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1.  Grin1 Receptor Deletion within CRF Neurons Enhances Fear Memory 
PLoS ONE  2014;9(10):e111009.
Corticotropin releasing factor (CRF) dysregulation is implicated in mood and anxiety disorders such as posttraumatic stress disorder (PTSD). CRF is expressed in areas engaged in fear and anxiety processing including the central amygdala (CeA). Complicating our ability to study the contribution of CRF-containing neurons to fear and anxiety behavior is the wide variety of cell types in which CRF is expressed. To manipulate specific subpopulations of CRF containing neurons, our lab has developed a mouse with a Cre recombinase gene driven by a CRF promoter (CRFp3.0Cre) (Martin et al., 2010). In these studies, mice that have the gene that encodes NR1 (Grin1) flanked by loxP sites (floxed) were crossed with our previously developed CRFp3.0Cre mouse to selectively disrupt Grin1 within CRF containing neurons (Cre+/fGrin1+). We find that disruption of Grin1 in CRF neurons did not affect baseline levels of anxiety, locomotion, pain sensitivity or exploration of a novel object. However, baseline expression of Grin1 was decreased in Cre+/fGrin1+ mice as measured by RTPCR. Cre+/fGrin1+ mice showed enhanced auditory fear acquisition and retention without showing any significant effect on fear extinction. We measured Gria1, the gene that encodes AMPAR1 and the CREB activator Creb1 in the amygdala of Cre+/fGrin1+ mice after fear conditioning. Both Gria1 and Creb1 were enhanced in the amygdala after training. To determine if the Grin1-expressing CRF neurons within the CeA are responsible for the enhancement of fear memory in adults, we infused a lentivirus with Cre driven by a CRF promoter (LV pCRF-Cre/fGrin1+) into the CeA of floxed Grin1 mice. Cre driven deletion of Grin1 specifically within CRF expressing cells in the CeA also resulted in enhanced fear memory acquisition and retention. Altogether, these findings suggest that selective disruption of Grin1 within CeA CRF neurons strongly enhances fear memory.
PMCID: PMC4207780  PMID: 25340785
2.  Remembering Another Aspect of Forgetting 
Although forgetting is most often thought of in terms of declines in performance (response loss or impairment), another class of memory phenomena, the forgetting of stimulus attributes, has begun to attract experimental attention. In non-human animals, the loss of memory for stimulus features is reflected in the flattening of stimulus generalization gradients as well as in the attenuation of the disrupting effect of a shift in context at testing. In both cases, a delay between the learning episode and testing results in increased responding in the presence of previously ineffective stimuli. Thus, previously discriminable cues become more functionally interchangeable. The implications of the forgetting of attributes for some theoretical issues of memory loss and for methodological strategies have been noted earlier. However, relatively little is known about the neurobiological mechanisms underlying stimulus attribute forgetting, and why some memories are maintained while others are not. In this paper we review the evidence for the forgetting of stimulus attributes, discuss recent findings identifying neurobiological underpinnings of forgetting and generalization of fear responses, and discuss relevant clinical implications of fear generalization.
PMCID: PMC3365651  PMID: 22675315
memory; fear; stimulus attributes; amygdala; hippocampus; context; generalization
3.  Thy1-Expressing Neurons in the Basolateral Amygdala May Mediate Fear Inhibition 
The Journal of Neuroscience  2013;33(25):10396-10404.
Research has identified distinct neuronal circuits within the basolateral amygdala (BLA) that differentially mediate fear expression versus inhibition; however, molecular markers of these populations remain unknown. Here we examine whether optogenetic activation of a cellular subpopulation, which may correlate with the physiologically identified extinction neurons in the BLA, would differentially support fear conditioning versus fear inhibition/extinction. We first molecularly characterized Thy1-channelrhodopsin-2 (Thy1-ChR2-EYFP)-expressing neurons as a subpopulation of glutamatergic pyramidal neurons within the BLA. Optogenetic stimulation of these neurons inhibited a subpopulation of medial central amygdala neurons and shunted excitation from the lateral amygdala. Brief activation of these neurons during fear training disrupted later fear memory in male mice. Optogenetic activation during unreinforced stimulus exposure enhanced extinction retention, but had no effect on fear expression, locomotion, or open-field behavior. Together, these data suggest that the Thy1-expressing subpopulation of BLA pyramidal neurons provide an important molecular and pharmacological target for inhibiting fear and enhancing extinction and for furthering our understanding of the molecular mechanisms of fear processing.
PMCID: PMC3685835  PMID: 23785152
4.  Spike-Timing Precision and Neuronal Synchrony Are Enhanced by an Interaction between Synaptic Inhibition and Membrane Oscillations in the Amygdala 
PLoS ONE  2012;7(4):e35320.
