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.

Age and Alzheimer's disease gene expression profiles reversed by the glutamate modulator riluzole.

Alzheimer's disease (AD) and age-related cognitive decline represent a growing health burden and involve the hippocampus, a vulnerable brain region implicated in learning and memory. To understand the molecular effects of aging on the hippocampus, this study characterized the gene expression changes associated with aging in rodents using RNA-sequencing (RNA-seq). The glutamate modulator, riluzole, which was recently shown to improve memory performance in aged rats, prevented many of the hippocampal age-related gene expression changes. A comparison of the effects of riluzole in rats against human AD data sets revealed that many of the gene changes in AD are reversed by riluzole. Expression changes identified by RNA-Seq were validated by qRT-PCR open arrays. Riluzole is known to increase the glutamate transporter EAAT2's ability to scavenge excess glutamate, regulating synaptic transmission. RNA-seq and immunohistochemistry confirmed an increase in EAAT2 expression in hippocampus, identifying a possible mechanism underlying the improved memory function after riluzole treatment.

Hippocampal gene expression changes underlying stress sensitization and recovery.

Chronic and acute stressors have been linked to changes in hippocampal function and anxiety-like behaviors. Both produce changes in gene expression, but the extent to which these changes endure beyond the end of stress remains poorly understood. As an essential first step to characterize abnormal patterns of gene expression after stress, this study demonstrates how chronic restraint stress (CRS) modulates gene expression in response to a novel stressor in the hippocampus of wild-type mice and the extent to which these changes last beyond the end of CRS. Male C57/bl6 mice were subjected to (1) a forced swim test (FST), (2) corticosterone (Cort) or vehicle injections, (3) CRS for 21 days and then a FST, or (4) allowed to recover 21 days after CRS and subjected to FST. Hippocampal mRNA was extracted and used to generate cDNA libraries for microarray hybridization. Naive acute stressors (FST and vehicle injection) altered similar sets of genes, but Cort treatment produced a profile that was distinct from both FST and vehicle. Exposure to a novel stress after CRS activated substantially more and different genes than naive exposure. Most genes increased by CRS were decreased after recovery but many remained altered and did not return to baseline. Pathway analysis identified significant clusters of differentially expressed genes across conditions, most notably the nuclear factor kappa-light-chain-enhancer of B cells (NF-κB) pathway. Quantitative reverse transcription-PCR (qRT-PCR) validated changes from the microarrays in known stress-induced genes and confirmed alterations in the NF-κB pathway genes, Nfkbia, RelA and Nfkb1. FST increased anxiety-like behavior in both the naive and recovery from CRS conditions, but not in mice 24h subsequent to their CRS exposure. These findings suggest that the effects of naive stress are distinct from Cort elevation, and that a history of stress exposure can permanently alter gene expression patterns in the hippocampus and the behavioral response to a novel stressor. These findings establish a baseline profile of normal recovery and adaptation to stress. Importantly, they will serve as a conceptual basis to facilitate the future study of the cellular and regional basis of gene expression changes that lead to impaired recovery from stress, such as those that occur in mood and anxiety disorders.

Lithium's role in neural plasticity and its implications for mood disorders.

OBJECTIVE: Lithium (Li) is often an effective treatment for mood disorders, especially bipolar disorder (BPD), and can mitigate the effects of stress on the brain by modulating several pathways to facilitate neural plasticity. This review seeks to summarize what is known about the molecular mechanisms underlying Li's actions in the brain in response to stress, particularly how Li is able to facilitate plasticity through regulation of the glutamate system and cytoskeletal components. METHOD: The authors conducted an extensive search of the published literature using several search terms, including Li, plasticity, and stress. Relevant articles were retrieved, and their bibliographies consulted to expand the number of articles reviewed. The most relevant articles from both the clinical and preclinical literature were examined in detail. RESULTS: Chronic stress results in morphological and functional remodeling in specific brain regions where structural differences have been associated with mood disorders, such as BPD. Li has been shown to block stress-induced changes and facilitate neural plasticity. The onset of mood disorders may reflect an inability of the brain to properly respond after stress, where changes in certain regions may become 'locked in' when plasticity is lost. Li can enhance plasticity through several molecular mechanisms, which have been characterized in animal models. Further, the expanding number of clinical imaging studies has provided evidence that these mechanisms may be at work in the human brain. CONCLUSION: This work supports the hypothesis that Li is able to improve clinical symptoms by facilitating neural plasticity and thereby helps to 'unlock' the brain from its maladaptive state in patients with mood disorders.

