Extinction and Retrieval + Extinction of Conditioned Fear Differentially Activate Medial Prefrontal Cortex and Amygdala in Rats

Pairing a previously neutral conditioned stimulus (CS; e.g., a tone) to an aversive unconditioned stimulus (US; e.g., a footshock) leads to associative learning such that the tone alone comes to elicit a conditioned response (e.g., freezing). We have previously shown that an extinction session that occurs within the reconsolidation window (termed retrieval+extinction) attenuates fear responding and prevents the return of fear in Pavlovian fear conditioning (Monfils et al., 2009). To date, the mechanisms that explain the different behavioral outcomes between standard extinction and retrieval+extinction remain poorly understood. Here we sought to examine the differential temporal engagement of specific neural systems by these 2 approaches using Arc catFISH (cellular compartment analysis of temporal activity using fluorescence in situ hybridization). Our results demonstrate that extinction and retrieval+extinction lead to differential patterns of expression, suggesting that they engage different networks. These findings provide insight into the neural mechanisms that allow extinction during reconsolidation to prevent the return of fear in rats

Treatment Strategies Targeting Excess Hippocampal Activity Benefit Aged Rats with Cognitive Impairment

Excess neural activity in the CA3 region of the hippocampus has been linked to memory impairment in aged rats. We tested whether interventions aimed at reducing this excess activity would improve memory performance. Aged (24 to 28 months old) male Long–Evans rats were characterized in a spatial memory task known to depend on the functional integrity of the hippocampus, such that aged rats with identified memory impairment were used in a series of experiments. Overexpression of the inhibitory neuropeptide Y 13–36 in the CA3 via adeno-associated viral transduction was found to improve hippocampal-dependent long-term memory in aged rats, which had been characterized with impairment. Subsequent experiments with two commonly used antiepileptic agents, sodium valproate and levetiracetam, similarly produced dose-dependent memory improvement in such aged rats. Improved spatial memory with low doses of these agents was observed in both appetitve and aversive spatial tasks. The benefits of these different modalities of treatment are consistent with the concept that excess activity in the CA3 region of the hippocampus is a dysfunctional condition that may have a key role underlying age-related impairment in hippocampal-dependent memory processes. Because increased hippocampal activation occurs in age-related memory impairment in humans as observed in functional neuroimaging, the current findings also suggest that low doses of certain antiepileptic drugs in cognitively impaired elderly humans may have therapeutic potential and point to novel targets for this indication

Therapeutic Liabilities of in Vivo Viral Vector Tropism: Adeno-Associated Virus Vectors, NMDAR1 Antisense, and Focal Seizure Sensitivity

The N-methyl-d-aspartic acid (NMDA) receptor provides a potential target for gene therapy of focal seizure disorders. To test this approach, we cloned a 729-bp NMDA receptor (NMDAR1) cDNA fragment in the antisense orientation into adeno-associated virus (AAV) vectors, where expression was driven by either a tetracycline-off regulatable promoter (AAV-tTAK-NR1A) or a cytomegalovirus (CMV) promoter (AAV-CMV-NR1A). After infection of primary cultured cortical neurons with recombinant AAV-tTAK-NR1A, patch clamp studies found a significant decrease in maximal NMDA-evoked currents, indicative of a decrease in the number of NMDA receptors. Similarly, infusion of AAV-tTAK-NR1A (1 μl) into the rat temporal cortex significantly decreased NMDAR1-like immunoreactivity in layer V pyramidal cells. When AAV-tTAK-NR1A vectors were infused into the seizure-sensitive site of the rat inferior collicular cortex, the seizure sensitivity increased significantly over a period of 4 weeks. However, collicular infusion of AAV-CMV-NR1A vectors caused the opposite effect, a significant decrease in seizure sensitivity. Subsequent collicular coinfusion of vector encoding green fluorescent protein (GFP) driven by the tetracycline-off promoter (AAV-tTAK-GFP) and vector encoding β-galactosidase driven by the CMV promoter (AAV-CMV-LacZ) transduced distinct neuronal populations with only partial overlap. Thus, differing transduction ratios of inhibitory interneurons to primary output neurons likely account for the divergent seizure influences. Although AAV vector-derived NMDAR1 antisense can influence NMDA receptor function both in vitro and in vivo, promoter-related tropic differences dramatically alter the physiological outcome of this receptor-based gene therapy

Behaviorally Activated mRNA Expression Profiles Produce Signatures of Learning and Enhanced Inhibition in Aged Rats with Preserved Memory

