Max Fletcher, Ph.D., Assistant Professor
- Anatomy and Neurobiology

Research Highlights

The primary focus of my lab is on understanding the neural mechanisms underlying olfactory system experience-induced plasticity and how it can lead to enhanced perception. Current work is concentrated on the first stage of central olfactory processing, the olfactory bulb. Here, the anatomical organization of olfactory receptor neuron input from the nose allows odorant information to be transformed into an odorant specific spatial map of glomerular neuronal activity. These maps can be directly visualized in vivo using optical imaging techniques in transgenic mouse lines expressing genetically encoded indicators of neuronal activity.

Under normal conditions, the map for a given odorant remains stable over time and displays little variability. However, these maps have the potential to be altered by both intrinsic inhibitory circuits within the bulb as well as by centrifugal input from several learning-related neuromodulatory regions of the brain that project into the olfactory bulb. Through multi-day imaging experiments in both awake and anesthetized mice, we are investigating how these circuits are involved in driving learning-induced changes in olfactory bulb odor representations. Our current work is focused on comparing odor maps in the same animal before and after olfactory associative conditioning. Under these circumstances, we find that olfactory associative conditioning leads to significant changes in the map representing the trained odor. We are currently investigating the role centrifugal neuromodulation plays in this plasticity by observing odor responses during direct activation of neuromodulatory brain regions and pharmacological manipulation. The results of these experiments will help further our understanding of the role plasticity plays in shaping early neural responses to salient sensory stimuli.

A: Dorsal view of an olfactory bulb glomerular odor response to methyl valerate before and after associative conditioning to methyl valerate in a GCaMP2 mouse. Each hotspot represents the average odor-evoked change in fluorescence detected from individual glomeruli. Following conditioning, the odor-evoked responses from several glomeruli are significantly altered (white arrows). B: Overall, associative conditioning alters the amplitude of individual glomerular responses in a predictable manner, with the weakest responding glomerulus (A) significantly enhanced and the strongest glomerulus (B) significantly depressed following conditioning.

Additionally, we are also interested in how the neuronal plasticity we observe following associative conditioning can lead to enhanced olfactory acuity and perception, a process known as perceptual learning. Using a simple olfactory fear-conditioning paradigm, we can quantify how well mice have learned an odor-fear association as well as how much they generalize what they have learned to other structurally and qualitatively similar odorants. Using this paradigm in combination with targeted pharmacological manipulations, we seek to understand both the mechanisms and brain regions involved in the acquisition and expression of this type of learning.

Recent Publications

1. Nagayama, S., Zeng, S., Xiong, W., Fletcher, M.L., Masurkar, A.V., Davis, D.J., Pieribone, V.A., Chen, W.R. (2007) In vivo simultaneous tracing and Ca2+ imaging of local neuronal circuits. Neuron 53: 789-803.
2. Fletcher, M.L., Masurkar A.V., Xing, J., Imamura, F., Xiong, W., Nagayama, S., Mutoh, H., Greer, C.A., Knopfel, T., and Chen, W.R. (2009) Optical imaging of olfactory bulb postsynaptic odor representation in the glomerular layer of the mouse olfactory bulb. Journal of Neurophysiology 102: 817-830.
3. Nagayama, S., Enerva, A., Fletcher, M.L., Masurkar, A.V., Igarashi, K., Mori, K., and Chen, W.R. (2010) Differential axonal projections of mitral and tufted cells in the mouse main olfactory bulb system Front. Neural Circuits 4:120. doi:10.3389/fncir.2010.00120
4. Fletcher, M.L. and Chen, W.R. (2010) Neural correlates of olfactory learning: critical role of centrifugal neuromodulation Learning and Memory 17:561-570.

Scott Heldt, Ph.D., Assistant Professor
- Anatomy and Neurobiology

Research Highlights

Current projects in our laboratory focus on studying the role of gamma-aminobutyric acid (GABA) transmission in fear and the behavioral and pharmacological function of GABAA receptor subtypes.

