Regulation of Extinction-Related Plasticity by Opioid Receptors in the Ventrolateral Periaqueductal Gray Matter

Recent work has led to a better understanding of the neural mechanisms underlying the extinction of Pavlovian fear conditioning. Long-term synaptic changes in the medial prefrontal cortex (mPFC) are critical for extinction learning, but very little is currently known about how the mPFC and other brain areas interact during extinction. The current study examined the effect of drugs that impair the extinction of fear conditioning on the activation of the extracellular-related kinase/mitogen-activated protein kinase (ERK/MAPK) in brain regions that likely participate in the consolidation of extinction learning. Inhibitors of opioid and N-methyl-d-aspartic acid (NMDA) receptors were applied to the ventrolateral periaqueductal gray matter (vlPAG) and amygdala shortly before extinction training. Results from these experiments show that blocking opioid receptors in the vlPAG prevented the formation of extinction memory, whereas NMDA receptor blockade had no effect. Conversely, blocking NMDA receptors in the amygdala disrupted the formation of fear extinction memory, but opioid receptor blockade in the same brain area did not. Subsequent experiments tested the effect of these drug treatments on the activation of the ERK/MAPK signaling pathway in various brain regions following extinction training. Only opioid receptor blockade in the vlPAG disrupted ERK phosphorylation in the mPFC and amygdala. These data support the idea that opiodergic signaling derived from the vlPAG affects plasticity across the brain circuit responsible for the formation of extinction memory

Time-Dependent Expression of Arc and Zif268 after Acquisition of Fear Conditioning

Memory consolidation requires transcription and translation of new protein. Arc, an effector immediate early gene, and zif268, a regulatory transcription factor, have been implicated in synaptic plasticity underlying learning and memory. This study explored the temporal expression profiles of these proteins in the rat hippocampus following fear conditioning. We observed a time-dependent increase of Arc protein in the dorsal hippocampus 30-to-90-minute post training, returning to basal levels at 4 h. Zif268 protein levels, however, gradually increased at 30-minute post training before peaking in expression at 60 minute. The timing of hippocampal Arc and zif268 expression coincides with the critical period for protein synthesis-dependent memory consolidation following fear conditioning. However, the expression of Arc protein appears to be driven by context exploration, whereas, zif268 expression may be more specifically related to associative learning. These findings suggest that altered Arc and zif268 expression are related to neural plasticity during the formation of fear memory

Grin1 receptor deletion within CRF neurons enhances fear memory.

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

Effects of post-training hippocampal injections of midazolam on fear conditioning

Benzodiazepines have been useful tools for investigating mechanisms underlying learning and memory. The present set of experiments investigates the role of hippocampal GABA(A)/benzodiazepine receptors in memory consolidation using Pavlovian fear conditioning. Rats were prepared with cannulae aimed at the dorsal hippocampus and trained with a series of white noise–shock pairings. In the first experiment, animals received intrahippocampal infusion of midazolam or vehicle immediately or 3 h after training. Then, 24 h later, freezing to the training context and the white noise were measured independently. Results show infusion of midazolam immediately, but not 3 h, after training selectively attenuates contextual fear conditioning. In the second experiment, animals received intrahippocampal infusions of an antisense oligodeoxynucleotide (ODN) targeting the α5 subunit of the GABA(A) receptor or a missense control for several days prior to training and testing. Immediately after training, animals received an infusion of either midazolam or vehicle. Western blots conducted after testing showed a significant decrease in α5-containing GABA(A) receptor protein. This reduction did not alter the effectiveness of midazolam immediately after training at impairing context fear memory. Therefore, α5-containing GABA(A) receptors may not contribute to the effects of midazolam on context fear conditioning when given immediately post-training

In situ hybridization conducted on sections from (A) Cre+/^f^{<i>Grin1</i>+} mice show Cre expression in CRF containing areas (<i>Left,</i> BNST; <i>Right,</i> CeA).

<p>Representative expression of (B) <i>CRF</i> in Cre−/^{f<i>Grin1</i>+} (<i>Left</i>, black arrowheads) and Cre+/^{f<i>Grin1</i>+} (<i>Right</i>, white arrowheads) PVN, CeA and BNST are indicated. Anterior commissure (AC) is indicated for orientation purposes. (C) <i>Grin1</i> in Cre−/^{f<i>Grin1</i>+} (<i>Left</i>, black arrow) and Cre+/^{f<i>Grin1</i>+} (<i>Right</i>, white arrow) mouse is shown. (D) Image of PCR from Cre+/^{f<i>Grin1</i>+} or Cre−/^{f<i>Grin1</i>+} mice (280 bp) compared to wildtype (WT, 180 bp) mouse. E. Detailed depiction of central amygdala and BNST dissection sites. Inset shows CRF expression images from CRF-Cre ROSA LacZ+ mice to indicate overlap of punch area with CRF expression<b>.</b> F. RTPCR shows a significant difference in baseline expression of Grin1 in the amygdala, but not in the (G) BNST in Cre+/^{f<i>Grin1</i>+} mice compared to Cre−/^{f<i>Grin1</i>+} controls. (H) RTPCR conducted on tissue from the amygdala or from the BNST showed no significant difference in <i>CRF</i> expression (I) <i>Grin1</i> in Cre+/^{f<i>Grin1</i>+} mice compared to Cre−/^{f<i>Grin1</i>+} controls.</p

Directed virus against CRF infused into the amygdala of ‘floxed’ <i>Grin1</i> mice significantly disrupts <i>GRIN1</i> with no significant effects on CRF.

