The BRAIN Initiative: developing technology to catalyse neuroscience discovery.

The evolution of the field of neuroscience has been propelled by the advent of novel technological capabilities, and the pace at which these capabilities are being developed has accelerated dramatically in the past decade. Capitalizing on this momentum, the United States launched the Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative to develop and apply new tools and technologies for revolutionizing our understanding of the brain. In this article, we review the scientific vision for this initiative set forth by the National Institutes of Health and discuss its implications for the future of neuroscience research.

Photoreceptor coupling mediated by connexin36 in the primate retina.

Photoreceptors are coupled via gap junctions in many mammalian species. Cone-to-cone coupling is thought to improve sensitivity and signal-to-noise ratio, while rod-to-cone coupling provides an alternative rod pathway active under twilight or mesopic conditions (Smith et al., 1986; DeVries et al.

Histamine receptors of cones and horizontal cells in Old World monkey retinas.

In primates the retina receives input from histaminergic neurons in the posterior hypothalamus that are active during the day. In order to understand how this input contributes to information processing in Old World monkey retinas, we have been localizing histamine receptors (HR) and studying the effects of histamine on the neurons that express them. Previously, we localized HR3 to the tips of ON bipolar cell dendrites and showed that histamine hyperpolarizes the cells via this receptor.

Ion channels and ionotropic receptors in human embryonic stem cell derived neural progenitors.

Human neural progenitor cells differentiated from human embryonic stem cells offer a potential cell source for studying neurodegenerative diseases and for drug screening assays. Previously, we demonstrated that human neural progenitors could be maintained in a proliferative state with the addition of leukemia inhibitory factor and basic fibroblast growth factor. Here we demonstrate that 96 h after removal of basic fibroblast growth factor the neural progenitor cell culture was significantly altered and cell replication halted.

Analysis of transcriptional profiles and functional clustering of global cerebellar gene expression in PCD3J mice.

The Purkinje cell degeneration (PCD) mutant mouse is characterized by a degeneration of cerebellar Purkinje cells and progressive ataxia. To identify the molecular mechanisms that lead to the death of Purkinje neurons in PCD mice, we used Affymetrix microarray technology to compare cerebellar gene expression profiles in pcd3J mutant mice 14 days of age (prior to Purkinje cell loss) to unaffected littermates. Microarray analysis, Ingenuity Pathway Analysis (IPA) and expression analysis systematic explorer (EASE) software were used to identify biological and molecular pathways implicated in the progression of Purkinje cell degeneration.

Ion channel compartments in photoreceptors: evidence from salamander rods with intact and ablated terminals.

Vertebrate photoreceptors are highly polarized sensory cells in which several different ionic currents have been characterized. In the present study we used whole cell voltage-clamp and optical imaging techniques, the former combined with microsurgical manipulations, and simultaneous recording of membrane current and intracellular calcium signals to investigate the spatial distribution of ion channels within isolated salamander rods. In recordings from intact rods with visible terminals, evidence for five previously identified ionic currents was obtained.

Fluoxetine inhibits calcium-activated currents of salamander rod photoreceptor somata and presynaptic terminals via modulation of intracellular calcium dynamics.

Purpose: In order to isolate voltage-gated calcium currents in rods retaining intact axons and presynaptic terminals, it is first necessary to identify specific blockers of the large calcium-dependent chloride current, ICl(Ca), which obscures them. Based upon previous reports of its efficacy as an inhibitor of a volume regulated chloride channel (VRAC), a calcium-dependent chloride channel, and the cystic fibrosis transmembrane conductance regulator (CFTR), we investigated whether the serotonin reuptake inhibitor, fluoxetine hydrochloride, could act as a specific blocker for ICl(Ca) in salamander rod photoreceptor terminals, without affecting other aspects of rod physiology.

Methods: Intact rod photoreceptors retaining axons and presynaptic terminals were enzymatically dissociated from salamander retinae.

Imaging of Ca2+ dynamics within the presynaptic terminals of salamander rod photoreceptors.

Although the overall importance of Ca(2+) as a mediator of cell signaling and neurotransmitter release has long been appreciated, the details of Ca(2+) dynamics within the inner segments of vertebrate rod photoreceptors are just beginning to be elucidated. Even less is known regarding Ca(2+) dynamics within the rod presynaptic terminal compartment. Using fura-2 to report changes in intracellular Ca(2+), we imaged the responses of enzymatically dissociated salamander rod photoreceptors retaining intact axons and presynaptic terminals stimulated with a brief depolarizing puff of KCl (30 mM pipette concentration).

