Molecular mechanisms mediating a deficit in recall of fear extinction in adult mice exposed to cocaine in utero.

Prenatal cocaine exposure has been shown to alter cognitive processes of exposed individuals, presumed to be a result of long-lasting molecular alterations in the brain. In adult prenatal cocaine exposed (PCOC) mice we have identified a deficit in recall of fear extinction, a behavior that is dependent on the medial prefrontal cortex (mPFC) and the hippocampus. While we observed no change in the constitutive expression of brain derived neurotrophic factor (BDNF) protein and mRNA in the mPFC and hippocampus of adult PCOC mice, we observed blunted BDNF signaling in the mPFC of adult PCOC mice after fear extinction compared to the control animals. Specifically, during the consolidation phase of the extinction memory, we observed a decrease in BDNF protein and it's phospho-TrkB receptor expression. Interestingly, at this same time point there was a significant increase in total Bdnf mRNA levels in the mPFC of PCOC mice as compared with controls. In the Bdnf gene, we identified decreased constitutive binding of the transcription factors, MeCP2 and P-CREB at the promoters of Bdnf exons I and IV in the mPFC of PCOC mice, that unlike control mice remained unchanged when measured during the behavior. Finally, bilateral infusion of recombinant BDNF protein into the infralimbic subdivision of the mPFC during the consolidation phase of the extinction memory rescued the behavioral deficit in PCOC mice. In conclusion, these findings extend our knowledge of the neurobiologic impact of prenatal cocaine exposure on the mPFC of mice, which may lead to improved clinical recognition and treatment of exposed individuals

Exogenous infusion of BDNF protein into the IL of PCOC mice normalizes the deficit in recall of an extinguished cue-conditioned fear.

<p>(<b>a</b>) Overview of the experimental paradigm. Guide cannulae, targeting the infralimbic subdivision of the mPFC bilaterally were surgically implanted into the mice after which they were allowed to recover for 5-7 days before behavioral testing. Immediately after the animals went through extinction training session 2 on day 3 of the behavioral paradigm, vehicle (0.9% saline; VEH) or recombinant BDNF protein (BDNF) was infused into the infralimbic cortex and animals were tested 24 hours later for extinction recall. (<b>b</b>) Percent freezing to the tone. On day 1, both prenatal treatment groups showed significant levels of acquisition with no difference in freezing between PSAL and PCOC mice. On days 2 and 3, both prenatal treatment groups showed significant within-session extinction with no difference in freezing between PSAL and PCOC mice. Immediately after day 3, recombinant BDNF or VEH was infused into the IL bilaterally. On day 4, PCOC VEH mice showed significantly increased freezing compared to PSAL VEH mice demonstrating a deficit in extinction recall (*p<0.05). However, PCOC BDNF mice showed significantly decreased freezing compared to PCOC VEH mice indicating a rescue of the behavioral deficit (††p<0.01). Error bars represent the mean ± SEM (PSAL VEH n=11 from 11 litters, BDNF n=9 from 9 litters; PCOC VEH n=9 from 9 litters; BDNF n=11 from 11 litters). <b>c</b>) Schematic representation of the guide cannula placements in animals that were used for behavioral testing. (<b>d</b>) A representative image of guide cannulae targeting the IL bilaterally.</p

Altered <i>Bdnf</i> mRNA expression in the mPFC of prenatal cocaine exposed mice during fear extinction.

<p>(<b>a</b>) There was no change in <i>Bdnf</i> exon IX mRNA levels in the mPFC at baseline. However, at day 3 there was significantly increased BDNF exon IX mRNA levels in the mPFC of PCOC mice compared to PSAL mice. (<b>b</b>) There was no change in BDNF exon IX mRNA levels in the hippocampus at baseline or on day 3. (<b>c</b>) There was no change in BDNF exon I mRNA in the mPFC of PCOC mice at baseline or on day 3. (<b>d</b>) In the hippocampus of PSAL mice, there were higher levels of BDNF exon I mRNA levels on day 3 relative to their levels at baseline. (<b>e</b>) There was decreased BDNF exon IV mRNA levels in the mPFC of PSAL mice on day 3 compared to its levels at baseline. (<b>f</b>) There was no change in BDNF exon IV mRNA levels in the hippocampus at baseline or day 3. (*p<0.05, PCOC vs. PSAL; †p<0.05, ††p<0.01 vs. baseline of that prenatal treatment). Error bars represent the mean ± SEM (Baseline: PSAL n=12 from 12 litters, PCOC n=14 from 14 litters; Day 3: PSAL n=9 from 9 litters, PCOC n=10 from 10 litters).</p

