Colocalization of Protein Kinase A with Adenylyl Cyclase Enhances Protein Kinase A Activity during Induction of Long-Lasting Long-Term-Potentiation

The ability of neurons to differentially respond to specific temporal and spatial input patterns underlies information storage in neural circuits. One means of achieving spatial specificity is to restrict signaling molecules to particular subcellular compartments using anchoring molecules such as A-Kinase Anchoring Proteins (AKAPs). Disruption of protein kinase A (PKA) anchoring to AKAPs impairs a PKA-dependent form of long term potentiation (LTP) in the hippocampus. To investigate the role of localized PKA signaling in LTP, we developed a stochastic reaction-diffusion model of the signaling pathways leading to PKA activation in CA1 pyramidal neurons. Simulations investigated whether the role of anchoring is to locate kinases near molecules that activate them, or near their target molecules. The results show that anchoring PKA with adenylyl cyclase (which produces cAMP that activates PKA) produces significantly greater PKA activity, and phosphorylation of both inhibitor-1 and AMPA receptor GluR1 subunit on S845, than when PKA is anchored apart from adenylyl cyclase. The spatial microdomain of cAMP was smaller than that of PKA suggesting that anchoring PKA near its source of cAMP is critical because inactivation by phosphodiesterase limits diffusion of cAMP. The prediction that the role of anchoring is to colocalize PKA near adenylyl cyclase was confirmed by experimentally rescuing the deficit in LTP produced by disruption of PKA anchoring using phosphodiesterase inhibitors. Additional experiments confirm the model prediction that disruption of anchoring impairs S845 phosphorylation produced by forskolin-induced synaptic potentiation. Collectively, these results show that locating PKA near adenylyl cyclase is a critical function of anchoring

Regulation of Neuromodulator Receptor Efficacy- Implications for Whole-Neuron and Synaptic Plasticity

Membrane receptors for neuromodulators (NM) are highly regulated in their distribution and efficacy - a phenomenon which influences the individual cell's response to central signals of NM release. Even though NM receptor regulation is implicated in the pharmacological action of many drugs, and is also known to be influenced by various environmental factors, its functional consequences and modes of action are not well understood. In this paper we summarize relevant experimental evidence on NM receptor regulation (specifically dopamine D1 and D2 receptors) in order to explore its significance for neural and synaptic plasticity. We identify the relevant components of NM receptor regulation (receptor phosphorylation, receptor trafficking and sensitization of second-messenger pathways) gained from studies on cultured cells. Key principles in the regulation and control of short-term plasticity (sensitization) are identified, and a model is presented which employs direct and indirect feedback regulation of receptor efficacy. We also discuss long-term plasticity which involves shifts in receptor sensitivity and loss of responsivity to NM signals. Finally, we discuss the implications of NM receptor regulation for models of brain plasticity and memorization.We emphasize that a realistic model of brain plasticity will have to go beyond Hebbian models of long-term potentiation and depression to include plasticity in the distribution and efficacy of NM receptors

Investigation Of The Spatiotemporal Dynamics Of Camp And Pka Signaling And The Role Of Hcn4 Subunits In Anxiety-Related Behavior And Memory

In the hippocampus, long-term memory and synaptic plasticity occur through a series of coordinated intracellular signaling cascades that strengthen and stabilize subsets of synaptic connections while leaving thousands of others unaltered. Therefore, understanding how molecular signals are accurately transmitted is critical to understanding how hippocampal neurons store information. Molecules like cAMP and protein kinase A are critical components of memory and plasticity, but it is unclear how these diffusible signals are dynamically regulated to achieve the spatial and temporal specificity that underlies pathway-specific plasticity. Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels are ion channels that are modulated by cAMP and are known to regulate the spatial and temporal dynamics of excitatory postsynaptic potentials. HCN1 and HCN2 subunits have been implicated in memory, plasticity and anxiety-related behaviors, but the role for HCN4 subunits remains untested. In Chapter 1, I review the role of cAMP signaling in hippocampal synaptic plasticity and memory consolidation with emphasis on the molecular mechanisms regulating cAMP, PKA and HCN channels. In Chapter 2, I combine live two-photon imaging of genetically-encoded fluorescent FRET sensors and computational modeling to investigate the molecular mechanisms regulating the spatiotemporal dynamics of cAMP and PKA activity in hippocampal neurons during stimulation of β-adrenergic receptors. Results suggest that the ratio between adenylyl cyclase and phosphodiesterase-4 scales with neuronal compartment size to maintain basal cAMP levels and produce rapid-onset, high-amplitude cAMP transients in small compartments. Conversely, imaging experiments show that PKA activity is greater in large neuronal compartments and modeling suggests that compartmental differences in PKA activity depend on the concentration of protein phosphatase and not on the concentration of PKA substrates or PKA holoenzyme. In Chapter 3, I use recombinant adeno-associated viruses and shRNA-mediated silencing of HCN4 subunits to examine their role in anxiety, memory, and contextual fear extinction. Results from a battery of behavioral assays suggest that reduction of HCN4 subunits increases anxiety-related behavior, but does not affect object-location memory or contextual fear conditioning. Together, my thesis work provides novel insight into the molecular mechanism regulating the spatiotemporal dynamics of cAMP/PKA signaling and provides suggests a role for HCN4 subunits in anxiety-related behavior