Identification and light-dependent translocation of a cone-specific antigen, cone arrestin, recognized by monoclonal antibody 7G6.

Purpose: To elucidate the antigen recognized by monoclonal antibody (mAb) 7G6, a widely used cone-specific marker.

Methods: 7G6 immunocytochemistry was performed on sections of human, primate, and bovine retina. The antigen was immunoprecipitated from human retinal lysates and purified with protein G.

Role of hyperpolarization-activated currents for the intrinsic dynamics of isolated retinal neurons.

The intrinsic dynamics of bipolar cells and rod photoreceptors isolated from tiger salamanders were studied by a patch-clamp technique combined with estimation of effective impulse responses across a range of mean membrane voltages. An increase in external K(+) reduces the gain and speeds the response in bipolar cells near and below resting potential. High external K(+) enhances the inward rectification of membrane potential, an effect mediated by a fast, hyperpolarization-activated, inwardly rectifying potassium current (K(IR)).

Relation between potassium-channel kinetics and the intrinsic dynamics in isolated retinal bipolar cells.

Characterization of the intrinsic dynamics of isolated retinal bipolar cells by a whole-cell patch-clamp technique combined with estimation of effective impulse responses across a range of mean injected currents reveals strikingly adaptive behavior. At resting potential, bipolar cells' effective impulse response is slow, high gain, and low pass. Depolarization speeds up response, decreases gain, and, in most cells, induces bandpass behavior.

Cone properties of retinal margin cells in the monkey (Macaca mulatta).

Purpose: To characterize a cell population in the monkey retinal margin that was labeled with a cone-specific antibody and to determine the presence of additional markers.

Methods: Retinal whole-mount preparations from infant and adult rhesus monkeys (Macaca mulatta) were immunolabeled by incubation overnight with the primary antibodies 7G6, a cone-specific antibody; SV2, a synaptic-vesicle antibody; and opsin antibodies that recognize either the short or long/middle wavelength-sensitive opsins.

Results: The retinal margin cells labeled by 7G6 lay within 1 mm of the ora serrata and differed from 7G6-labeled cones in the central retina.

Mechanisms of glutamate metabolic signaling in retinal glial (Müller) cells.

Retinal Müller (glial) cells metabolize glucose to lactate, which is preferentially taken up by photoreceptor neurons as fuel for their oxidative metabolism. We explored whether lactate supply to neurons is a glial function controlled by neuronal signals. For this, we used subcellular fluorescence imaging and either amperometric or optical biosensors to monitor metabolic responses simultaneously from mitochondrial and cytosolic compartments of individual Müller cells from salamander retina.

Trafficking of molecules and metabolic signals in the retina.

Photoreceptors need the support of pigment epithelial (PE) and Müller glial cells in order to maintain visual sensitivity and neurotransmitter resynthesis. In rod outer segments (ROS), all-trans-retinal is transformed to all-trans-retinol by retinol dehydrogenase using NADPH. NADPH is restored in ROS by the pentose phosphate pathway utilizing high amounts of glucose supplied by choriocapillaries.

The intrinsic dynamics of retinal bipolar cells isolated from tiger salamander.

We studied how intrinsic membrane properties affect the gain and temporal pattern of response in bipolar cells dissociated from retinae of tiger salamanders. Currents specified by a pseudorandom binary sequence, an m-sequence, superimposed on various means, were injected into the cells. From the resultant membrane voltage response for each mean current, impulse responses were estimated.

Immunohistochemical analysis of the neurotrophins BDNF and NT-3 and their receptors trk B, trk C, and p75 in the developing chick retina.

The neurotrophins are trophic and mitogenic factors critical for the development of specific classes of neurons in the central and peripheral nervous systems. In the retina, BDNF and NT-3 have been shown to promote the survival of differentiated ganglion cells (Rodriguez-Tebar et al., 1989; De La Rosa et al.

Early emergence of photoreceptor mosaicism in the primate retina revealed by a novel cone-specific monoclonal antibody.

