The Emergence of Hippocampal Function in Contextual Fear Conditioning in a Rodent Model

Jenn Foster (Neuroscience-Honors)

Advisor: Mike Burman

The dorsal hippocampus is required for auditory fear conditioning only when a temporal gap of sufficient length is inserted between the conditioned stimulus and the unconditioned stimulus (long trace conditioning), but not at shorter intervals. When the temporal gap is too short, it is unknown which part of the brain processes the association. To examine the role of the prefrontal cortex in short trace auditory fear conditioning, fear conditioning was conducted using either a short (2-s) or long (10-s) trace interval on animals who had received either dorsal hippocampus lesions or both dorsal hippocampus and prefrontal cortex lesions. While lesioning the dorsal hippocampus did reduce fear to a tone on long trace intervals, the combination lesions did not affect fear on the short trace interval, implying that that the prefrontal cortex is not involved in acquisition of the association.

Chlordiazepoxide’s Anxiolytic Effects Depend on Previous Experience and Differentially Affect the Anxiety of Rats on the Elevated Plus Maze and the Successive Alleys Test

Elissa Maunus (Neuroscience)

Advisor: Mike Burman

The biological substrates of anxiety are a complex mix of neural and endocrine systems.  To study a response that is so intricate in nature it is important to use simple animal models of anxiety.  However, although it is accepted that animal models are reliable measures of anxiety, it is unclear whether these different behavioral tasks assess common or distinct aspects of anxiety, and are therefore affected by the same treatment. To examine this question this experiment compares the effects of two controversial doses of chlordiazepoxide (CDP) (3 mg/kg, 8 mg/kg) on rats’ behavior on an elevated plus maze (EPM), a successive alleys test, and a hyponeophagia task.  Locomotion was also measured in order to ensure that CDP’s effects on anxiety behavior was not a result of the drug’s effects on general activity.  While both doses of CDP decreased anxiety on the EPM, this effect was the strongest when rats had previously participated in the successive alleys test.  CDP failed to significantly affect anxiety on the successive alleys test and the hyponeophagia test. Results that previous exposure to the successive alleys test attenuates the anxiolytic effects of chlordiazepoxide on the EPM are consistent with a phenomenon known as “one-trial tolerance commonly demonstrated when animals participate in multiple trials of the EPM. The EPM’s failure to elicit this pattern of behavior on the successive alleys test implies that these two tasks may not be equivalent measures of anxiety, perhaps due to spatial and constructional differences between the two apparatuses or due to differences in the availability of behavioral choices that each maze presents.  Knowledge of differences between the two animal models is a valuable asset to the interpretation of past research that utilizes these animal models, and is critical to the formation of new research, both of which have the potential to influence the development of more effective treatment plans for individuals suffering from anxiety disorders.

Investigating the Distribution of Neuropeptide Phenylalanine (NPF) within the Alimentary System and CNS of the Pond Snail Helisoma trivolvis and Determining its Impact on the Buccal Feeding Circuitry

Greg Sousa (Biology)

