Antidepressant efficacy in a motivational task and behavioral despair assay are associated with altered limbic pERK1/2, including restored pERK1/2 in the dentate gyrus after stress-related insult.

The medial prefrontal cortex (mPFC) has been implicated in the extinction of emotional memories, including conditioned fear. Here we show that ventral hippocampal (vHPC) projections to the infralimbic (IL) cortex recruit parvalbumin (PV)-expressing interneurons to counter the expression of extinguished fear and promote fear relapse. Whole-cell recordings ex vivo revealed that optogenetic activation of vHPC input to amygdala projecting pyramidal neurons in the IL is dominated by feed-forward inhibition. Selectively silencing PV- but not somatostatin (SOM)-expressing interneurons in the IL eliminated vHPC-mediated inhibition. In behaving rats, pharmacogenetic activation of vHPC→IL projections impairs extinction recall, whereas silencing IL projectors diminishes fear renewal. Intra-IL infusion of GABA receptor agonists or antagonists, respectively, reproduced these effects. Together, these experiments reveal a novel circuit mechanism for the contextual control of fear, and indicate that vHPC-mediated inhibition of IL is an essential neural substrate for fear relapse.

The activity-regulated cytoskeletal-associated protein (Arc/Arg3.1) is an immediate early gene that has been widely implicated in hippocampal-dependent learning and memory and is believed to play an integral role in synapse-specific plasticity. Here, we examined the role of Arc/Arg3.1 in amygdala-dependent Pavlovian fear conditioning. We first examined the regulation of Arc/Arg3.

Recent studies have shown that nitric oxide (NO) signaling plays a crucial role in memory consolidation of Pavlovian fear conditioning and in synaptic plasticity in the lateral amygdala (LA). In the present experiments, we examined the role of the cGMP-dependent protein kinase (PKG), a downstream effector of NO, in fear memory consolidation and long-term potentiation (LTP) at thalamic and cortical input pathways to the LA. In behavioral experiments, rats given intra-LA infusions of either the PKG inhibitor Rp-8-Br-PET-cGMPS or the PKG activator 8-Br-cGMP exhibited dose-dependent impairments or enhancements of fear memory consolidation, respectively. In slice electrophysiology experiments, bath application of Rp-8-Br-PET-cGMPS or the guanylyl cyclase inhibitor LY83583 impaired LTP at thalamic, but not cortical inputs to the LA, while bath application of 8-Br-cGMP or the guanylyl cyclase activator YC-1 resulted in enhanced LTP at thalamic inputs to the LA. Interestingly, YC-1-induced enhancement of LTP in the LA was reversed by concurrent application of the MEK inhibitor U0126, suggesting that the NO-cGMP-PKG signaling pathway may promote synaptic plasticity and fear memory formation in the LA, in part by activating the ERK/MAPK signaling cascade. As a test of this hypothesis, we next showed that rats given intra-LA infusion of the PKG inhibitor Rp-8-Br-PET-cGMPS or the PKG activator 8-Br-cGMP exhibit impaired or enhanced activation, respectively, of ERK/MAPK in the LA after fear conditioning. Collectively, our findings suggest that an NO-cGMP-PKG-dependent form of synaptic plasticity at thalamic input synapses to the LA may underlie memory consolidation of Pavlovian fear conditioning, in part, via activation of the ERK/MAPK signaling cascade.Nitric oxide (NO) signaling has been widely implicated in synaptic plasticity and memory formation (Schuman and Madison 1991;Bredt and Snyder 1992;Chapman et al. 1992;Bohme et al. 1993;Zhuo et al. 1994;Bernabeu et al. 1995;Arancio et al. 1996;Doyle et al. 1996;Holscher et al. 1996;Suzuki et al. 1996;Zou et al. 1998;Ko and Kelly 1999;Lu et al. 1999). A highly soluble gas generated by the conversion of L-arginine to L-citrulline by the Ca 2+ -regulated enzyme nitric oxide synthase (NOS), NO is known to have a variety of effects both pre-and postsynaptically. One immediate downstream effector of NO, for example, is soluble guanylyl cyclase (sGC) (Bredt and Snyder 1992;Son et al. 1998;Denninger and Marletta 1999;Arancio et al. 2001). This enzyme directly leads to the formation of cyclic-GMP, and in turn, to the activation of the cGMP-dependent protein kinase (PKG). PKG, in turn, can have a number of effects, including targeting and mobilization of synaptic vesicles in the presynaptic cell, leading to enhanced transmitter release (Hawkins et al. 1993) and also to activation of protein kinase signaling cascades in the postsynaptic cell, leading to activation of transcription and translation that are critical for long-term synaptic plasticity and memory formation (Lu et al. 1999;Chi...

The Neuronal PAS domain protein 4 (Npas4) is a neuronal activity-dependent immediate early gene that has recently been identified as a transcription factor which regulates the transcription of genes that control inhibitory synapse development and synaptic plasticity. The role Npas4 in learning and memory, however, is currently unknown. Here, we systematically examine the role of Npas4 in auditory Pavlovian fear conditioning, an amygdala-dependent form of emotional learning. In our first series of experiments, we show that Npas4 mRNA and protein are regulated in the rat lateral nucleus of the amygdala (LA) in a learning-dependent manner. Further, knockdown of Npas4 protein in the LA via adeno-associated viral (AAV) mediated gene delivery of RNAi was observed to impair fear memory formation, while innate fear and the expression of fear memory were not affected. In our second series of experiments, we show that Npas4 protein is regulated in the LA by retrieval of an auditory fear memory and that knockdown of Npas4 in the LA impairs retention of a reactivated, but not a non-reactivated, fear memory. Collectively, our findings provide the first comprehensive look at the functional role of Npas4 in learning and memory.