We have examined the emergence of the photoreceptor mosaic in fetal macaque monkeys by using a novel monoclonal antibody, 7G6, that recognizes all cones in the adult primate retina. In the fetal retina, however, between embryonic (E) day 80 and E130, some opsin-positive cones were not labeled by 7G6. Double-labeling experiments revealed that though long and middle wavelength-sensitive fetal cones are 7G6-positive, a subset of short wavelength-sensitive cones are delayed in their acquisition of 7G6 immunoreactivity.

Ionic conductances of monkey solitary cone inner segments.

1. The membrane properties of cone inner segments dissociated enzymatically from monkey retina were studied under voltage-clamp conditions using patch pipettes in the whole-cell clamp configuration. 2.

Process outgrowth and synaptic varicosity formation by adult photoreceptors in vitro.

To assess the regenerative capability of the photoreceptor synapse, we have isolated and cultured photoreceptors from the mature salamander retina. Both rod and cone photoreceptors were able to regenerate processes within 3 d of plating. Cells extended numerous actin-containing filopodia as well as a few neuritic processes.

Monoclonal antibody substrates facilitate long-term regenerative growth of adult primate retinal neurons.

A culture system for adult monkey and human retinal cells was developed utilizing monoclonal antibodies as substrates. Monoclonal antibodies were made using monkey retinal membranes as immunogen, and screening for the ability to bind and facilitate growth of dissociated retinal neurons when used as a culture substrate. Two antibodies, MR1 and 9B5, which recognize immunohistochemically distinct cell surface antigens, were found to work effectively as a culture substrate for dissociated monkey retinal cells.

Induction of class I major histocompatibility complex antigens on adult primate retinal neurons.

The expression of class I major histocompatibility complex (MHC) antigens was examined on adult monkey and human retinal cells following injury caused by dissociation. In a panning assay, neurons and glia demonstrated a rapid induction of class I MHC antigens following enzymatic dissociation, and expression was sustained on regenerating neurons as well as on glia in culture. Class I MHC antigen expression was also enhanced following optic nerve crush of monkey eyes in vivo.

Glutamate and 2-amino-4-phosphonobutyrate evoke an increase in potassium conductance in retinal bipolar cells.

Although there is general agreement that L-glutamate can produce a depolarizing inward current to account for the hyperpolarizing (OFF) bipolar cell response, the conductance mechanism underlying the depolarizing (ON) response has been difficult to establish satisfactorily. To investigate the ionic bases of the center responses, we studied the whole-cell currents controlled by L-glutamate and its analogues in solitary bipolar cells from salamander retina. We report here two groups of isolated bipolar cells: one group responded to L-glutamate with the previously described inward current [Attwell, D.

Fibroblast behavior at aqueous interfaces with perfluorocarbon, silicone, and fluorosilicone liquids.

Perfluorocarbon, silicone, and fluorosilicone liquids with potential for use as vitreous substitutes in the management of complex retinal detachment were evaluated for surface reactivity by assessing the behavior of anchorage-dependent fibroblasts plated at the phase boundary between these compounds and culture medium. Low-viscosity perfluorcarbons were alumina-treated to remove polar impurities. On perfluorodecalin, perfluorodimethylcyclohexane, perfluorotrimethylcyclohexane, perfluoroethylcyclohexane, perfluorooctane, perfluoroperhydrophenanthrene, perfluoromethyladamantane, perfluorodimethyladamantane, the highly viscous perfluoropolyether liquids Krytox TLF7067 and 6354, and dimethylsiloxane liquids of a variety of viscosities, most cells did not attach; the few that did attach exhibited minimal spreading behavior and did not achieve the flattened spindle-shape morphology which is a prerequisite to normal proliferative activity.

Immunolabelling by a newt retinal pigment epithelium antibody during retinal development and regeneration.

The binding of RPE-1, a mouse monoclonal antibody selective for newt retinal pigment epithelium, was followed in eyes undergoing embryonic development and retinal regeneration. Using the indirect immunofluorescence technique on frozen sections, we observed bright and continuous labelling exclusively in the retinal pigment epithelium (RPE) of normal adult newts, but labelling became diminished near the ora serrata region and stopped abruptly at the ciliary margin. During development, labelling was not detected in the retinal pigment epithelium (RPE) until the formation of photoreceptor outer segments and was not observed in any other ocular tissue.