The basolateral complex of the amygdala (BLA) is a critical component of the neural circuit regulating fear learning. During fear learning and recall, the amygdala and other brain regions, including the hippocampus and prefrontal cortex, exhibit phase-locked oscillations in the high delta/low theta frequency band (∼2–6 Hz) that have been shown to contribute to the learning process. Network oscillations are commonly generated by inhibitory synaptic input that coordinates action potentials in groups of neurons. In the rat BLA, principal neurons spontaneously receive synchronized, inhibitory input in the form of compound, rhythmic, inhibitory postsynaptic potentials (IPSPs), likely originating from burst-firing parvalbumin interneurons. Here we investigated the role of compound IPSPs in the rat and rhesus macaque BLA in regulating action potential synchrony and spike-timing precision. Furthermore, because principal neurons exhibit intrinsic oscillatory properties and resonance between 4 and 5 Hz, in the same frequency band observed during fear, we investigated whether compound IPSPs and intrinsic oscillations interact to promote rhythmic activity in the BLA at this frequency. Using whole-cell patch clamp in brain slices, we demonstrate that compound IPSPs, which occur spontaneously and are synchronized across principal neurons in both the rat and primate BLA, significantly improve spike-timing precision in BLA principal neurons for a window of ∼300 ms following each IPSP. We also show that compound IPSPs coordinate the firing of pairs of BLA principal neurons, and significantly improve spike synchrony for a window of ∼130 ms. Compound IPSPs enhance a 5 Hz calcium-dependent membrane potential oscillation (MPO) in these neurons, likely contributing to the improvement in spike-timing precision and synchronization of spiking. Activation of the cAMP-PKA signaling cascade enhanced the MPO, and inhibition of this cascade blocked the MPO. We discuss these results in the context of spike-timing dependent plasticity and modulation by neurotransmitters important for fear learning, such as dopamine.
PMCID: PMC3338510  PMID: 22563382
5.  A transcriptomic analysis of type I-III neurons in the bed nucleus of the stria terminalis 
The activity of neurons in the anterolateral cell group of the bed nucleus of the stria terminalis (BNSTALG) plays a critical role in anxiety- and stress-related behaviors. Histochemical studies have suggested that multiple distinct neuronal phenotypes exist in the BNSTALG. Consistent with this observation, the physiological properties of BNSTALG neurons are also heterogeneous, and three distinct cell types can be defined (Type I–III) based primarily on their expression of four key membrane currents, namely Ih, IA, IT, and IK(IR). Significantly, all four channels are multimeric proteins and can comprise of more than one pore-forming α subunit. Hence, differential expression of α subunits may further diversify the neuronal population. However, nothing is known about the relative expression of these ion channel α subunits in BNSTALG neurons.
We have addressed this lacuna by combining whole cell patch clamp recording together with single cell reverse transcriptase polymerase chain reaction (scRT-PCR) to assess the mRNA transcript expression for each of the subunits for the four key ion channels in Type I-III neurons of the BNSTALG. Here, cytosolic mRNA from single neurons was probed for the expression of transcripts for each of the α subunits of Ih (HCN1- HCN4), IT (Cav3.1- Cav3.3), IA (Kv1.4, Kv3.4, Kv4.1- Kv 4.3) and IK(IR) (Kir2.1-Kir2.4).
An unbiased hierarchical cluster analysis followed by discriminant function analysis revealed that a positive correlation exists between the physiological and genetic phenotype of BNSTALG neurons. Thus, the analysis segregated BNSTALG neurons into 3 distinct groups, based on their α subunit mRNA expression profile, which positively correlated with our existing electrophysiological classification (Type I–III). Furthermore, analysis of mRNA transcript expression in Type I –Type III neurons suggested that, whereas Type I and III neurons appear to represent genetically homologous cell populations, Type II neurons may be further subdivided into three genetically distinct subgroups. These data not only validate our original classification scheme, but further refine the classification at the molecular level, and thus identifies novel targets for potential disruption and/or pharmacotherapeutic intervention in stress-related anxiety-like behaviors.
PMCID: PMC3066260  PMID: 21310239
Bed nucleus of the stria terminalis; ion channels; α subunits; patch clamp recording; single cell reverse transcriptase polymerase chain reaction
6.  A novel transgenic mouse for gene-targeting within cells that express corticotropin-releasing factor 
Biological psychiatry  2010;67(12):1212-1216.
Corticotropin-releasing factor (CRF) orchestrates the mammalian endocrine, autonomic, and behavioral stress response and has been implicated in the pathophysiology of illnesses ranging from irritable bowel syndrome to mood and anxiety disorders. CRF is produced and released from a variety of cell types, making it difficult to distinguish the specific role of CRF from other neurotransmitters with which it colocalizes. To clarify the basic biology of the CRF neuron, we must be able to manipulate selectively CRFergic cells. Here we describe a novel transgenic mouse using 3.0Kb of the CRF promoter to drive expression of Cre-recombinase (CRFp3.0Cre). Crossing CRFp3.0Cre with a fluorescent reporter strain results in Cre-dependent GFP expression within CRF-producing cells. Thus CRF cells can be identified for single-cell PCR and electrophysiological procedures. Furthermore, the CRFp3.0Cre transgenic can be combined with other available mouse strains containing a “floxed” gene of interest to allow unparalleled detailed analysis of the CRF system.
PMCID: PMC3039842  PMID: 20303068

Results 1-6 (6)