The role of transforming growth factor beta in the generation of suppression: an interaction between CD8+ T and NK cells.

CD8+ T cells have suppressor effector functions, but the mechanisms involved in the generation of this activity are poorly understood. We report that natural killer (NK) cells have an important role in the acquisition of this function. CD8+ cells induce NK cells to produce transforming growth factor-beta (TGF-beta) which, in turn, stimulates CD8+ T cells to become suppressors of antibody production. Using a monocyte-dependent and -independent method to induce antibody production, we first observed that the addition of NK cells to CD8+ cells was required for optimal suppression. Next, we determined that the interaction of CD8+ T cells with NK cells resulted in a striking increase NK cell TGF-beta mRNA and its production. This cytokine appeared to be involved in the induction of T suppressor cell activity since: (a) anti-TGF-beta 1 completely abrogated the suppression of immunoglobulin G synthesis; (b) TGF-beta 1 could substitute for NK cells in inducing CD8+ T cells to develop suppressor activity; and (c) a short exposure of T cells to TGF-beta 1 in the absence of B cells was sufficient for the generation of suppressor activity by CD8+ T cells. Interferon gamma did not have this property. These studies provide strong evidence that in addition to its suppressive properties, TGF-beta is involved in the generation of CD8+ T suppressor effector cells. Because NK cell function is decreased in many autoimmune diseases, these cells may fail to interact properly with these individuals' CD8+ cells in generating suppressors of aggressive anti-self responses.

Clinical pharmacology education: looking into the future.

The numbers of specialists who are practicing Clinical Pharmacology is declining. This raises questions about the nature of the discipline and the roles of those who practice Clinical Pharmacology. Numbers and qualifications of successful trainees in North American accredited Clinical Pharmacology training programs are presented. Questions are posed that require discussion by a forum of academic, regulatory, and industry-based scientists to enable Clinical Pharmacology training programs to meet the clinical care, drug development, and drug policy needs in Western countries in the next decade.

Lymphocytes expressing type 3 complement receptors proliferate in response to interleukin 2 and are the precursors of lymphokine-activated killer cells.

In the absence of antigenic or mitogenic stimulation, certain peripheral blood lymphocytes exhibit proliferative and lymphokine-activated killer (LAK) cell activities when cultured with recombinant IL-2. Both activities were found to be an exclusive property of lymphocytes expressing type 3 complement receptors (CR3) identified by anti-CD11 monoclonal antibodies. CD11+ lymphocytes were then fractionated into three subsets by two-color flow cytometry. These included CD16+ cells, which display distinctive Fc receptors for IgG (CD16). Using anti-CD5, the CD11+ CD16- lymphocytes were separated into non-T cell and T cell subsets. The two non-T cell subsets (CD11+ CD16+ and CD11+ CD16- CD5-), but not the T cell subset (CD11+ CD16- CD5+), could proliferate in response to IL-2. Both CD11+ non-T cell subsets, but not the CD11+ T cell subset, had the capacity to mediate natural killer cell activity. However, all three CD11+ lymphocyte subsets were capable of generating LAK activity. These findings are consistent with the concept that two signals are required to stimulate T cells to proliferate. However, at least a small subset of blood T cells can be activated by IL-2 to become LAK cells.

Lymphocyte subset alterations in nodes regional to human melanoma.