<p>(<b>A</b>) Intended infusion site of CRF driven Cre virus into the central amygdala of floxed <i>Grin1</i> mice (LV pCRF-Cre/^{f<i>Grin1</i>+}) is illustrated on the <i>Left </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111009#pone.0111009-Paxinos1" target="_blank">[<i>53</i>]</a>. The <i>upper right</i> panel shows <i>CRF</i> expression in the CeA, <i>lower right</i> panel shows <i>Grin1</i> expression levels in the CeA. <i>Image credit</i>: Allen Institute for Brain Science <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111009#pone.0111009-Hawrylycz1" target="_blank">[54]</a>. (<b>B</b>) (<i>Left</i>) Representative Cre <i>in situ</i> shows representative Cre expression in the CeA of a LV pCRF-Cre/^{f<i>Grin1</i>+} infused mouse. <i>(Right)</i> Representative GFP infection in the CeA of LV pCRF-GFP/^{f<i>Grin1</i>+} infused mouse is shown. RTPCR conducted on f<i>Grin1</i> mice that received unilateral CeA infusion of LV pCRF-Cre virus show a significant decrease in (<b>C</b>) <i>Grin1</i>, but not (<b>D</b>) <i>CRF</i> expression in the CeA. Dual fluorescent <i>in situ</i> hybridization was conducted on tissue from the CeA of f<i>Grin1</i> mice that received CeA infusion of LV pCRF-Cre virus. We show relative expression of <i>CRF</i> (<b>E, F</b>), <i>Grin1</i> (<b>G, H</b>), the overlay of <i>CRF</i> and <i>Grin1</i> (<b>I, J</b>) and Hoescht staining (<b>K, L</b>) of cell nuclei in the control (<i>Left</i> panels) and Infected (R<i>ight</i> panels) hemispheres.</p

<i>Grin1</i> deletion in CRF-containing neurons does not affect locomotion, anxiety-like behavior, or pain reactivity.

<p>No significant differences were found between Cre−/^{f<i>Grin1</i>+}and Cre+/^{f<i>Grin1</i>+} mice in anxiety or locomotion as shown by (A) open field time spent in center and (B) open field distance travelled. (C) Time spent in the open arms was not significantly different between Cre−/^{f<i>Grin1</i>+}and Cre+/^{f<i>Grin1</i>+} mice in a plus maze test of anxiety. D. Distance traveled was also not significantly different in the plus maze test. No difference in (E) shock reactivity was found, indicating that mice showed no difference in sensitivity to pain. No differences in activity were seen between Cre−/^{f<i>Grin1</i>+}and Cre+/^{f<i>Grin1</i>+} mice in (F) the novel object task, indicating no difference in novelty seeking behavior.</p

Results from RTPCR of amygdala or BNST after fear conditioning.

<p>(<b>A</b>) Shows a detailed depiction of dissection sites in central amygdala and BNST. The inset shows CRF expression image the amygdala of CRF-Cre ROSA LacZ+ mice to indicate overlap of the sampling area with CRF expression<b>.</b> Cre+/^{f<i>Grin1</i>+} mice show a significant increase in (B) <i>Gria1</i> and (C) <i>Creb1</i> in the amygdala after fear conditioning compared to fear conditioned Cre−/^{f<i>Grin1</i>+}. No difference between these groups is seen when (D) <i>Gria1</i>, (E) <i>Creb1</i> or (F) <i>Grin1</i> are measured in the BNST after fear conditioning. Inset shows CRF expression images from CRF-Cre ROSA LacZ+ mice in the respective area to indicate overlap of punch with CRF expression<b>.</b></p

Directed virus against CRF infused into the amygdala of ‘floxed’ <i>Grin1</i> mice resulted in enhanced fear memory and delayed fear extinction.

<p>Infusion of CRF driven Cre virus into the central amygdala of floxed <i>Grin1</i> mice (LV pCRF-Cre/^{f<i>Grin1</i>+}) significantly (<b>A</b>) enhanced auditory fear conditioning compared to those that received control virus (LV-pCRF-GFP/^{f<i>Grin1</i>+}) and (<b>B</b>) retention (1^st 5 trials of extinction) tested the following day. A significant disruption was found during (<b>C</b>) the extinction training session as well as during (<b>D</b>) extinction testing. (<b>E</b>) Tests of anxiety-like behavior using the open field test following extinction showed no differences in anxiety behavior in the (<b>F</b>) time in center or (<b>G</b>) total distance traveled in virus infused mice.</p

Cre+/^f^{<i>Grin1</i>+} mice show enhanced fear memory consolidation and retention.

<p>(<b>A</b>) Percent freezing during auditory fear conditioning over tone- shock pairings and (<b>B</b>) Cre+/^{f<i>Grin1</i>+} mice shows significantly more freezing during the 1^st six trials of fear extinction (also considered the fear retention test) suggesting an increase in fear memory consolidation. <b>C</b>) Average freezing behavior over the entire fear extinction session averaged into bins of 3 trials shows significantly disrupted fear extinction over the 30 extinction trials (<b>D</b>) during the extinction test conducted the following day Cre+/^{f<i>Grin1</i>+} mice do not show a significant difference from controls.</p