Role of cyclic nucleotides and their downstream signaling cascades in memory function:Being at the right time at the right spot

A plethora of studies indicate the important role of cAMP and cGMP cascades in neuronal plasticity and memory function. As a result, altered cyclic nucleotide signaling has been implicated in the pathophysiology of mnemonic dysfunction encountered in several diseases. In the present review we provide a wide overview of studies regarding the involvement of cyclic nucleotides, as well as their upstream and downstream molecules, in physiological and pathological mnemonic processes. Next, we discuss the regulation of the intracellular concentration of cyclic nucleotides via phosphodiesterases, the enzymes that degrade cAMP and/or cGMP, and via A-kinase-anchoring proteins that refine signal compartmentalization of cAMP signaling. We also provide an overview of the available data pointing to the existence of specific time windows in cyclic nucleotide signaling during neuroplasticity and memory formation and the significance to target these specific time phases for improving memory formation. Finally, we highlight the importance of emerging imaging tools like Förster resonance energy transfer imaging and optogenetics in detecting, measuring and manipulating the action of cyclic nucleotide signaling cascades

Investigation Of The Molecular Mechanisms Of Synaptic Tagging And Capture

Memory formation is continuously influenced by past, present, and future experiences. Memories linked to events that require more attention or involve emotional arousal are more persistent than ordinary memories. Information from multiple inputs that consist of memory is integrated in the hippocampus, a brain region responsible for memory storage. As a form of hippocampal long-term potentiation, pathway-specific synaptic tagging and capture (STC) has been proposed as a synaptic model of memory because it illustrates the interaction of two independent sets of synapses. This pathway-specificity is a remarkable property of neuronal signaling because it requires highly coordinated cellular signaling only at the activated synapses. However, elucidating the mechanism that is responsible for this specificity is a big challenge in the field. In my dissertation, I focused on PKA anchoring and RNA-binding proteins because they can contribute to STC through compartmentalization of PKA signaling and regulation of dendritic expression of RNAs, respectively. In Chapter 1, I review the mechanism of STC and discuss how compartmentalized PKA signaling contributes to STC. PKA is involved in the process of STC by orchestrating the activity of synaptic molecules and by mediating gene expression. In Chapter 2, I combine genetic and pharmacological approaches to determine the role of PKA anchoring in STC and memory. The results from electrophysiological, biochemical and behavioral experiments suggest that presynaptically anchored PKA contributes to STC and memory by regulating the size of the readily releasable pool of synaptic vesicles. In Chapter 3, I perform genetic and viral approaches to define whether an RNA-binding protein translin (also known as testes-brain RNA-binding protein, TBRBP) is involved in STC and memory. The data from electrophysiological, behavioral and gene expression studies suggest that translin mediates STC and memory via RNA processing. Taken together, my thesis work provides evidence that presynaptic PKA anchoring-mediated synaptic vesicle release and postsynaptic processing of specific RNAs by translin are critical for STC and memory

Genetic manipulation of cyclic nucleotide signaling during hippocampal neuroplasticity and memory formation

Decades of research have underscored the importance of cyclic nucleotide signaling in memory formation and synaptic plasticity. In recent years, several new genetic techniques have expanded the neuroscience toolbox, allowing researchers to measure and modulate cyclic nucleotide gradients with high spatiotemporal resolution. Here, we will provide an overview of studies using genetic approaches to interrogate the role cyclic nucleotide signaling plays in hippocampus-dependent memory processes and synaptic plasticity. Particular attention is given to genetic techniques that measure real-time changes in cyclic nucleotide levels as well as newly-developed genetic strategies to transiently manipulate cyclic nucleotide signaling in a subcellular compartment-specific manner with high temporal resolution

Calcium-Stimulated Adenylyl Cyclases are Critical Modulators of Fear Learning and Experience-Dependent Plasticity