The lymphocyte subpopulations in tumor-draining lymph nodes of melanoma patients were determined using two-color flow cytometry. Data were analyzed according to: (a) the staging of the melanoma; (b) whether or not the nodes contained tumor; and (c) their distance from the primary tumor. Compared with Stage I patients (without metastasis), uninvolved nodes of stage II patients (with nodal metastases) had a significant decrease in helper/inducer (CD4+) T-cells (P less than 0.001), with a corresponding increase in cytotoxic/suppressor (CD8+) cells (P less than 0.001) and Leu 19+ natural killer (CD56+) cells (P less than 0.01). In some patients the presence of tumor within a node was associated with a large decrease in CD3+ total T-cells, whereas in others tumor involvement had little influence on lymphocyte phenotypes. When analyzed by distance from the primary tumor, nodes closest to tumor in Stage I patients contained a smaller percentage of CD19+ B-cells. In Stage II, tumor-free nodes nearest to tumor showed an increase in CD19+ cells, but statistical significance was not reached. CD56+ natural killer cells increased progressively in nodes near tumor and were more numerous in Stage II uninvolved nodes compared with Stage I nodes. Alterations in phenotypically defined lymph node lymphocytes occur in nodes regional to melanoma as the disease progresses, as growth of metastases occurs, and in tumor-free nodes nearest to tumor. These alterations may be essential to the establishment and progression of metastases.

Depression of theophylline elimination by erythromycin.

The elimination of a single oral dose of theophylline was studied in 12 healthy subjects before and after 250 mg erythromycin every 8 hr for 10 days. Serum theophylline elimination half-life rose from 4.79 +/- 0.43 hr before to 7.53 +/- 0.71 hr after erythromycin. Theophylline clearance decreased from a mean of 91.6 +/- 27.0 to 54.8 +/- 10.0 ml/kg/hr after erythromycin and the mean apparent volumes of distribution were much the same before and after the antibiotic. The excretion of theophylline and its metabolites was studied in the urine of three of the subjects. In each case the amount of 3-methylxanthine and 1,3-dimethyluric acid decreased after antibiotic. Adjustments of the theophylline dosage may be necessary for patients who take theophylline and erythromycin concurrently to minimize the risk of theophylline toxicity.

Role of NK cells and TGF-beta in the regulation of T-cell-dependent antibody production in health and autoimmune disease.

Natural killer (NK) cells are a third lymphocyte population especially important in innate immunity. NK cells may also have an important role in the regulation of acquired immunity. These lymphocytes spontaneously produce large amounts of both active and latent transforming growth factor-beta (TGF-beta). NK-cell-derived TGF-beta1 enabled activated CD8(+) T cells to inhibit antibody production by blocking the induction of this response. Production of lymphocyte-derived TGF-beta is decreased in systemic lupus erythematosus. Insufficient levels of this cytokine in SLE and other autoimmune diseases may contribute to defective T regulatory cell function characteristic of this and other autoimmune diseases. NK cells are found in mucosal tissues and the TGF-beta spontaneously released by these cells could contribute to the usual tolerogenic response of T cells to antigens presented at these sites. Thus, in addition to its well known immunosuppressive effects, TGF-beta could have an equally important role in the generation of regulatory T cells.

Transforming growth factor beta enhances the expression of CD154 (CD40L) and production of tumor necrosis factor alpha by human T lymphocytes.

Activation of lymphocytes in the presence of transforming growth factor beta (TGFbeta) can impair or enhance their functional activity. We have found that TGFbeta is important in the generation of lymphocytes, which are capable of suppressing antibody production. To better understand how this cytokine affects lymphocyte activity, we looked at the expression of early activation events of T cells stimulated in the presence or absence of TGFbeta. The results show that TGFbeta enhances the expression of CD154 (CD40L), TNFR2 and the production of TNFalpha. These findings clarify the co-stimulatory effects of TGFbeta that enhance T lymphocyte activation.

Age-related differences in sensitivity to the antinociceptive effects of opioids in male rats. Influence of nociceptive intensity and intrinsic efficacy at the mu receptor.