Advisor: Nancy Kleckner

Neuropeptide phenylalanine (NPF), the invertebrate neuropeptide tyrosine (NPY) homologue, modulates feeding and feeding-related behaviors in a number of invertebrate organisms.  Recently, it was determined that Aplysia californica NPF (Apca-NPF) reconfigures the buccal central pattern generator (CPG) to facilitate the transition to a satiety state (Jing et al., 2007).  Similarly, neurons within the Buccal A Cluster (BAC) of the freshwater pond snail, Helisoma trivolvis are thought to be involved in distention induced satiation and regurgitation (Arnett, 1996).  A subset of the BAC neurons become excited following esophageal distension and contribute to the termination of the protraction (S1) and hyper-retraction (S3) phases of the ‘standard’, tri-phasic feeding cycle (Arnett, 1996).  BAC neuron stimulation is also implicated in the prolongation and inhibition of the S1 and S3 phases, respectively, and the subsequent transition to the bi-phasic (S1-S2) regurgitation rhythm (Arnett, 1996).  This thesis project has sought to determine the distribution of NPF-like molecules within the H. trivolvis alimentary system and central nervous system (CNS) and to ascertain the possible impacts of NPF on buccal motor function via its actions within the BAC.  A polyclonal antibody generated against the carboxy terminus of Apca-NPF was utilized to assess the distribution of NPF-like molecules within the alimentary system and CNS.  Numerous immunoreactive processes and somata were also observed throughout the alimentary system.  Immunopositive somata were observed throughout the CNS, including a bilaterally symmetric cell cluster located on the caudal surface of the buccal ganglia.  Preabsorption of the primary antiserum with Lymnaea stagnalis NPF (Lyst-NPF) completely abolished all labeling.  The whole CNS from H. trivolvis was isolated and pinned to Sylgard dishes for intracellular recordings. Simultaneous recordings from candidate BAC neurons and S3 phase motor neuron B19 revealed that 100 nM Lyst-NPF elicits BAC neuron excitatory postsynaptic potentials (EPSPs) or action potential discharges that are coordinated in time with inhibitory post-synaptic potentials (IPSPs) in B19 that appear to disrupt/modify firing during the hyper-retraction phase.  This suggests that NPF could be involved in the transition to the satiation and/or the regurgitation rhythms.

Characterization of a novel α-GluR5/6/7-immunoreactive cell group in the buccal ganglia of the pond snail, Helisoma trivolvis.

Mimi Gallo (Neuroscience-Honors)

Advisor: Nancy Kleckner

A modifiable central pattern generator (CPG) composed of neurons divided intothree distinct subunits controls the three-part motor feeding behavior of Helisoma trivolvis. The subunits S1, S2, and S3 correspond to protraction, retraction, and hyperretraction, respectively, of the dentated radula. The subunits interact as independent conditional oscillators, becoming active when released from inhibition or when directly stimulated, and thus producing a cyclic feeding pattern. Glutamate is known to control the CPG through inhibition of phase 1 and 3 subunits during excitation of S2. This indicates that distinct glutamate receptors must be present to provide for the divergent responses to glutamate. Glutamate receptor antibodies provide a method to assess which glutamate receptors are present in the network. Probing with an antibody that recognizes α-GluR5/6/7 subunits revealed cells whose involvement with the feeding circuitry has not yet been characterized. The goal of this study is to characterize these α-GluR5/6/7immunoreactive (IR) cells with respect to physiology, pharmacology, and involvement with the feeding CPG. Further immunocytochemistry with a different antibody that recognizes α-GluR6/7 subunits labeled a subset of the α-GluR5/6/7 IR cells, lending support to the binding specificity of the α-GluR5/6/7 antibody. Serendipitously it was discovered that there is extensive double-labeling of α-GluR5/6/7 and an antibody made against Lymnaea stagnalis neuropeptide phenylalanine (NPF), the invertebrate homolog of neuropeptide tyrosine (NPY). This suggests that α-GluR5/6/7 IR cells should release NPF when stimulated to fire. Simultaneous electrophysiological recordings were made from cells within the α-GluR5/6/7 IR group to characterize their pharmacology and involvement with the CPG. Targeting accuracy was ensured by iontophoretic injection of Neurobiotin in the targeted cell and co-processing for the α-GluR5/6/7 antibody. α-GluR5/6/7 IR cell morphologies resemble that of the buccal A cluster (BAC) cells. GluR5/6/7 IR cells were depolarized by kainate but insensitive to glutamate and quisqualate. α-GluR5/6/7 IR cells generate sub-threshold slow excitatory post-synaptic potentials (GR EPSPs) that correspond with excitation of S2 motor neurons and, logically, S2-stimulated hyperpolarizations of S3 motor neurons. Interestingly, similar slow and phasic EPSPs are seen in the BAC cells which, when stimulated, shut down phase three of feeding. These preliminary data suggest a possible modulatory role of α-GluR5/6/7 IR neurons in the feeding CPG that converges with the satiation/regurgitation behaviors initiated by the BAC neuron firing.

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