1. The role of the amygdala and GABA transmission in fear conditioning: In the laboratory, we study the circuits underlying fear by using Pavlovian conditioning procedures in which an initially neutral conditioned stimulus (CS), such as a tone, is repeatedly paired with an aversive unconditioned stimulus (US) such as a shock. After a few CS-US pairings, presentation of the CS alone induces physiological and behavioral responses similar to those seen in normal and abnormal states of fear and anxiety. The results of numerous studies have demonstrated that changes in fast glutamatergic transmission within the amygdala play an important role in the formation of emotional memories associated with the acquisition and extinction of conditioned fear. However, there are converging lines of evidence suggesting that changes in gamma-aminobutyric acid (GABA) transmission is also involved in these processes, as highlighted by the fact that patients suffering from anxiety disorders are commonly treated with benzodiazepines (BZs), which mediate their actions via GABAA receptors (GABAARs).
   
   Recently, our lab examined training-induced changes in the GABAergic system by measuring the mRNA levels of GABA-related genes in the amygdala after the acquisition and extinction of Pavlovian fear. We found that mRNA levels of GABA-related genes and the total number of BZ-GABAARs decreased following fear conditioning. In contrast, fear extinction increased the expression levels of GABA-related genes. These changes suggest that Pavlovian conditioning is capable of altering GABAAR subunit composition and potentially modifying the number and/or ratio of discrete functional BZ-GABAAR subtypes in the amygdala.
   
   We are currently examining the acquisition and extinction of conditioned fear in mice with knocked down expression of GABAgeric genes in the amygdala using a lentiviral (LV)-based RNA interference strategy to locally induce loss-of-function. As an example, we recently examined fear responses in mice with knocked down expression of the GABA synthesizing enzyme, GAD67, in the amygdala. In vivo studies showed that a reduction of GAD67 in the amygdala produced no changes in motor activity levels, the acquisition of fear, or basal anxiety levels. However, reduced amygdala GAD67 levels disrupted the extinction of conditioned fear. In addition, when compared to control mice, diazepam was ineffective in reducing anxiety in the elevated plus maze in mice with reduced amygdala GAD67. These results suggest GABA synthesis within the amygdala may help mediate the extinction of fear and anxiolytic effects of BZ.
2. Functional role of local GABAA receptor subtypes: Our understanding of the behavioral and pharmacological function of GABAARs has been greatly advanced by the development and use of mice with altered expression of receptor subtypes. These mice have revealed that the genetic alterations of individual GABAAR subunits results in distinct changes in their phenotypic behavior and response to BZs and/or site-specific ligands. While the use of these transgenic mice has become an increasingly invaluable tool, these strategies produce broad changes in GABAAR function throughout the neuroaxis and do little to identify the effects of such changes in specific brain regions. To overcome this limitation, our lab has been utilizing site-specific, temporally-inducible, knockdown approaches to investigate the role of GABAAR subunits in regionally defined areas of the brain.
   
   For example, past studies using transgenic mice possessing mutations of the α1 subunit of the GABAAR indicate that the anti-convulsant and sedative actions of BZs are, in part, mediated by these α1-GABAARs. However, the degree to which these BZ actions are mediated by distinct brain regions is presently unknown. Recently, we examined the sedative and anticonvulsant effects of BZ, as well as baseline behaviors, in mice lacking α1-GABAARs in the amygdala using α1-GABAA inducible knockout mice. These mice possess loxP sites on both sides of the α1 exon encoding an essential transmembrane domain which can be deleted in the presence of Cre recombinase (CRE). To induce CRE-mediated gene deletion in a localized fashion, α1-GABAAR inducible mice received bilateral microinjections of a CRE- producing lentivirus. We found that amygdala-specific reduction of α1 receptor subunits does affect baseline locomotion or measures of anxiety. However, site-specific deletion did disrupt the normal motor sedative as well as the anticonvulsive effects of two distinct BZ-site ligands, diazepam and zolpidem. Together these findings demonstrate that amygdala expression of the α1-GABAAR subunit is required for normal BZ effects on sedation, locomotion, and seizure inhibition, but not for anxiolysis.

Michael Hankins, Cary Small, Yudong Gao

Startle, conditioning and activity chambers

Automated conditioned
freezing chambers

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Neuroscience Institute
University of Tennessee Health Science Center
875 Monroe Ave, Suite 426
Memphis, TN 38163
Phone: (901) 448-5960
Fax: (901) 448-4685

Physical Address
426 Wittenborg Anatomy Building

Director:
William E. Armstrong, Ph.D.

Co-Director:
Anton J. Reiner, Ph.D.

Administrative Services Assistant:
Shannon Guyot

Website:
Brandy Fleming, M.S.