Decreased constitutive MeCP2 and P-CREB binding at <i>Bdnf</i> exon I and IV promoters in the mPFC of prenatal cocaine exposed mice.

<p>(<b>a</b>, <b>c</b>, <b>e</b>, <b>g</b>) In the mPFC of PCOC mice there was decreased binding of MeCP2 (a,c) and P-CREB (e,g) at the promoters of <i>Bdnf</i> exons I and IV at baseline. On day 3 compared to baseline, there was lower MeCP2 (a, c) and P-CREB (e, g) binding at the <i>Bdnf</i> exon I and IV promoters in PSAL mice but not PCOC mice. (<b>b</b>, <b>d</b>, <b>f</b>, <b>h</b>) In the hippocampus at day 3 compared to baseline, there were higher levels of MeCP2 and P-CREB binding at the promoter of <i>Bdnf</i> exons I and IV in PSAL and PCOC mice. (*p<0.05, **p<0.01 PCOC vs. PSAL; ††p<0.01, †††p<0.001 vs. baseline of that prenatal treatment). Error bars represent the mean ± SEM (Baseline: MeCP2: PSAL n=11 from 11 litters, PCOC n=12 from 12 litters; P-CREB: PSAL n=8 from 8 litters, PCOC n=8 from 8 litters; Day 3: MeCP2: PSAL n=14 from 14 litters, PCOC n=14 from 14 litters; P-CREB: PSAL n=14 from 14 litters, PCOC n=14 from 14 litters).</p

Deficit in recall of an extinguished cue-conditioned fear in prenatal cocaine exposed mice.

<p>(<b>a</b>) Percent freezing to the tone. On day 1, mice were fear conditioned with five 30 sec tones that co-terminated with a 1 sec 0.7mA shock. On days 2 and 3, mice received extinction training in a novel environment and on day 4 were tested for extinction recall. Both prenatal treatment groups showed significant levels of acquisition on day 1. On days 2 and 3, all animals showed significant within-session extinction with no difference in freezing between PSAL and PCOC mice. At extinction testing on day 4, PCOC mice showed a deficit in recall of fear extinction (*p<0.05). Error bars represent the mean ± SEM (PSAL n=9 from 9 litters; PCOC n=9 from 9 litters). (<b>b</b>) Overview of the experimental paradigm. Brain tissue for molecular experiments was collected at three different time points from separate cohorts of animals. For analysis of constitutive levels of MeCP2 and P-CREB binding, Bdnf mRNA and protein expression, and phosphorylated TrkB analyses, tissue was collected in adult PSAL and PCOC animals that were not subjected to any behavioral testing (baseline). For molecular analyses during the consolidation phase of the extinction memory, tissue was collected immediately after extinction training on day 3 (Day 3) or immediately after the test for extinction recall on day 4 (Day 4).</p

Decreased mature BDNF protein and phosphorylated TrkB protein levels in the mPFC of prenatal cocaine exposed mice during fear extinction.