Electroconvulsive shock (ECS) is the most effective treatment for depression, but the mechanism underlying the therapeutic action of this treatment is still unknown. To better understand the molecular changes that may be necessary for the clinical effectiveness of ECS we have combined the technologies of gene expression profiling using cDNA microarrays with T7-based RNA amplification and laser microdissection to identify regulated genes in the dentate gyrus granule cell layer of the hippocampus. We have identified genes previously reported to be up-regulated following ECS, including brain-derived neurotrophic factor, neuropeptide Y, and thyrotrophin releasing hormone, as well as several novel genes. Notably, we have identified additional genes that are known to be involved in neuroprotection, such as growth arrest DNA damage inducible beta (Gadd45beta), and the excitatory amino acid transporter-1 (EAAC1/Slc1A1). In addition, via in situ hybridization we show that EAAC1 is specifically up-regulated in the dentate gyrus, but not in other hippocampal subfields. This study demonstrates the utility of microarray analysis of microdissected subregions of limbic brain regions and identifies novel ECS-regulated genes. Keywords: EAAC1, glutamate transporter, laser microdissection, microarray, neuroprotection, RNA amplification.Depression is a devastating illness that affects 12-15% of people at some point in their lives (Kessler et al. 2003). Current chemical treatments are only partially effective, alleviating symptoms for only approximately 65% of depressed individuals, and require weeks or months of administration to achieve a therapeutic response. Electroconvulsive shock (ECS) is an extremely effective, nonchemical treatment for depression, often efficacious in cases that are resistant to chemical antidepressants.Despite the clinical usefulness of ECS, the mechanisms underlying the therapeutic actions of this treatment are currently unknown. Previous reports have documented gene expression changes in the hippocampus Altar et al. 2004) and other brain regions (Yamada et al. 2002) following ECS that could contribute to adaptive changes underlying the therapeutic actions of ECS. Although there has been progress made using these traditional gene expression profiling techniques, these data sets still have shortcomings because they represent detectable changes that occur within large subregions of the brain (e.g. hippocampus or frontal cortex) constituting multiple cell types and independently acting neuronal circuits. This could result in dilution of important gene expression changes, underscoring the importance of utilizing methods that can increase signals from discrete brain regions and/or cell types. Recent advances in laser microdissection methodologies coupled with T7-based RNA amplification and gene expression detection technologies have made it feasible to determine gene expression profiles from discrete regions of the brain as well as from single cells (Bonaventure et al. 2002;Kelz et al. 2002;Kamme et al....

J. Neurochem. (2010) 112, 636–650. Abstract Most recent studies aimed at defining the cellular and molecular mechanisms of Pavlovian fear conditioning have focused on protein kinase signaling pathways and the transcription factor cAMP‐response element binding protein (CREB) that promote fear memory consolidation in the lateral nucleus of the amygdala (LA). Despite this progress, there still remains a paucity of information regarding the genes downstream of CREB that are required for long‐term fear memory formation in the LA. We have adopted a strategy of using microarray technology to initially identify genes induced within the dentate gyrus following in vivo long‐term potentiation (LTP) followed by analysis of whether these same genes are also regulated by fear conditioning within the LA. In the present study, we first identified 34 plasticity‐associated genes that are induced within 30 min following LTP induction utilizing a combination of DNA microarray, qRT‐PCR, and in situ hybridization. To determine whether these genes are also induced in the LA following Pavlovian fear conditioning, we next exposed rats to an auditory fear conditioning protocol or to control conditions that do not support fear learning followed by qRT‐PCR on mRNA from microdissected LA samples. Finally, we asked whether identified genes induced by fear learning in the LA are downstream of the extracellular‐regulated kinase/mitogen‐activated protein kinase signaling cascade. Collectively, our findings reveal a comprehensive list of genes that represent the first wave of transcription following both LTP induction and fear conditioning that largely belong to a class of genes referred to as ‘neuronal activity dependent genes’ that are likely calcium, extracellular‐regulated kinase/mitogen‐activated protein kinase, and CREB‐dependent.

We report that the paired homeodomain transcription factor Pax6 is imported into the nucleus by the Karyopherin ␤ family member Karyopherin 13 (Kap13). Pax6 was identified as a potential cargo for Kap13 by a yeast two-hybrid screen. Direct binding of Pax6 to Kap13 was subsequently confirmed by in vitro assays with recombinant proteins, and binding in vivo was shown by coimmunoprecipitation. Ran-dependent import of Pax6 by Kap13 was shown to occur by using a digitonin-permeabilized cells assay. Kap13 binds to Pax6 via a nuclear localization sequence (NLS), which is located within a segment of 80 amino acid residues that includes the homeodomain. Kap13 showed reduced binding to Pax6 when either region located at each end of the homeodomain (208 to 214 and 261 to 267) was deleted. The paired-type homeodomain transcription factor family includes more than 20 members. All members contain a region similar to the NLS found in Pax6 and are therefore likely to be imported by Kap13. We confirmed this hypothesis for Pax3 and Crx, which bind to and are imported by Kap13.