Potential Regulation of AMP-Activated Protein Kinase Pathway in Stress Induced Synaptic Plasticity in the Basolateral Complex of the Amygdala Open Access

Liu, Wei (2013)

Permanent URL: https://etd.library.emory.edu/concern/etds/cj82k747v?locale=en
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Abstract

The basolateral complex of the amygdala (BLC) is known to be involved in the regulation of emotion, and over excitation of the BLC is thought to underlie the development of mood disorders. An imbalance in the activity of two neuronal subpopulations in the BLC, namely excitatory, glutamatergic, principal neurons and inhibitory, GABAergic, interneurons can cause the amygdala to become hyper-excitable. Chronic stress is known to cause such a change and facilitate a type of synaptic plasticity, long term potentiation (LTP) in the BLC. Moreover, LTP formation in the BLC is calcium-dependent, and requires activation of dopamine D1 receptors that are coupled to the adenylate cyclase, cAMP, and protein kinase A second messenger cascade. Similar to stress, unpublished in vitro patch clamp studies in the Rainnie lab showed that rolipram, a phosphodiesterase type 4 (PDE4) inhibitor that blocks the hydrolysis of cAMP to 5'AMP, decreased the threshold for LTP induction and action potential generation, thereby increasing the excitability of BLC principal neurons. Significantly, increasing intracellular cAMP levels did not mimic the effect of rolipram, whereas increasing 5'AMP levels did. Thus, we reasoned chronic stress disrupts a baseline homeostatic regulatory system that detects 5'AMP levels and shifts induction thresholds to maintain homeostasis in the BLC. We proposed that AMP-activated protein kinase (AMPK), which is known to maintain homeostasis and conserve energy in the periphery, acts as a central metabolic sensor by detecting AMP:ATP levels in BLC neurons. Here, we used a multidisciplinary approach employing RT-PCR, Western blots, and immunohistochemistry to create a baseline profile of the AMPK signaling cascade in the BLC, and examined protein expression level changes following repeated restraint stress (RRS) and food restriction (FR) protocols. We report that principal neurons of the BLC express two upstream activators of AMPK, LKB1 and CaMKKβ, as well as AMPK itself, and downstream target Kv2.1, which is known to alter the threshold for action potential firing upon activation. Moreover, using selective AMPK activators we further confirmed the role of AMPK as a functional regulator for energy conservation in neurons under baseline conditions, and showed RRS can cause a persistent perturbation of neuronal homeostasis.

Table of Contents

Introduction...1

The basolateral complex of the amygdala: Anatomy and its role in fear memory formation...1

The molecular mechanism underlying fear memory formation: Long term potentiation (LTP) and the involvement of second messenger signaling cascades...4

Homeostasis: potential homeostatic mechanisms underlying stress induced fear and anxiety...7

The role of second messenger systems in stress-induced synaptic plasticity in fear and anxiety circuits...8

AMP kinase: the potential homeostatic sensor in the amygdala that regulates synaptic plasticity and fear and anxiety...11

Specific aims...13

Methods...15

Results...23

Baseline profile for AMPK and related cascades in the BLC...23

Electrophysiological study: effect of AMPK activation on synaptic plasticity in BLC principal neurons from control animals...28

Effect of repeated restraint stress on protein expression levels of AMPK and related cascades in the BLC...28

Electrophysiology study: effect of AMPK activation on synaptic plasticity in BLC principal neurons from stressed animals...29

Effect of food restriction on protein expression levels of AMPK and related cascades in the BLC...30

Electrophysiology study: effect of AMPK activation on synaptic plasticity in BLC principal neurons from food restricted animals...30

Discussion...32

The presence of AMPK and related cascades in the control BLC...33

Differential expression of AMPK and related cascades in control BLC principal neurons and interneurons...34

Examination of the functional role of AMPK in the BLC from control animals...36

Effects of chronic stress on AMPK and related cascades...37

Effects of food restriction on AMPK and related cascades...40

Metformin and AMPK...42

Future Directions...43

Figure 1: A schematic of the neural circuits underlying auditory fear conditioning...46

Figure 2a: A summary of the cAMP/PKA second messenger cascade and AMP-activated protein kinase system in the BLC...47

Figure 2b: A summary of proposed AMP-activated protein kinase system in the BLC...48

Figure 3a: Rolipram causes down shift of LTP induction threshold...49

Figure 3b: Rolipram causes down shift of action potential generation threshold...50

Figure 4: Stress causes down shift of LTP induction threshold...51

Table 1: Table of PCR primer sequences used in the study and resulting PCR product size...52

Figure 5: An example of a principle BLC neuron and a sample of its electrophysiological profile...53

Figure 6: Baseline RT-PCR results in whole BLC tissue...54

Table 2: Single cell RT-PCR results in BLC principal neurons...55

Figure 7: Baseline Western blot results in whole BLC tissue...56

Figure 8: Dual-immunofluorescence results on CaMKKβ...57

Figure 9: Dual-immunofluorescence results on LKB1...58

Figure 10: Dual-immunofluorescence results on p-AMPKα1,2...59

Figure 11: Dual-immunofluorescence results on CaMKIV...60

Figure 12: Immunofluorescence result on Kv2.1...61

Table 3: Summary of quantified percentage of specific neuronal subtypes that co-express AMPK and related signaling cascades...62

Table 4: AICAR causes up shift of action potential threshold...63

Figure 13: Effect of repeated restraint stress on protein expression levels of AMPK signaling cascade...64

Figure 14: Effect of repeated restraint stress on immunoreactive intensity levels of AMPK signaling cascade. (a, example of immunofluorescence staining; b quantified result)....65

Figure 15: AICAR blocks effect of stress on LTP induction threshold shift...66

Figure 16: Effect of food restriction on protein expression levels of AMPK signaling cascade...67

Table 5: Food restriction and 2-deoxy-D-glucose cause down shift of action potential generation threshold while 2-deoxy-D-glucose has no effect on BLC principal neurons obtained from food-restricted animals...69


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