Optimizing real time fMRI neurofeedback for therapeutic discovery and development.

While reducing the burden of brain disorders remains a top priority of organizations like the World Health Organization and National Institutes of Health, the development of novel, safe and effective treatments for brain disorders has been slow. In this paper, we describe the state of the science for an emerging technology, real time functional magnetic resonance imaging (rtfMRI) neurofeedback, in clinical neurotherapeutics. We review the scientific potential of rtfMRI and outline research strategies to optimize the development and application of rtfMRI neurofeedback as a next generation therapeutic tool. We propose that rtfMRI can be used to address a broad range of clinical problems by improving our understanding of brain-behavior relationships in order to develop more specific and effective interventions for individuals with brain disorders. We focus on the use of rtfMRI neurofeedback as a clinical neurotherapeutic tool to drive plasticity in brain function, cognition, and behavior. Our overall goal is for rtfMRI to advance personalized assessment and intervention approaches to enhance resilience and reduce morbidity by correcting maladaptive patterns of brain function in those with brain disorders.

Visualization of glucocorticoid receptor in the brain of green fluorescent protein-glucocorticoid receptor knockin mice.

Glucocorticoids exert various neuroendocrinological effects, including stress response, in the central nervous system via glucocorticoid receptor (GR). GRs are transported from the cytoplasm to the nucleus upon ligand binding, and then exert the transcriptional activity. Although it is important for unraveling the actual property of the GR in vivo, subcellular dynamics of the GR are still unclear within the brain tissue in which the neuronal circuitry is maintained. To address this issue, we generated green fluorescent protein (GFP)-GR knockin mice, whose GR has been replaced by a GFP-GR fusion protein that is functionally indistinguishable from endogenous GR. In fixed brain sections of the GFP-GR knockin mice, the distribution of the green fluorescence was similar to that of GR immunoreactivity. By subtracting autofluorescence using fluorescent emission fingerprinting method with confocal laser scanning microscope, nuclear localization of GFP-GR was identifiable in the hippocampal CA3 subregion, where subcellular localization of the GR has been unsolved compared with other areas. To examine the subcellular trafficking of GFP-GR in vivo, we performed adrenalectomy on the GFP-GR knockin mice. GFP-GR was translocated from the nucleus to the cytoplasm and neurites two days after adrenalectomy. Furthermore, laser scanning cytometry by which fluorescence intensity in situ can be quantitatively measured revealed the entire GFP-GR expression level was increased. We then examined the dynamic changes in the subcellular localization of GFP-GR in living hippocampal neurons both in dissociated culture and in tissue slices. GFP-GR was localized in not only the perikarya but also neurites in the absence of ligand, and nuclear translocation following ligand treatment was observed. This is the first report visualizing subcellular trafficking of the GR in the mouse brain in more physiological condition. The present results propose new avenues for the research of the GR dynamics both in vitro and in vivo.

Knock-ins and conditional knockouts: in vivo analysis of glucocorticoid receptor regulation and function.

To determine the cellular targets for glucocorticoid (GC) action, we have generated mice in which a green fluorescent protein-glucocorticoid receptor (GFP-GR) fusion gene is knocked into the endogenous GR locus. We found that GFP-GR function is indistinguishable from endogenous GR on both a cellular and systemic level. Furthermore, the green fluorescence intensity of the GFP-GR protein is proportional to its expression, allowing quantitation of GR expression in single living cells. We initiated our analysis of GR regulation in the thymus. Using multicolor flow cytometry, we found that GR expression is uniform among embryonic thymocyte subpopulations, but gradually "matures" over a three-week period after birth. In the adult, analysis of GFP-GR expression on RAG2-/- and HY T cell receptor (TCR) transgenic genetic backgrounds, showed that GR is induced to high levels in immature CD25+ CD4- CD8- thymocytes and down-regulated by activation of the pre-TCR during positive but not negative selection. Additionally, relative GR expression is dissociated from GC-induced apoptosis in vivo. These results implicate pre-TCR signaling as a mechanism for GR down-regulation and separate receptor abundance from susceptibility to apoptosis across thymocyte populations.

Vasopressin and developmental onset of flank marking behavior in golden hamsters.

Golden hamsters start displaying flank marking behavior (a form of scent marking) around postnatal day 20 (P-20). Because the behavior is dependent upon the central activity of arginine vasopressin (AVP), the present study was conducted to correlate this activation with changes in the vasopressinergic system. A first set of experiments was performed to compare flank marking activity between P-18 and P-22. A second set of experiments was performed to compare the density of AVP receptors between the age periods and assess responsiveness to AVP microinjection. Finally, a third set of experiments incorporated immunocytochemistry, radioimmunoassay, in situ hybridization, and Northern blot analysis to determine the location and numbers of AVP immunoreactive neurons and the level of mRNA correlating with the developmental onset of flank marking behavior. Our results show that flank marking develops between P-18 and P-22. Male and female hamsters do not display odor-induced flank marking anytime before P-19. However, all animals show odor-induced flank marking by P-22. The onset of flank marking does not appear to be associated with any change in AVP receptor binding in the anterior hypothalamus. Indeed, flank marking can be triggered in hamsters on P-18 by the microinjection of AVP in the anterior hypothalamus. This would suggest that the postsynaptic mechanisms contributing to the transduction of the AVP signal and the motor control of flank marking are intact prior to the onset of odor-induced flank marking. In contrast, AVP levels in the hypothalamus and pituitary increase by two to threefold between P-18 and P-22, suggesting that changes in AVP synthesis and release from presynaptic sites may contribute to the onset of flank marking. Interestingly, there is no change in AVP mRNA between P-18 and P-22, which raises questions about posttranslational processing during this developmental period. These results suggest that heightened synthesis and release of AVP between P-18 and P-22 may contribute to the developmental onset of flank marking.

