain: Long Evans-Tg(ChAT-Cre)5.1Deis).
3.2. Chronic Control of Cholinergic and Dopaminergic Cross-Talk in the Limbic Circuitry.
Memory consolidation is the phenomenon by which a newly formed memory transitions from a fragile state to a stable, long-term state. Hippocampus temporally encodes representations of spatial context-dependent experiences (Knierim, 2003) and these memory traces are functionally strengthened in the cortical areas for long-term recollection (Nadel and Moscovitch, 1997; Kitamura et al., 2009; Tayler et al., 2013; Denny et al., 2014). The defining feature of consolidation is a finite time window that begins immediately after learning, during which a memory is susceptible to disruptions, such as protein synthesis inhibition, resulting in retrograde amnesia. I plan to apply the engram methodology to the test the network substrate for episodic memory consolidation and retrograde amnesia. My subsequent research aims target the investigation of dopaminergic optogenetic and pharmacological control of neuromodulation in animal model of depression.
(i) Does selective dopaminergic optogenetic stimulation improves the engram representation in control animals (RQ7)?
(ii) Does selective dopaminergic stimulation improves the engram representation in animal model of depression (RQ8)?
(iii) Does chronic pharmacological agonist treatment improves the consolidation of episodic memory in control and depression models (RQ9)?
(iv) Does chronic dopaminergic optogenetic stimulation up/downregulates the cholinergic innervation in septal/pedunculopontine nuclei (RQ10)?
(v) Is there a direct correlation between degree of optogenetic or pharmacological effect on the engram representation and the degree of depressive behaviour (RQ11)?
(vi) Are the electrophysiological, pharmacological and behavioural data related to misbalanced innervation of dopaminergic and cholinergic circuits during memory consolidation (RQ12)?
To achieve these goals I plan to implement a methodological innovation: engram circuitry identification. Currently, I collaborate with Prof Karl Deisseroth (Stanford University) for the investigation of engram cells plasticity in transgenic rats. I am designing experimental setup to label memory engram cells with adeno-associated viruses under the control of a tetracycline-responsive element-containing promoter. For each engram experiment, I will inject one of these viruses into the target brain region of c-fos-tTA transgenic rat, which express tTA under the control of a c-fos promoter.
The latest advances in optogenetic tools allow us to investigate the functional role of neuronal subtypes in behavioral contexts. Due to successful collaboration with Prof Boyden (MIT, Boston) I have developed innovative optogenetic tools in parallel with electrophysiological recordings in the context of rodent spatial behavior.
3.3. Interrogation of the Circuits Mediating both Amnesic and Depressive Dysfunction.
If we ultimately aim to enhance the memories for declarative events for the treatment of neurodegenerative we need to uncover 1) how the context-dependent episodes rewire the brain connectivity and 2) how we can manipulate this process. Current theories propose that memory of an event is represented by a population of neurons, referred to as engram (Ramirez et al., 2013; Josselyn et al., 2015; Tonegawa et al., 2015). Engrams engage overlapping neuronal populations for different memories (Cai et al., 2016; Rashid et al., 2016), such as the encoding of a single spatial environment with differing contextual emotional valences (Hsiang et al., 2014; Ye et al., 2016).
Despite recent advances in localizing and manipulating single engrams, it is still unclear which neurons remap to encode fearful experience and which neurons preserve their spatial fields. I have recently examined the rules governing aversion-induced place field remapping (Mamad et al., 2017b). We showed that the place cells remapping follows spatial and temporal pattern, which depends on the aversive stimulus perception.
(i) Does chronic stress lead to reduced plasticity of single neurons and engram representation (RQ13)?
(ii) Is chronic stress related to concurrent change of the hippocampus-dependent spatial learning and the acquisition of reward-related context (RQ14)?
(iii) Does chronic suppression of the neuronal connectivity within engram circuits related to enhanced propensity to depressive behaviour (RQ15)?
(iv) Does synaptic plasticity in hippocampal circuitry represent the encoding and retrieval of reinforcement and aversive episodes (RQ16)?
(v) Does pharmacological up/downregulation of dopaminergic and cholinergic projections to hippocampal circuitry improves the behavioural response to chronic stress (RQ17)?
(vi) Do the electrophysiological, pharmacological and behavioural data show correlates of engram pathophysiology in amnestic and depressive symptoms (RQ18)?
To achieve these goals I plan to implement a methodological innovation: two-colour calcium imaging in behaving rodents. I have applied with another collaborator with Dr Tomas Ryan (MIT, TCD) for the funding of miniaturized fluorescence microscope for live two-colour brain imaging. The research plan involves optogenetic manipulation of engram cells in different hippocampal areas and the regulation of the signal transmission by the neuromodulatroty subcortical inputs.
If the funding is successful I will be able to relate to behavior the following measurements in behaving animals: fluctuations in calcium levels correlating with events such as action potential generation, dendritic morphology, exocytosis of neurotransmitters, changes in synaptic plasticity, and gene transcription, in parallel with optogenetically-induced silencing or activation of large population of selected neurons. The study the population dynamics in hippocampal neurons is one of the most powerful tools for understanding the encoding and retrieval of memory. I have developed optogenetic closed-loop control of the hippocampal population response to aversive inputs mediated by amygdala.
4. Overview and Feasibility.
The field of electrophysiology has developed in last 50 years the backbone of neurocognitive understandings about the brain function. These fundamental findings brought the Nobel Prize 2014 for Physiology to the founders of the place and the grids cells. Recent progress in neuroscience techniques will allow us to translate the fundamental neuroscience knowledge into applications that treat neurocognitive disorders. Experiments performed so far only in vitro are now able to be tested in freely behaving animals, where we could relate neuronal communication to cognitive processes. The convergence between neuromodulatory models and behavioral research is finally possible due to 1) major advances in long-term multiple units recordings, 2) tremendous growth of anatomical and physiological data about episodic memory circuits, 3) recent development of the biological and computational tools for the evaluation of the signal processing in vivo and in silico. My research brings all three areas into focus, enabling our aim to describe how neuromodulatory mechanisms work together to augment network representations of previous experiences for the treatment of neurodegenerative diseases.