Priorities in stress research: a view from the U.S. National Institute of Mental Health.

The mission of the National Institute of Mental Health is to transform the understanding and treatment of mental illnesses through basic and clinical research, paving the way for prevention, recovery, and cure. In consultation with a broad range of experts, the NIMH has identified a set of priorities for stress biology research aimed squarely at creating the basic and clinical knowledge bases for reducing and alleviating mental health burden across the lifespan. Here, we discuss these priority areas in stress biology research, which include: understanding the heterogeneity of stressors and outcomes; refining and expanding the experimental systems used to study stress and its effects; embracing and exploiting the complexity of the stress response; and prioritizing translational studies that seek to test mechanistic hypotheses in human beings.

NIMH initiatives to facilitate collaborations among industry, academia, and government for the discovery and clinical testing of novel models and drugs for psychiatric disorders.

There is an urgent need to transform basic research discoveries into tools for treatment and prevention of mental illnesses. This article presents an overview of the National Institute of Mental Health (NIMH) programs and resources to address the challenges and opportunities in psychiatric drug development starting at the point of discovery through the early phases of translational research. We summarize NIMH and selected National Institutes of Health (NIH) efforts to stimulate translation of basic and clinical neuroscience findings into novel targets, models, compounds, and strategies for the development of innovative therapeutics for psychiatric disorders.

Preclinical models: status of basic research in depression.

Approximately one half-century ago several classes of medications, discovered by serendipity, were introduced for the treatment of depression and bipolar disorder. These highly effective medications revolutionized our approach to mood disorders and helped launch the modern era of psychiatry. Yet our progress since those serendipitous discoveries has been disappointing.

AP2-like cis element is required for calretinin gene promoter activity in cells of neuronal phenotype differentiated from multipotent human cell line DEV.

Calretinin (CR) is an EF-hand calcium binding protein expressed at high level in neurons. To identify regulatory elements in CR gene promoter, cultured rat cortical cells were transfected with constructs containing its 5'-end deletion mutants and the luciferase reporter gene. A fragment ending at -115 bp upstream of the transcription start site had high promoter activity and was able to induce expression of luciferase specifically in neuronal cells of cortical cultures.

Summary of a National Institute of Mental Health workshop: developing animal models of anxiety disorders.

Rationale: There exists a wide range of animal models and measures designed to assess anxiety or fearfulness. However, the relationship between these models and clinical anxiety symptoms and syndromes is unclear. The National Institute of Mental Health convened a workshop to discuss the relationship between existing behavioral models of anxiety and the clinical profile of anxiety disorders.

The mouse calretinin gene promoter region: structural and functional components.

The 5' flanking region of the mouse calretinin gene was cloned and a 1.8 kbp region adjacent to exon 1 was sequenced. Putative upstream promoter and enhancer elements were identified, including appropriately positioned TATA and CAAT boxes (positions -50 and -68, respectively).

Calretinin expression in the chick brainstem auditory nuclei develops and is maintained independently of cochlear nerve input.

The expression of the calcium-binding protein calretinin (CR) in the chick brainstem auditory nuclei angularis (NA), laminaris (NL), and magnocelularis (NM) was studied during normal development and after deafening by surgical removal of the otocyst (embryonic precursor of the inner ear) or columella (middle ear ossicle). CR mRNA was localized by in situ hybridization by using a radiolabeled oligonucleotide chick CR probe. CR immunoreactivity (CR-IR) was localized on adjacent tissue sections.

Calretinin mRNA and immunoreactivity in the medullary reticular formation of the rat: colocalization with glutamate receptors.

Calretinin-positive cells were identified in the medullary reticular formation of the rat by both immunohistochemistry and in situ hybridization histochemistry. In addition, double immunocytochemical labeling was used to examine the degree of colocalization of calretinin with GluR2/R3, GluR4 and GluR5-7 glutamate receptor subtypes. Results indicated regional variation in calretinin expression across reticular formation regions with the exception of the largest cells which were mostly calretinin-positive.

Cellular expression of MAP 2 kinase in rat brain.