Yolk sac tumors and alpha-fetoprotein in first year of life.

Serum alpha-fetoprotein (AFP) levels are normally elevated in the first eight months of life. This is important information when using AFP as a tumor marker for patients in this age group. We report a case of a one-month-old boy with a yolk sac tumor of the testis. After radical orchiectomy and a negative workup for metastatic disease, his AFP level dropped, but remained mildly elevated over the normal range for six months. Using a half-life of five days it was predicted that it would fall into this range in eighty days. Although the elevated levels suggested residual tumor, they did continue to decline each month which led us to pursue some other explanation for this pattern. A literature search revealed the AFP levels are normally elevated in this age group. In retrospect, our patient's AFP was within this range less than sixty days after surgery. Dissemination of knowledge that the normal AFP ranges in young infants are higher than those in older patients will improve the clinical usefulness of AFP as a tumor marker in this age group.

Testosterone modulation of N-acetylation in mouse kidney.

N-acetylation participates in the biotransformation of hydrazine drugs and arylamine carcinogens to cytotoxic and carcinogenic products. Differences in acetylation capacity expressed in several mammalian species, including humans and mice, are associated with differences in toxicity and carcinogenicity from these chemicals. The present study examines the influence of genotype, age and sex on kidney N-acetyltransferase (NAT) activity in C57BL/6J (B6) and A/J inbred mouse strains using p-amino-benzoic acid (PABA) as a substrate. There were no strain differences in kidney PABA NAT activity. However, within these strains, males have greater kidney NAT activity than females. A 2-fold increase in kidney NAT activity of males was evident by 30 days postnatally and persisted into maturity (> 200 days after birth), whereas the kidney NAT activity of females remained unchanged. Castration reduced male kidney NAT to female levels, whereas testosterone replacement restored original levels of activity. Ovariectomized females exhibited the same enzyme activity as intact females. Testosterone increased kidney NAT activity in females, but not in intact males. Estradiol decreased kidney NAT in males, but had no effect on female NAT activity. The data suggest that the increase in kidney NAT activity in male mice that accompanies development is under androgenic control. This idea is further supported by our finding that the kidney NAT activity of androgen-insensitive tfm/y mice is significantly less than the activity of either females or males sharing the same genetic background. These observations may explain, in part, the higher susceptibility of male mice to 2-acetylaminofluorene mutagenicity and carcinogenicity.

Efficacy of self-care measures for perimenstrual syndrome (PMS).

More than 90% of women are affected by one or more of the signs and symptoms of perimenstrual syndrome (PMS), which causes disruptions at work and in family relations and can be economically costly. Self-care measures relating to lifestyle modifications, most particularly nutrition and exercise, can be helpful in preventing and/or alleviating the number and severity of symptoms. This quasi-experimental study of a pre-test and post-test design showed a statistically significant increase in self-care measures for PMS with a significant decrease in symptoms, thus impacting on the control of PMS.

Heated lymphocytes express HLA-DR antigens despite their inability to stimulate in MLC.

We have utilized serological techniques and mixed lymphocyte culture (MLC) reactions to examine HLA-DR and HLA-D expression by heated (45 degrees C for 1 h) lymphocytes in order to study the functional relationship of these antigens. Heated lymphocytes do not stimulate proliferation of allogeneic lymphocytes in MLC, yet they express HLA-DR antigens. The fraction of peripheral blood lymphocytes (PBL) expressing DR is not altered by heating, nor is the staining intensity altered as detected by fluorescence microscopy. Alloantisera to "B cell alloantigens" recognize HLA-DR determinants on heated cells without any detectable change in either specificity or quantitative cytotoxic effects. Flow cytometry with monoclonal antibody demonstrates only minimal decrease in HLA-DR expression after heating. Thus stimulation in MLC requires more of the stimulating cell than the mere expression of HLA-DR.

A unique pharmacogenetic expression of the N-acetylation polymorphism in the inbred hamster.

Liver N-acetyltransferase (NAT) preparations (105,000 x g cytosol) were obtained from both sexes of 26 strains of inbred hamsters. Liver NAT activity levels were determined for six arylamine substrates; isoniazid, p-aminobenzoic acid (PABA), p-aminosalicylic acid (PAS), sulfamethazine (SMZ), procainamide and 2-aminofluorene. The N-acetylation of isoniazid, SMZ, procainamide and 2-aminofluorene exhibited a monomorphic expression in the hamster as the genetic variation in NAT activity levels between hamster strains was only about 2-fold for each substrate. In contrast, the N-acetylation of PABA and PAS showed a polymorphic expression in the hamster. Two inbred hamster lines (Bio 1.5 and Bio 82.73) had over 400-fold lower PABA NAT and over 20-fold lower PAS NAT activity levels than did the other inbred strains. In addition, the genetically determined N-acetylation differences between the rapid and slow acetylator hamster strains were also demonstrated in vivo for PABA but not for SMZ. Comparison of Michaelis-Menten kinetic constants of PABA NAT activity in a rapid and slow acetylator strain showed a 20-fold lower Km in the rapid acetylator strain suggesting an intrinsic structural difference in rapid and slow acetylator hamster liver NAT. Thus, the pharmacogenetic expression of the N-acetylation polymorphism is quite unique in the inbred hamster, in that PABA and PAS are genetically polymorphic substrates, whereas isoniazid, SMZ, procainamide and 2-aminofluorene are monomorphic, in direct contrast to man and rabbit.