Translational profiling of stress-induced neuroplasticity in the CA3 pyramidal neurons of BDNF Val66Met mice.

Genetic susceptibility and environmental factors (such as stress) can interact to affect the likelihood of developing a mood disorder. Stress-induced changes in the hippocampus have been implicated in mood disorders, and mutations in several genes have now been associated with increased risk, such as brain-derived neurotrophic factor (BDNF). The hippocampus has important anatomical subdivisions, and pyramidal neurons of the vulnerable CA3 region show significant remodeling after chronic stress, but the mechanisms underlying their unique plasticity remain unknown. This study characterizes stress-induced changes in the in vivo translating mRNA of this cell population using a CA3-specific enhanced green fluorescent protein (EGFP) reporter fused to the L10a large ribosomal subunit (EGFPL10a). RNA-sequencing after isolation of polysome-bound mRNAs allows for cell-type-specific, genome-wide characterization of translational changes after stress. The data demonstrate that acute and chronic stress produce unique translational profiles and that the stress history of the animal can alter future reactivity of CA3 neurons. CA3-specific EGFPL10a mice were then crossed to the stress-susceptible BDNF Val66Met mouse line to characterize how a known genetic susceptibility alters both baseline translational profiles and the reactivity of CA3 neurons to stress. Not only do Met allele carriers exhibit distinct levels of baseline translation in genes implicated in ion channel function and cytoskeletal regulation, but they also activate a stress response profile that is highly dissimilar from wild-type mice. Closer examination of genes implicated in the mechanisms of neuroplasticity, such as the NMDA and AMPA subunits and the BDNF pathway, reveal how wild-type mice upregulate many of these genes in response to stress, but Met allele carriers fail to do so. These profiles provide a roadmap of stress-induced changes in a genetically homogenous population of hippocampal neurons and illustrate the profound effects of gene-environment interactions on the translational profile of these cells.

Reprogramming axonal behavior by axon-specific viral transduction.

The treatment of axonal disorders, such as diseases associated with axonal injury and degeneration, is limited by the inability to directly target therapeutic protein expression to injured axons. Current gene therapy approaches rely on infection and transcription of viral genes in the cell body. Here, we describe an approach to target gene expression selectively to axons. Using a genetically engineered mouse containing epitope-labeled ribosomes, we find that neurons in adult animals contain ribosomes in distal axons. To use axonal ribosomes to alter local protein expression, we utilized a Sindbis virus containing an RNA genome that has been modified so that it can be directly used as a template for translation. Selective application of this virus to axons leads to local translation of heterologous proteins. Furthermore, we demonstrate that selective axonal protein expression can be used to modify axonal signaling in cultured neurons, enabling axons to grow over inhibitory substrates typically encountered following axonal injury. We also show that this viral approach also can be used to achieve heterologous expression in axons of living animals, indicating that this approach can be used to alter the axonal proteome in vivo. Together, these data identify a novel strategy to manipulate protein expression in axons, and provides a novel approach for using gene therapies for disorders of axonal function.

Extinction training regulates tyrosine hydroxylase during withdrawal from cocaine self-administration.

Chronic exposure to drugs of abuse is known to modulate tyrosine hydroxylase (TH) levels in the mesolimbic dopamine system. In this study, 12 d of cocaine self-administration in rats (4 hr/d) reduced TH immunoreactivity by 29% in the nucleus accumbens (NAc) shell, but not core, after a 1 week withdrawal period. In contrast, TH immunoreactivity in the NAc was completely restored in animals that experienced extinction training (4 hr/d) during the same withdrawal period. Extinction training also increased TH levels in the ventral tegmental area (VTA) by 45%, whereas TH was not altered in the VTA by cocaine withdrawal alone. Thus, extinction-induced normalization of NAc TH levels could involve increased TH synthesis, stability, and/or transport from the VTA to the NAc. A similar extinction training regimen failed to alter TH levels in the NAc or VTA of rats trained to self-administer sucrose pellets, indicating that TH regulation in cocaine-trained animals is not a generalized effect of extinction learning per se. Rather, these data suggest that neuroadaptative responses during cocaine withdrawal ultimately are determined by a complex interaction between chronic drug exposure and drug-seeking experience. The ability of extinction training to restore NAc TH levels is hypothesized to accelerate recovery from dopamine depletion and anhedonia during cocaine withdrawal.

Correlation of the hydrolysis of benzoyl-arginine naphthylamide (BANA) by plaque with clinical parameters and subgingival levels of spirochetes in periodontal patients.

Recent studies have shown that the extent of hydrolysis by plaque of the trypsin substrate, N-benzoyl-DL-arginine-2-naphthylamide (BANA), correlates with the numbers and proportions of spirochetes in subgingival plaque samples, and appears to be an indicator of clinical disease. In this study, BANA hydrolysis by subgingival plaque was evaluated in a blind manner for its ability to reflect both clinical parameters and subgingival levels of bacteria and spirochetes. Subgingival plaque samples were collected from periodontally healthy and diseased sites in 23 untreated periodontal patients and in 13 treated and maintained periodontal patients. In untreated patients, BANA hydrolysis was statistically associated with the total number of spirochetes and bacteria in the plaque sample, but in the treated patients BANA hydrolysis was statistically associated only with the spirochetes. Most BANA-positive reactions in both patient groups were from the sites which were clinically diseased and high in spirochetes. The majority of the negative reactions for BANA hydrolysis in both patient groups was among the sites which were periodontally healthy and low in spirochetes. Specificity and sensitivity of the test were above 80% for disease status in untreated patients. The predictive value of a positive and negative test was above 83%. Slightly lower sensitivity, specificity, and predictive values were found in the treated group. The BANA reaction appears to be an accurate and simple indicator of both clinical disease status and plaque levels of spirochetes in individual tooth sites in untreated and treated periodontal patients.

Effect of periodontal therapy on alveolar bone as measured by subtraction radiography.

Changes in the periodontal alveolar bone are often evaluated by comparing a series of radiographs taken over time. This investigation used a technique that allowed the image registration to be geometrically standardized each time a radiograph was taken. Radiographs of 24 patients from an ongoing double-blind, clinical study using metronidazole were obtained: (1) before any treatment, (2) at the completion of scaling and root planing and surgery (when performed) and (3) during the maintenance phase. One hundred six (106) paired comparisons were analyzed by subtraction radiography using a computerized system. Of these, 95 (89%) exhibited a minimal degree of geometric distortion and could be successfully substracted. Most areas (67%) showed no change in bone structure following periodontal treatment. Bone gain was noted in 12% of the sites examined, while bone loss was seen in 21% of the sites. This bone loss was statistically associated with sites that had received some form of surgical treatment.