RATIONALE: Despite the widespread popularity of opioid analgesics, significant differences in the potency and effectiveness of these drugs are often observed across age groups. OBJECTIVES: The purpose of this investigation was to examine age-related differences in sensitivity to the antinociceptive effects of mu opioids and to identify the conditions under which these differences are most apparent. METHODS: In a warm-water tail-withdrawal procedure, young (3 months) and aged (24 months) male rats were habituated to restraint and the latencies to remove their tails from 50 degrees C (low nociceptive intensity) and 55 degrees C (high nociceptive intensity) water were measured. Opioids possessing a range of intrinsic efficacy at the mu receptor (morphine, levorphanol, buprenorphine, butorphanol, nalbuphine, nalorphine) were examined. RESULTS: Young and aged rats were equally sensitive to the antinociceptive effects of morphine, levorphanol, and buprenorphine when tested at the low nociceptive intensity. When these drugs were tested at the high nociceptive intensity, differences between the two age groups became apparent, such that aged rats were significantly more sensitive to the antinociceptive effects of these drugs than young rats. Differences between age groups were most apparent when butorphanol, nalbuphine, and nalorphine were tested, in that each of these drugs produced maximal levels of antinociception in aged rats under conditions in which they failed to produce antinociceptive activity in young rats. Under conditions in which lower efficacy opioids failed to produce antinociceptive activity in young rats, they antagonized the effects of morphine in drug combination tests. CONCLUSIONS: These data may be taken as evidence that aged male rats are more sensitive to the antinociceptive effects of mu opioids than young male rats, and that age-related differences in opioid sensitivity are most apparent when lower efficacy opioids and higher nociceptive intensities are employed during behavioral testing.

The role of iron-induced hippocampal peroxidation in acute epileptogenesis.

Intracortical injection of iron salts causes lipid peroxidation, focal edema, necrosis, gliosis, and the development of behavioral and electrographic seizures. Tocopherol pretreatment prevents the histopathologic perturbations associated with iron injection, and appears to accelerate the resolution of focal accumulation of peroxidation products. In this experiment, rats were pretreated with 500 mg/kg DL-alpha-tocopherol acetate prior to the injection of 3 microliter of 100 mM FeCl2 into the dorsal hippocampus, or induction of convulsive seizures by s.c. injection of 0.8 mg/100 g bicucullin. Tocopherol pretreatment prevented the occurrence of convulsive seizures in a significant number of iron-salts injected animals. Lipid peroxidation measured in the dissected hippocampus was significantly increased in untreated rats developing iron-induced seizures and in rats treated with tocopherol, but developing convulsive seizures. Tocopherol failed to prevent bicucullin-induced seizures. Further, convulsive seizures induced by bicucullin failed to alter hippocampal fluorescence levels. Hence, we concluded that the epileptogenic effects of hippocampal injection of iron salts appear to be related to the induction of peroxidation of neural lipids within the injection site.

The Dalhousie University experience of training residents in many small communities.

BACKGROUND: Dalhousie University Faculty of Medicine provides a fully integrated postgraduate medical education program that prepares both primary care and other specialist physicians. Training takes place in many urban and rural communities. METHOD: In 1991-92 reviews were undertaken of the practice locations of (1) the 200 graduates of primary care residencies between 1987 and 1991 who were licensed to practice in one of the four Atlantic provinces and (2) the 371 graduates of other specialty residences between 1981 and 1991, regardless of practice location but including only those programs with more than ten graduates. The graduates of primary care programs had received training in either one-year rotating internships or two-year family medicine programs. RESULTS: Of the primary care trainees, 130 (65%) remained in practice in Atlantic Canada; of these trainees, 74 (57%) had practices in rural locations. For the other specialist trainees, 50% or more of those with practices in the Atlantic provinces practiced in rural locations in the following specialties: general surgery, internal medicine, ophthalmology, and otolaryngology. CONCLUSION: Postgraduate medical education at Dalhousie is somewhat unusual in that all first-year trainees are required to spend time in a small community as part of their training; hence, students who choose programs at Dalhousie often do so because of their interest in experience in a variety of communities, including those outside metropolitan areas. Still, it was surprising that graduates of some non-primary-care specialty programs, in addition to the graduates of the primary care programs, tended to choose rural practice locations. The site of postgraduate training appears to be one of the important factors in the selection of practice locations.

Improving the quality of health care through contracting: a study of health authority practice.

