LEARNING IMPULSE CONTROL IN A NOVEL ANIMAL MODEL: SYNAPTIC, CELLULAR, AND PHARMACOLOGICAL SUBSTRATES

Impulse control, an executive process that restrains inappropriate actions, is impaired in numerous psychiatric conditions. This thesis reports three experiments that utilized a novel animal model of impulse control, the response inhibition (RI) task, to examine the substrates that underlie learning this task. In the first experiment, rats were trained to withhold responding on the RI task, and then euthanized for electrophysiological testing. Training in the RI task increased the AMPA/NMDA ratio at the synapses of pyramidal neurons in the prelimbic, but not infralimbic, region of the medial prefrontal cortex. This enhancement paralleled performance as subjects underwent acquisition and extinction of the inhibitory response. AMPA/NMDA was elevated only in neurons that project to the ventral striatum. Thus, this experiment identified a synaptic correlate of impulse control. In the second experiment, a separate group of rats were trained in the RI task prior to electrophysiological testing. Training in the RI task produced a decrease in membrane excitability in prelimbic, but not infralimbic, neurons as measured by maximal spiking evoked in response to increasing current injection. Importantly, this decrease was strongly correlated with successful inhibition in the task. Fortuitously, subjects trained in an operant control condition showed elevated infralimbic, but not prelimbic, excitability, which was produced by learning an anticipatory signal that predicted imminent reward availability. These experiments revealed two cellular correlates of performance, corresponding to learning two different associations under distinct task conditions. In the final experiment, rats were trained on the RI task under three conditions: Short (4-s), long (60-s), or unpredictable (1-s to 60-s) premature phases. These conditions produced distinct errors on the RI task. Interestingly, amphetamine increased premature responding in the short and long conditions, but decreased premature responding in the unpredictable condition. This dissociation may arise from interactions between amphetamine and underlying cognitive processes, such as attention, timing, and conditioned avoidance. In summary, this thesis showed that learning to inhibit a response produces distinct synaptic, cellular, and pharmacological changes. It is hoped that these advances will provide a starting point for future therapeutic interventions of disorders of impulse control.

A structural basis for different antifreeze protein roles

Antifreeze proteins (AFPs) are produced by a variety of organisms to either protect them from freezing or help them tolerate being frozen. Recent structural work has shown that AFPs bind to ice using ordered surface waters on a particular surface of the protein called the ice-binding site (IBS). These 'anchored clathrate' waters fuse to particular planes of an ice crystal and hence irreversibly bind the AFP to its ligand. An AFP isolated from the perennial ryegrass, Lolium perenne (LpAFP) was previously modelled as a right-handed beta helix with two proposed IBSs. Steric mutagenesis, where small side chains were replaced with larger ones, determined that only one of the putative IBSs was responsible for binding ice. The mutagenesis work also partly validated the fold of the computer-generated model of this AFP. In order to determine the structure of the protein, LpAFP was crystallized and solved to 1.4 Å resolution. The protein folds as an untwisted left-handed beta-helix, of opposite handedness to the model. The IBS identified by mutagenesis is remarkably flat, but less regular than the IBS of most other AFPs. Furthermore, several of the residues constituting the IBS are in multiple conformations. This irregularity may explain why LpAFP causes less thermal hysteresis than many other AFPs. Its imperfect IBS is also argued to be responsible for LpAFP's heightened ice-recrystallization inhibition activity. The structure of LpAFP is the first for a plant AFP and for a protein responsible for providing freeze tolerance rather than freeze resistance. To help understand what constitutes an IBS, a non-ice-binding homologue of type III AFP, sialic acid synthase (SAS), was engineered for ice binding. Point mutations were made to the germinal IBS of SAS to mimic key features seen in type III AFP. The crystal structures of some of the mutant proteins showed that the potential IBS became less charged and flatter as the mutations progressed, and ice affinity was gained. This proof-of-principle study highlights some of the difficulties in AFP engineering.