<div><p>Aging is often associated with cognitive decline, but many elderly individuals maintain a high level of function throughout life. Here we studied outbred rats, which also exhibit individual differences across a spectrum of outcomes that includes both preserved and impaired spatial memory. Previous work in this model identified the CA3 subfield of the hippocampus as a region critically affected by age and integral to differing cognitive outcomes. Earlier microarray profiling revealed distinct gene expression profiles in the CA3 region, under basal conditions, for aged rats with intact memory and those with impairment. Because prominent age-related deficits within the CA3 occur during neural encoding of new information, here we used microarray analysis to gain a broad perspective of the aged CA3 transcriptome under activated conditions. Behaviorally-induced CA3 expression profiles differentiated aged rats with intact memory from those with impaired memory. In the activated profile, we observed substantial numbers of genes (greater than 1000) exhibiting increased expression in aged unimpaired rats relative to aged impaired, including many involved in synaptic plasticity and memory mechanisms. This unimpaired aged profile also overlapped significantly with a learning induced gene profile previously acquired in young adults. Alongside the increased transcripts common to both young learning and aged rats with preserved memory, many transcripts behaviorally-activated in the current study had previously been identified as repressed in the aged unimpaired phenotype in basal expression. A further distinct feature of the activated profile of aged rats with intact memory is the increased expression of an ensemble of genes involved in inhibitory synapse function, which could control the phenotype of neural hyperexcitability found in the CA3 region of aged impaired rats. These data support the conclusion that aged subjects with preserved memory recruit adaptive mechanisms to retain tight control over excitability under both basal and activated conditions. </p> </div

Behaviorally activated expression profiles are distinct from basal expression.

<p>SAM d-statistic values derived from basal CA3 gene expression comparison between AU and AI rats [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083674#B14" target="_blank">14</a>] were plotted against AU-act v AI-act SAM d-statistic values for each probeset. Each dot represents a single probeset (N>15,000 probesets). Correlation r-value and accompanying p-value are indicated on the graph. A slight negative correlation indicates negligible similarity between the basal and activated expression profiles.</p

Spatial training induces differential AU and AI expression profiles.

<p><b>A</b>. CA3 gene expression MDS (distance = 1 – r) analysis of AU and AI spatially trained rats shows clustering of AU-S subjects. Each point within the graph represents the array for a single subject colored by cognitive phenotype, as indicated. The distance between points is an indication of relative similarity of mRNA profiles. AU arrays (red) tend to cluster together, at least partially segregated from AI arrays (blue). Green arrow points to an AI rat with borderline LI (LI=254); LI range for AU is 240 or lower. <b>B</b>. SAM d-statistic density plot comparing AU-S expression to AI-S, shows large numbers of genes differentially expressed between groups. The dashed black line represents the expected, random d-statistic density distribution while the black solid curve represents the distribution observed AU-S/AI-S comparison. Numbers of genes differentially expressed at an FDR = 0.1 (depicted by red dashed lines) are indicated on each graph. The gray colored area under solid curve represents the numbers of probesets that meet an FDR of 0.1. Positive SAM d-stat values indicate increased gene expression in AU-S with most of the differentially expressed genes increased in AU. <b>C</b>. SAM d-statistic density plot comparing AU-NS expression to AI-NS, while similar in shape to B., shows few genes differentially expressed between groups. The dashed black line represents the expected, random d-statistic density distribution while the black solid curve represents the distribution observed AU-NS/AI-NS comparison. Numbers of genes differentially expressed at an FDR = 0.1 (depicted by red dashed lines) are indicated on each graph. Positive SAM d-stat values indicate increased gene expression in AU-NS. </p

Learning genes are increased in AU relative to AI with behavioral experience.

<p><b>A</b>. SAM d-statistic values derived from young learning (LA) v control (CTL) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083674#B18" target="_blank">18</a>] were plotted against AU-act v AI-act SAM d-statistic values for each probeset. Each dot represents a single probeset (N>7,000 probesets). The positive correlation indicates significant similarity between young learning and aged activated expression profiles. <b>B</b>. CA3 genes that were significantly modulated by a learning episode in young rats [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083674#B18" target="_blank">18</a>] were tested for differential expression between AU-act and AI-act rats from the current study. Positive d-stat values indicate increased expression in AU-act subjects. Genes observed to increase expression with learning in young rats (red line) were also significantly increased in AU-act compared to AI-act subjects (N=189; p=7.7 x10^-44). Black line represents the distribution of all genes. <b>C</b>. Similar to B., learning-activated genes in young rats were examined with respect to differences in AU and AI rats under basal conditions (red line), revealing a significant decrease in AU rats compared to AI (N=173; p=0.0067). In B and C, genes observed to decrease expression with learning in young rats (blue line) did not have a significantly different distribution in aged rats with behavioral activation (blue line shown in B, N=81; p=0.12), although the distribution was marginally decreased in AU relative to AI rats under basal conditions (blue line shown in C, N=81; p=0.067). <b>D</b>., <b>E</b>. Genes known to be increased with LTP [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083674#B18" target="_blank">18</a>] were also tested for differential expression between AU and AI rats in (<b>D</b>) activated and (<b>E</b>) basal microarray datasets. Similar to learning induced genes, LTP genes (red line) were significantly increased in AU-act rats (N=54; p=0.03) and significantly decreased in AU rats under basal conditions (N=44; p=0.0056). The black line in panels B and D is equivalent to the solid black line in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083674#pone-0083674-g003" target="_blank">Figure 3B</a>.</p