Stress can exacerbate psychiatric disease, often resulting in cognitive deficits. Consequently, a better understanding of what modulates stress-facilitated memory processing will help identify new targets for possible therapeutic intervention. Recent evidence suggests a role of the Ca2+-stimulated adenylyl cyclases: AC), AC1 and AC8, in modulating fear memory. Ca2+-stimulated AC activity couples neuronal activity and intracellular Ca2+ increases to the production of cAMP, and therefore, can very tightly regulate signal transduction after learning; yet, the details by which this occurs are not well understood. In this dissertation, I first investigated the temporal and regional importance of Ca2+-stimulated AC activity during different stages of memory processing using the tetracycline-off system, which allowed me to produce AC8 Rescue mice with forebrain-specific inducible expression of AC8 on an AC1 and AC8 double knockout: DKO) background. The results showed that forebrain Ca2+-stimulated AC activity was necessary to modulate long-term memory on several learning paradigms, and more specifically, that it was necessary during memory consolidation and retention. This finding is further supported by an overall decrease in transcriptional changes in DKO mice across several time points after conditioned fear: CF) learning, but most strikingly, at periods when memory consolidation and retention should be occurring. Since transcriptional changes are often dictated by synaptic activity and AC1 and AC8 are both localized at the synapse, I examined synaptic activity in DKO mice. Initial analysis of synaptic protein abundance in hippocampal cell cultures revealed decreased SV2 levels in DKO mice, but this can be rescued by infection with an AC8 lentivirus. Moreover, DKO mice also display synaptic deficits after learning as measured by p-synapsin. The CA1 LTP results coincide with the above data as DKO mice, but not AC8 Rescue mice, show impaired LTP. Finally, WT mice show changes in CF memory strength that is dependent on prior environmental exposure, but DKO mice do not, suggesting that Ca2+-stimulated AC activity modulates plasticity at the behavioral level as well. From these studies, I have observed a critical role for Ca2+-stimulated AC activity in modulating the consolidation and retention of fear memory and experience-dependent plasticity

Absence of Ca2+-stimulated adenylyl cyclases leads to reduced synaptic plasticity and impaired experience-dependent fear memory

Ca2+-stimulated adenylyl cyclase (AC) 1 and 8 are two genes that have been shown to play critical roles in fear memory. AC1 and AC8 couple neuronal activity and intracellular Ca2+ increases to the production of cyclic adenosine monophosphate and are localized synaptically, suggesting that Ca2+-stimulated ACs may modulate synaptic plasticity. Here, we first established that Ca2+-stimulated ACs modulate protein markers of synaptic activity at baseline and after learning. Primary hippocampal cell cultures showed that AC1/AC8 double-knockout (DKO) mice have reduced SV2, a synaptic vesicle protein, abundance along their dendritic processes, and this reduction can be rescued through lentivirus delivery of AC8 to the DKO cells. Additionally, phospho-synapsin, a protein implicated in the regulation of neurotransmitter release at the synapse, is decreased in vivo 1 h after conditioned fear (CF) training in DKO mice. Importantly, additional experiments showed that long-term potentiation deficits present in DKO mice are rescued by acutely replacing AC8 in the forebrain, further supporting the idea that Ca2+-stimulated AC activity is a crucial modulator of synaptic plasticity. Previous studies have demonstrated that memory is continually modulated by gene–environment interactions. The last set of experiments evaluated the effects of knocking out AC1 and AC8 genes on experience-dependent changes in CF memory. We showed that the strength of CF memory in wild-type mice is determined by previous environment, minimal or enriched, whereas memory in DKO mice is unaffected. Thus, overall these results show that AC1 and AC8 modulate markers of synaptic activity and help integrate environmental information to modulate fear memory

Mechanism Of Gaba\u3csub\u3eb\u3c/sub\u3e Receptor-Activated Increases In L-Type Calcium Current In The Neonatal Mammalian Hippocampus

Activation of the metabotropic GABAB receptor has most commonly been demonstrated to produce inhibitory effects on neurons, including the attenuation of voltage-dependent calcium current. However, during the early neonatal period in mammalian development, activation of GABAB receptors leads to an enhancement of calcium current through a specific class of calcium channels, termed L-type channels, (because they conduct Long-lasting current) . This response peaks at 7 days postnatal, and is only demonstrated in a subset of cells. In the work presented here, the signal transduction pathway of GABAB receptor-mediated increase of L-type current is described. GABAB receptors couple to G proteins, traditionally believed to be Gαi/o. However, previous data from the laboratory suggested that the enhancing effect observed was not due to Gαi/o, but a different G protein not previously described in GABAB receptor signaling. Indeed, when the Gαq G protein was knocked down in cell culture, the enhancement of L-type channels was no longer observed. These data suggest that GABAB receptors couple to Gαq(/sub\u3e G proteins to mediate calcium current enhancement. Protein kinase C (PKC) had previously been demonstrated as a requisite member of this pathway. Furthermore, there was precedence for PKC to work through calcium/calmodulin-dependent kinase II (CaMKII) to enhance L-type current. However, the isozyme of PKC was not known, nor was the involvement of CaMKII on L-type current enhancement. Confocal imaging analysis suggests PKCα is the isozyme that is activated by GABAB receptor activation, and pharmacological studies indicate CaMKII is not a participant in this pathway. In seeking to inhibit CaMKII signaling, highly specific pharmacological inhibitors are often required. However, several inhibitors that were thought to be specific initially demonstrate nonspecific effects. A newly synthesized molecule, CK59, has been described to potently inhibit CaMKII activity (IC50 \u3c 10 μm). However, data presented here describe off-target effects of CK59, specifically its ability to inhibit voltage-gated calcium channels. Treatment of cells with CK59 significantly reduced calcium influx in depolarized neurons, whereas other CaMKII inhibitors did not change calcium influx. Thus, CK59 is not a useful tool when studying the interplay between voltage-caged calcium channels and CaMKII signaling