Changing strengths of learned associations: neuronal ensemble mechanism

The ability to learn and remember the changing meanings about learned associations of biologically significant experiences and sensory stimuli is crucial for survival. For example, animals respond to external cues that predict the availability of natural rewards (e.g. food). However, they will learn to inhibit responding to these cues if the same cues fail to predict their availability. For many decades, a major goal of neuroscience has been to reveal the precise location and physiological nature of representations about these associations in the brain. Recent studies implicate the role of a minority of sparsely distributed, activated neurons called 'neuronal ensembles' in forming mental representations about learned associations about salient experiences and associated environmental cues. Although it is established that these ensembles play a causal role in mediating these associations, we do not fully understand the mechanisms that are involved in their formation. Also, little is known about what happens to these neurons once the original meaning of the learned associations change, such as when a cue no longer predicts food availability. A likely site where these changes occur to encode the meaning of these associations at neuronal ensembles is at the synapse, the key sites of neuronal communication. It is thought that enduring changes at the synapse akin to 'neuronal rewiring' underlie the formation of new associative representations. However, the precise nature of this rewiring process, especially with regards to how neuronal ensembles establish and maintain these associations, is largely unknown due to the technical challenges in identifying and recording from activated neurons.

The aim of this grant is to elucidate the mechanisms that modulate the strength of learned associations between natural rewards and associated environmental cues at the synaptic and local circuit level in neuronal ensembles. We will accomplish this by using an animal model of associative learning, where a mouse learns how to associate a tone cue with sucrose delivery. Once this association forms, a tone cue elicits a conditioned approach response towards the sucrose delivery receptacle. Conversely, this response is inhibited following a procedure called 'extinction learning' when the animal learns that the cue does not predict sucrose delivery. It is widely believed for this type of learning that the original associative memory is not erased but rather suppressed, since exposure to sucrose can re-trigger the approach response. At the neuronal level, a sucrose predictive cue elicits activation of a minority of neurons in the prefrontal cortex. This brain area is important for motivated behaviours, such as securing food sources, and these activated neurons are believed to encode information about sucrose and its associated cues. We will selectively record from these neurons using transgenic mice in which these activated neurons can be identified and physiologically characterised. We will examine structural alterations at their synapses to reveal the changes in neuronal wiring, after mice have learned that the cue predicts or no longer predicts sucrose delivery. We will also examine changes in 'connectivity' or the ability of one ensemble member to communicate with another, a process that may facilitate activation of the entire ensemble representation. Finally, we will characterize the behavioural role of these neurons that undergo rewiring by silencing their activity and testing this effect on conditioned approach responses, and whether the mice can relearn the original conditioning task.

Understanding these neuronal mechanisms will shed light on basic associative learning processes that are integral to healthy cognitive functioning. Moreover, it will reveal specific alterations at synapses on behaviourally relevant neurons and could potentially reveal new therapeutic targets.

A minority of sparsely distributed, activated neurons called 'neuronal ensembles' mediate learned associations about biologically significant experiences and associated environmental cues in the prefrontal cortex. During Pavlovian conditioning, animals learn how to associate a cue (e.g. tone) with a reward (e.g. sucrose). We recently observed that following this procedure, the presentation of a sucrose-associated cue eliicts neuronal ensemble activity in this area, as indicated by increased expression of the neural activity marker, c-Fos. The aim of this grant is to elucidate the mechanisms that establish and maintain learned associations at the synaptic and local circuit level in prefrontal cortex neuronal ensembles during acquisition of a Pavlovian association (i.e. cue predicts sucrose) and extinction (i.e. cue no longer predicts sucrose). Also, we will determine the behavioural role of these neurons. These goals will be accomplished using innovative transgenic approaches that selectively characterise and silence activated neurons that express c-Fos. These techniques differ from conventional techniques that characterise or manipulate neurons regardless of neuronal activity history, and hence do not directly examine learning relevant changes. First, we will selectively record from the sucrose cue activated neurons during acquisition to reveal the synaptic and local connectivity properties that establish appetitive associations. Next, we will record from these neurons following extinction learning to determine the dynamic nature of these appetitive representations when these associations are thought to be suppressed, but not erased. Finally, we will determine if these neuronal ensembles are necessary for eliciting conditioned reponses and for reconditioning. Many neuroscientists will expoit these findings by determining whether similar neuronal rewiring mechanisms occur in other brain areas or in other forms of associative learning (e.g. addiction, fear).

Who will benefit from this research?
This project will reveal key neuronal wiring processes at the synapse that help establish and maintain mental representations about natural rewards (e.g. sucrose) and the stimuli that surround them. These wiring processes are important to understand mechanisms of memory storage and retrieval, which tend to go awry in old age. The findings from this fundamental research will have widespread appeal and benefit the following groups that have a vested interest in learning more about the mechanisms of associative learning and memory, and appetitive behaviours: 1) Academics, including researchers in psychology, pharmacology, and neuroscience; and the post-doctoral researcher and technician who will compose my research team 2) The pharmaceutical industry that are looking for new drug targets and PET tracers. 3) The general public, including the elderly that suffer from memory decline (e.g. Alzheimer's), and those who suffer from excessive food cravings when exposed to food-associated stimuli. 4) Non-profit research promotion groups, such as the European Association for the Study of Obesity (EASO), which use scientific evidence for effective anti-obesity educational campaigns.

How will they benefit from this research?
1) Academics: In terms of capacity-building impact, the post-doctoral research fellow and research technician will greatly benefit from learning the advanced, multi-disciplinary research approaches utilised here (e.g. neuronal ensemble inactivation, neuronal ensemble recordings using a confocal-imaging capable electrophysiology rig) and will be highly sought after in the in both academia (e.g. neuroscience research) and industry (e.g. pharmaceutical companies, neurophysiology/imaging companies). 2) The pharmaceutical industry: Our research may reveal novel excitatory and inhibitory receptors that are expressed on neurons activated by associative representations that play a role in appetitive behaviours. Thus, data from this research will help devise more targeted therapeutic approaches that selectively restore normal function in associative learning and appetitive behaviour relevant neurons instead of treating neurons as a uniform cluster. It may also generate novel ligands used in PET imaging to detect brain areas in humans that contain similar receptors. Such ligands may be used to detect the vulnerable brain areas for conditions such as excessive food cravings and maladaptive eating, and confirm the relevance of our pre-clinical findings. I plan to communicate my findings to these companies via medicinal chemistry researchers at Sussex with industry contacts that are capable of evaluating the translational value of my findings. Although it is a goal that may take many years to achieve, this selective approach may one day generate therapies that improve quality of life with lower side effects, such as restoring associative memories or reducing unwanted cue-induced eating while leaving normal cognitive functions intact. 3) The general public: Newly devised therapeutics could benefit the elderly who have a problem remembering information associated with important events or dieters who are trying to control their food cravings. The general public will benefit as a result since less money is spent on health care costs associated with medical conditions caused by cognitive decline or excessive eating. 4) Research promotion groups (EASO), may effectively campaign to the public about the neurobiological effects of increased sucrose consumption that contributes to obesity, by utilising our data on how neuronal representations of sucrose and associated stimuli persistently linger in the brain. They may also use these findings to inform public health policy makers. I have already contacted EASO and we plan to have regular meetings to discuss my research findings.