<p>(<b>a</b>) In the mPFC, there was significantly decreased mBDNF protein in PCOC mice compared to PSAL mice on day 3 and day 4. (<b>b</b>) In the hippocampus, there was no change in mBDNF protein levels at baseline, day 3 or day 4. (<b>c</b>) In the mPFC of PCOC mice compared to PSAL mice there was no change in total TrkB levels at baseline. However, at day 3 there was a trend (p=0.08) towards decreased TrkB protein levels in the mPFC of PCOC mice compared to PSAL mice. (<b>d</b>) There was no change in total TrkB protein levels in the hippocampus of PCOC mice at baseline or at day 3. (<b>e</b>) In the mPFC of PCOC mice compared to PSAL mice there was no change in P-Tyrosine protein levels at baseline but significantly decreased P-Tyrosine levels on day 3. (<b>f</b>) There was no change in P-Tyrosine protein levels in the hippocampus of PCOC mice at baseline or day 3. (*p<0.05, **p<0.01, ***p<0.001 PCOC vs. PSAL). Error bars represent the mean ± SEM (mBDNF - Baseline: PSAL n=9 from 9 litters, PCOC n=11 from 11 litters; Day 3: PSAL n=8 from 8 litters, PCOC n=8 from 8 litters; Day 4: PSAL n=8 from 8 litters; PCOC n=8 from 8 litters; TrkB/ P-Tyrosine - Baseline: PSAL n=8 from 8 litters, PCOC n=8 from 8 litters; Day 3: PSAL n=9 from 9 litters, PCOC n=8 from 8 litters).</p

D-cycloserine improves synaptic transmission in an animal mode of Rett syndrome

<div><p>Rett syndrome (RTT), a leading cause of intellectual disability in girls, is predominantly caused by mutations in the X-linked gene <i>MECP2</i>. Disruption of <i>Mecp2</i> in mice recapitulates major features of RTT, including neurobehavioral abnormalities, which can be reversed by re-expression of normal <i>Mecp2</i>. Thus, there is reason to believe that RTT could be amenable to therapeutic intervention throughout the lifespan of patients after the onset of symptoms. A common feature underlying neuropsychiatric disorders, including RTT, is altered synaptic function in the brain. Here, we show that <i>Mecp2</i>^{<i>tm1</i>.<i>1Jae/y</i>} mice display lower presynaptic function as assessed by paired pulse ratio, as well as decreased long term potentiation (LTP) at hippocampal Schaffer–collateral-CA1 synapses. Treatment of <i>Mecp2</i>^{<i>tm1</i>.<i>1Jae/y</i>} mice with D-cycloserine (DCS), an FDA-approved analog of the amino acid D-alanine with antibiotic and glycinergic activity, corrected the presynaptic but not LTP deficit without affecting deficient hippocampal BDNF levels. DCS treatment did, however, partially restore lower BDNF levels in the brain stem and striatum. Thus, treatment with DCS may mitigate the severity of some of the neurobehavioral symptoms experienced by patients with Rett syndrome.</p></div

Administration of DCS (20 mg/kg/day IP) to <i>Mecp2</i>^{<i>tm1</i>.<i>1Jae/y</i>} mice, beginning at 3 weeks of age, improved the breathing pattern and spontaneous locomotor activity, but did not affect neurological function, walking gate, tremors or grip strength compared to <i>Mecp2</i>^{<i>tm1</i>.<i>1Jae/y</i>} mice receiving vehicle (VEH) injection (n = 5 per group), at 18 weeks of age.

<p>Administration of DCS (20 mg/kg/day IP) to <i>Mecp2</i>^{<i>tm1</i>.<i>1Jae/y</i>} mice, beginning at 3 weeks of age, improved the breathing pattern and spontaneous locomotor activity, but did not affect neurological function, walking gate, tremors or grip strength compared to <i>Mecp2</i>^{<i>tm1</i>.<i>1Jae/y</i>} mice receiving vehicle (VEH) injection (n = 5 per group), at 18 weeks of age.</p

Administration of DCS (20 mg/kg/day IP) to <i>Mecp2</i>^{<i>tm1</i>.<i>1Jae/y</i>} mice did not alter body weight compared to <i>Mecp2</i>^{<i>tm1</i>.<i>1Jae/y</i>} mice receiving vehicle (VEH) injection (n = 5 per group).

<p>Mice were injected daily beginning at 3 weeks of age and continued throughout the life of the animal.</p