The cellular localization of microtubule-associated protein (MAP) 2 kinase mRNA in rat brain was examined by in situ hybridization histochemistry using a synthetic oligonucleotide probe. MAP 2 kinase was expressed in both neuronal and non-neuronal cells. Areas of high density of mRNA label by the MAP 2 kinase probe appeared to be associated with high cellular packing density.

Antibody recognition of calcium-binding proteins depends on their calcium-binding status.

Previous studies have revealed changes in immunohistochemical stains for calcium-binding proteins after manipulations that influence intracellular calcium. Cases have been revealed in which these changes in immunoreactivity were not correlated with changes in protein amounts. The present experiments examined whether these effects might be explained by changes in antiserum recognition due to calcium-induced changes in protein conformation.

Alteration in levels of expression of brain calbindin D-28k and calretinin mRNA in genetically epilepsy-prone rats.

Variations in the concentration of free calcium in neurons is believed to play a major role in regulating neuronal excitability. Because calcium-binding proteins such as calbindin D-28k and calretinin help to regulate intracellular calcium, we investigated the possibility that the expression of these proteins may be affected in genetically epilepsy-prone rats (GEPRs). The mRNA levels of both proteins were compared across several brain regions using in situ hybridization histochemistry and Northern blot analysis with semiquantitation by optical density measures in autoradiograms from two GEPR strains that differ in the severity of audiogenic seizures (GEPR9 and GEPR3) and from Sprague-Dawley rats.

Distribution of calretinin, calbindin D28k, and parvalbumin in subcellular fractions of rat cerebellum: effects of calcium.

The distribution of calretinin, calbindin D28k, and parvalbumin was examined in subcellular fractions prepared from rat cerebellum and analyzed by immunoblot. Calretinin was also quantified by radioimmunoassay. As expected, all three soluble, EF-hand calcium-binding proteins were predominantly localized in the cytosolic fraction.

Localization of Ca(2+)-dependent conformational changes of calretinin by limited tryptic proteolysis.

Calretinin is an EF-hand Ca(2+)-binding protein expressed predominantly in some neurons. We have found that the tryptic digestion pattern of rat recombinant calretinin depends on Ca2+ concentration as determined by SDS/PAGE, amino-acid-sequence analysis and electrospray-ionization MS. Ca(2+)-saturated calretinin was cleaved between amino acids 60 and 61 to yield two fragments, which accumulated during cleavage.

Quadruple colocalization of calretinin, calcitonin gene-related peptide, vasoactive intestinal peptide, and substance P in fibers within the villi of the rat intestine.

Double-labeling immunofluorescent histochemistry demonstrates that calretinin, a calcium-binding protein, coexists with calcitonin gene-related peptide, vasoactive intestinal peptide, and substance P in the fibers innervating the lamina propria of the rat intestinal villi. An acetylcholinesterase histochemical stain revealed that the majority of calretinin-containing cells in the myenteric ganglia were cholinergic and that about one half of the submucosal calretinin-containing cells colocalized with acetylcholinesterase. In situ hybridization studies confirmed the presence of calretinin mRNA in the dorsal root ganglia, and a ribonuclease protection assay verified the presence of calretinin message in the intestine.

Effects of unilateral cochlea ablation on the distribution of calretinin mRNA and immunoreactivity in the guinea pig ventral cochlear nucleus.

The predominantly neuronal, calcium-binding protein calretinin is highly expressed in the guinea pig auditory system. Within the ventral cochlear nucleus (VCN), calretinin-positive auditory nerve fibers terminate on many calretinin-containing bushy, octopus, and multipolar cells. The abundance of calretinin in the cochlear nucleus provides an ideal system for examining the effects of altered neuronal input on the expression of this calcium-binding protein.

Ca(2+)-dependent and independent interactions of calretinin with hydrophobic resins.

The ability of rat calretinin to bind to hydrophobic resins in a Ca(2+)-dependent manner was examined. Both native calretinin present in cerebellum extract and purified recombinant calretinin bound similarly to hydrophobic resins such as phenyl-, hexyl-, octyl-, and W7-agarose. Hydrophobic interactions of calretinin were partially Ca(2+)-dependent since 1/3 of bound protein was released from the resins by EGTA under varied conditions.