OBJECTIVES: To investigate approaches of district health authorities to quality in contracting. DESIGN: Descriptive survey. SETTING: All district health authorities in one health region of England in a National Health Service accounting year. MATERIAL: 129 quality specifications used in contracting for services in six specialties (eight general quality specifications and 121 service specific quality specifications) MAIN MEASURES: Evaluation of the use of quality specifications; their scope and content in relation to established criteria of healthcare quality. RESULTS: Most district health authorities developed quality specifications which would be applicable to their local hospital. When purchasing care outside their boundaries they adopted the quality specifications developed by other health authorities. The service specific quality specifications were more limited in scope than the general quality specifications. The quality of clinical care was referred to in 75% of general and 43% of service specific quality specifications. Both types of specification considered quality issues in superficial and broad terms only. Established features of quality improvement were rarely included. Prerequisites to ensure provider accountability and satisfactory delivery of service specifications were not routinely included in contracts. CONCLUSION: Quality specifications within service contracts are commonly used by health authorities. This study shows that their use of this approach to quality improvement is inconsistent and unlikely to achieve desired quality goals. Continued reliance on the current approach is holding back a more fundamental debate on how to create effective management of quality improvement through the interaction between purchasers and providers of health care.

The problem of consent in emergency medicine research.

This paper outlines some of the ethical and practical dilemmas of securing true informed consent in resuscitation research in the prehospital or emergency department setting. Possible substitutes to such consent are discussed and evaluated. The Canadian Tri-Council Policy Statement guidelines for emergency medicine research are compared to the US Food and Drug Administration rules, and the former are assessed and critiqued. Modifications to the current Tri-Council guidelines are suggested.

Functional properties of CD8 positive lymphocyte subsets in systemic lupus erythematosus.

CD8+ lymphocytes comprise several cell subpopulations that differ phenotypically and functionally. Although the percentage of T cytotoxic/suppressor cells (CD3+ CD8+) is usually increased in patients with active SLE, these lymphocytes are unable to suppress immunoglobulin (Ig) synthesis. However, freshly prepared lymphocytes from patients with SLE contain CD8+ DR+ cells which spontaneously suppress lymphocyte production of mitogen induced interleukin 2 (IL-2). Furthermore, CD8+ Leu 11+ non-T cells which comprise only 5% of total lymphocytes are also potent suppressors of IL-2 production. At the present time it is not known whether CD8+ suppressors of Ig synthesis and CD8+ suppressors of IL-2 production represent different maturation stages of common precursor cells or represent true heterogeneity of CD8+ lymphocytes.

Dynamic plasticity: the role of glucocorticoids, brain-derived neurotrophic factor and other trophic factors.

Brain-derived neurotrophic factor (BDNF) is a secreted protein that has been linked to numerous aspects of plasticity in the central nervous system (CNS). Stress-induced remodeling of the hippocampus, prefrontal cortex and amygdala is coincident with changes in the levels of BDNF, which has been shown to act as a trophic factor facilitating the survival of existing and newly born neurons. Initially, hippocampal atrophy after chronic stress was associated with reduced BDNF, leading to the hypothesis that stress-related learning deficits resulted from suppressed hippocampal neurogenesis. However, recent evidence suggests that BDNF also plays a rapid and essential role in regulating synaptic plasticity, providing another mechanism through which BDNF can modulate learning and memory after a stressful event. Numerous reports have shown BDNF levels are highly dynamic in response to stress, and not only vary across brain regions but also fluctuate rapidly, both immediately after a stressor and over the course of a chronic stress paradigm. Yet, BDNF alone is not sufficient to effect many of the changes observed after stress. Glucocorticoids and other molecules have been shown to act in conjunction with BDNF to facilitate both the morphological and molecular changes that occur, particularly changes in spine density and gene expression. This review briefly summarizes the evidence supporting BDNF's role as a trophic factor modulating neuronal survival, and will primarily focus on the interactions between BDNF and other systems within the brain to facilitate synaptic plasticity. This growing body of evidence suggests a more nuanced role for BDNF in stress-related learning and memory, where it acts primarily as a facilitator of plasticity and is dependent upon the coactivation of glucocorticoids and other factors as the determinants of the final cellular response.