- Molecular Biology/Princeton Neuroscience Institute
- Computational Neuroscience
Animals are programmed to solve certain problems by learning predictive relationships among stimuli and storing information about those predictive relationships in a form that is stable for periods ranging from one to 7x105 hours. Remarkably complex associative learning has evolved in animals with neural circuits highly suited to biophysical analysis, such as the terrestrial garden slug Limax maximus. We study odor learning and odor information processing in Limax, as this animal displays robust and reliable one-trial odor conditioning and a variety of higher-order learning modes during olfactory learning. The central circuit which stores odor memories has oscillatory dynamics of its local field potential and propagates activity waves along its apical-basal axis. This dynamics arises from a network of coupled neuronal oscillators that have a gradient of excitability along the apical-basal axis. Oscillatory dynamics is widespread in mammalian cortical networks during sensory processing and motor command generation. Thus we study both synaptic events during learning and the computational function of oscillatory dynamics in the Limax olfactory circuit to shed light on the roles of learning and oscillations during cortical processing.
Our current studies of Limax odor memory formation and odor information processing aim to answer the following four questions: Can odor memory formation be imaged in the isolated nose-brain preparation? Does odor learning-specific uptake of Lucifer yellow reveal neurons that store an odor memory? What is the role of post-hatching neurogenesis in the odor memory storage network and how is it affected by olfactory experience and learning? What are the contributions to synaptic stabilization of genes activated by one-trial odor learning?
Imaging odor memory formation in vitro. Our previous imaging studies of odor responses in the odor memory circuit used naïve brains. We now have a procedure to train a naïve nose-brain preparation in vitro by pairing nerve shock with odor application. We measure action potential generation by identified withdrawal neurons as the output measure. Before conditioning an attractive odor does not activate withdrawal motoneurons while after pairing the odor with nerve shock the odor strongly activates the identified withdrawal motoneurons. We image the odor memory circuit after staining its neurons with a voltage-sensitive dye. We record a series of images with a CCD camera and analyze the patterns of neural activity in response to odor stimulation before and after conditioning. Odor application initially causes a collapse of the apical-basal phase gradient, which in our model of odor memory formation is a necessary precondition for synaptic modification. This can now be tested.
Learning-specific dye uptake. If a slug is given one-trial odor conditioning and then injected with the highly fluorescent dye Lucifer yellow, a band of neurons is found in the odor memory storage circuit containing Lucifer yellow in membrane bound vesicles. Animals given odor exposure alone or unpaired applications of odor and the aversive stimulus do not show neuronal uptake of Lucifer yellow in the odor memory circuit. We are performing imaging experiments to test the hypothesis that the Lucifer yellow containing neurons found after odor conditioning store the odor memory in the strengths of their synaptic connections. We also train the naïve nose-brain preparation in vitro by pairing nerve shock and odor application while applying drugs selectively to the odor learning circuit. This in vitro training technique with drug application restricted to the odor learning circuit allows us to block long-term memory formation and determine if dye uptake is also blocked. We also want to determine if learning-specific Lucifer yellow uptake occurs in mammalian systems.
Stimulus modulated neurogenesis. Mammals and mollusks add new olfactory receptors throughout life and new olfactory interneurons until adulthood, yet their olfactory systems appear to maintain a constant input-output relation. The odor memory storage circuit in Limax hatches with a zone of active neurogenesis that produces 80% of the neurons found in the adult circuit after hatching. We explore how odor experience and odor learning affect neurogenesis by labeling dividing neurons with bromodeoxyuridine (BrdU) and giving slugs varied odor experiences before developing the BrdU label immunocytochemically. The zone of neurogenesis at hatching is at the most apical position in the odor learning circuit, where activity waves originate. New neurons are added only on the apical side of the band of neurogenesis. Removal of one nose retards neurogenesis until the nose regenerates. We plan to image neurogenesis with 2-photon laser-scanning microscopy using fluorescent nucleotides applied to an in vitro nose-brain preparation which can learn odor-shock associations while the zone of neurogenesis is being imaged.
Odor learning-activated gene expression. Professor Yutaka Kirino and his collaborators at the University of Tokyo have just described (Genes To Cells 6:43, 2001) the activation by one-trial odor conditioning in Limax of a gene coding for an extracellular matrix protein. The gene is expressed in neurons of the odor memory storage circuit and makes a protein that is homologous to proteins found in zebrafish, mice and human. The gene product is secreted into extracellular space and may stabilize connections between neurons in the odor learning circuit. In collaboration with the Kirino laboratory we plan to assess the effect of the new learning-specific extracellular matrix protein on connections between odor memory storage neurons cultured in vitro and on long-term memory formation by the isolated nose-brain preparation trained in vitro.