Expression and rapid purification of recombinant rat calretinin: similarity to native rat calretinin.

Rat calretinin coding region was subcloned into a prokaryotic expression vector (pGEX). The glutathione-S-transferase:calretinin fusion protein produced in Escherichia coli was purified on a glutathione-Sepharose affinity column. Recombinant rat calretinin was cleaved on the column by thrombin, eluted, and purified to homogeneity using DEAE-cellulose chromatography.

Localization of calretinin mRNA in rat and guinea pig inner ear by in situ hybridization using radioactive and non-radioactive probes.

The localization of calretinin mRNA was studied in the rat and guinea pig inner ear by in situ hybridization, and compared to the distribution of the protein previously examined by immunocytochemistry. Radioactive and non-radioactive in situ hybridization (ISH) were performed using oligonucleotide probes labelled with 35S or digoxigenin. Radioactive ISH was more sensitive than non-radioactive ISH.

Calretinin distribution in the thalamus of the rat: immunohistochemical and in situ hybridization histochemical analyses.

The distribution of calretinin-containing cells was examined by in situ hybridization histochemistry and compared with the immunohistochemical mapping of calretinin in the thalamus of the rat. Results revealed a close correspondence between the immunohistochemical localization of cell bodies and the messenger RNA label produced by the calretinin oligonucleotide probe. Calretinin cells were most prominent in the midline (paraventricular, reuniens, rhomboid) and intralaminar (central medial, paracentral) nuclei and in a group of cells along the rostral central gray which appeared continuous with the caudal extent of the midline nuclei.

Identification of neuron subpopulations in the rat vestibular ganglion by calbindin-D 28K, calretinin and neurofilament proteins immunoreactivity.

Immunocytochemical and morphometric analyses were combined to demonstrate the presence of neuron subpopulations in the rat vestibular ganglion. Monoclonal antibodies reacting with neurofilament proteins (NF), calbindin-D 28K (CaBP) and calretinin (CaR) were used. Three subpopulations were identified: (1) CaBP- and CaR-positive neurons were the largest neurons (16%) and they were also highly NF-immunoreactive; (2) exclusively NF-positive neurons; (3) unlabelled neurons, representing about two-thirds of the population.

6-Hydroxydopamine induced impairment of Pavlovian conditioning in the rabbit.

This study employed bilateral, intraventricular injections of 6-hydroxydopamine (6-HDA) to examine the effects of monoamine depletion on Pavlovian conditioning of the rabbit's nictitating membrane response. 6-HDA produced dose-dependent and highly correlated decreases in the rate of acquisition of conditioned responses and in the telencephalic content of 5-HT, DA, and NE. At the highest dose of 6-HDA (1340 micrograms), 5-HT, DA, and NE were reduced by 42, 48, and 89%, respectively, and the number of trials required to achieve criterion acquisition was increased by 123%.

Calretinin, a neuronal calcium binding protein, inhibits phosphorylation of a 39 kDa synaptic membrane protein from rat brain cerebral cortex.

The neuronal calcium binding protein calretinin was studied for possible effects on brain protein phosphorylation. Calretinin (100 nM) inhibited the appearance of a calcium stimulated 39 kDa phosphoprotein within a synaptic membrane fraction following sucrose density centrifugation. Calmodulin or a specific protein kinase C inhibitor had no effect on either the phosphorylation of the 39 kDa protein or the inhibition produced by calretinin.

Identification and ultrastructural localization of a calretinin-like calcium-binding protein (protein 10) in the guinea pig and rat inner ear.

Calretinin has been identified as a brain specific calcium-binding protein which appears as a prominent protein in the cochlear nucleus. We identified and localized calretinin in the guinea pig and rat inner ear using polyclonal antibodies. Immunoblot analyses of guinea pig and rat auditory nerve homogenates revealed an immunoreactive band migrating with the same molecular weight as the purified protein, at Mr = 29 k.