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. Particular emphasis is given to its potential impact on the mapping and study of neural circuits, and how this knowledge will transform our understanding of the complexity of the human brain and its diverse array of behaviours, perceptions, thoughts and emotions.

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., 2002; Hornstein et al., 2005). Gap junctions are composed of connexins, and connexin36 (Cx36), the dominant neuronal connexin, is expressed in the outer plexiform layer. Primate (Macaca mulatta) cone pedicles, labeled with an antibody against cone arrestin (7G6) were connected by a network of fine processes called telodendria and, in double-labeled material, Cx36 plaques were located precisely at telodendrial contacts between cones, suggesting strongly they are Cx36 gap junctions. Each red/green cone made nonselective connections with neighboring red/green cones. In contrast, blue cone pedicles were smaller with relatively few short telodendria and they made only rare or equivocal Cx36 contacts with adjacent cones. There were also many smaller Cx36 plaques around the periphery of every cone pedicle and along a series of very fine telodendria that were too short to reach adjacent members of the cone pedicle mosaic. These small Cx36 plaques were closely aligned with nearly every rod spherule and may identify sites of rod-to-cone coupling, even though the identity of the rod connexin has not been established. We conclude that the matrix of cone telodendria is the substrate for photoreceptor coupling. Red/green cones were coupled indiscriminately but blue cones were rarely connected with other cones. All cone types, including blue cones, made gap junctions with surrounding rod spherules.

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. We raised antisera against synthetic peptides corresponding to an extracellular domain of HR1 between the 4th and 5th transmembrane domains and to an intracellular domain near the carboxyl terminus of HR2. Using these, we localized HR1 to horizontal cells and a small number of amacrine cells and localized HR2 to puncta closely associated with synaptic ribbons inside cone pedicles. Consistent with this, HR1 mRNA was detected in horizontal cell perikarya and primary dendrites and HR2 mRNA was found in cone inner segments. We studied the effect of 5 µM exogenous histamine on primate cones in macaque retinal slices. Histamine reduced I(h) at moderately hyperpolarized potentials, but not the maximal current. This would be expected to increase the operating range of cones and conserve ATP in bright, ambient light. Thus, all three major targets of histamine are in the outer plexiform layer, but the retinopetal axons containing histamine terminate in the inner plexiform layer. Taken together, the findings in these three studies suggest that histamine acts primarily via volume transmission in primate retina.

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. IPA analysis indicated that mutant pcd3J mice showed dysregulation of specific processes that may lead to Purkinje cell death, including several molecules known to control neuronal apoptosis such as Bad, CDK5 and PTEN. These findings demonstrate the usefulness of these powerful microarray analysis tools and have important implications for understanding the mechanisms of selective neuronal death and for developing therapeutic strategies to treat neurodegenerative disorders.

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. These include two Ca(2+)-dependent, i.e., a Ca(2+)-dependent chloride current [I(Cl(Ca))] and a large-conductance Ca(2+)- and voltage-dependent K(+) or BK current [I(K(Ca))], and three voltage-dependent currents, i.e., a delayed-rectifier type current [I(K(V))], a hyperpolarization-activated cation current (I(h)), and a dihydropyridine-sensitive L-type calcium current (I(Ca)). Of these, I(Cl(Ca)) was highly correlated with the presence of a terminal; rods with visible terminals expressed I(Cl(Ca)) without exception (n = 125), whereas approximately 71% of rods (40/56) without visible terminals lacked I(Cl(Ca)). More significantly, I(Cl(Ca)) was absent from all rods (n = 33) that had their terminals ablated, and recordings from the same cell before and after terminal ablation led, in all cases (n =10), to the loss of I(Cl(Ca)). In contrast, I(K(Ca)), I(K(V)), and I(h) remained largely intact after terminal ablation, suggesting that they arose principally from ion channels located in the soma and/or inner segment. The outward I(K)((Ca)) in terminal-ablated rods was reversibly suppressed on "puffing" a Ca(2+)-free extracellular solution over the soma and was appreciably enhanced by the L-type Ca(2+) channel agonist, Bay K 8644 (0.1-2 microM). These data indicate that rod photoreceptors possess discrete targeting mechanisms that preferentially sort ion channels mediating I(Cl(Ca)) to the terminal.

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). In the vast majority of cells, the response was a large increase in Ca(2+) levels in the terminal compartment, but not in the soma. In contrast, rods exhibited a substantial elevation in somatic Ca(2+) levels when depolarized with a brief puff of 100 mM KCl (pipette concentration). These data are consistent with previously reported differences in Ca(2+) buffering mechanisms within the somatic and terminal compartments. Additionally, they may reflect the presence of Ca(2+) channels having distinct properties within the membranes of the two compartments. Consistent with this hypothesis, fluorescent immunocytochemistry using an antibody against the L-type Ca(2+) channel Ca(v)1.2 (alpha1C) subunit and semiquantitative confocal microscopy revealed a high concentration of immunoreactivity in the membranes of terminals of intact rods compared with the somata. Further investigations using enzymatically dissociated preparations of intact rod photoreceptors retaining their presynaptic terminals will allow further testing of these and other hypotheses regarding the compartmentalized regulation of Ca(2+) dynamics within rod photoreceptors.

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. Under whole cell voltage clamp, depolarization-induced whole cell currents were recorded in the presence and absence of fluoxetine (10 and 50 microM pipette concentration) administered via a puffing pipette or in the bath (25 microM). Changes in intracellular free calcium levels were monitored as changes in fura-2 fluorescence following brief depolarization with high K+ (50 and 100 mM) administered via a puffing pipette in the presence and absence of fluoxetine (4 and 10 microM) in the bath. RESULTS: When puffed onto cells, fluoxetine inhibited ICl(Ca) in a dose-dependent fashion (50 microM=96% reduction; 10 microM=14% reduction). In addition to the reduction in amplitude of ICl(Ca), 4 microM fluoxetine (pipette concentration) significantly reduced the duration of ICl(Ca) (48% reduction). Fluoxetine also suppressed the calcium-activated potassium current, IK(Ca), to similar extents (50 microM=75% reduction; 10 microM=23% reduction) when puffed onto cells. Preincubation of rods with 25 microM fluoxetine in the bath significantly reduced outward currents at both 0 mV, where ICl(Ca) is negligible because ECl is about 0 mV and the bulk of the current is carried by IK(Ca), and at +40 mV, where the current is a combination of ICl(Ca) and IK(Ca). Parallel calcium imaging experiments with fura-2 revealed that preincubation of rods in 10 microM fluoxetine virtually eliminated the normal rise in intracellular free calcium in somatic (99.6% reduction) and terminal (98% reduction) compartments following brief depolarization with high K+ (100 mM pipette concentration). Cells preincubated in 4 microM fluoxetine, a therapeutically relevant concentration, showed smaller but significant reductions in Ca2+ elevations in both somatic (66% reduction) and terminal (36% reduction) compartments and even more significant reductions in the duration of sustained calcium levels of the terminal compartment (50% reduction) following brief depolarization with high K+ (50 mM pipette concentration). CONCLUSIONS: We conclude that in addition to blocking ICl(Ca), fluoxetine inhibits IK(Ca). We further conclude that the inhibition of both of these currents is the consequence of inhibition of the normal sustained elevation in intracellular calcium following depolarization and initial calcium influx. Combined, the data suggest that fluoxetine may have multiple sites of action in rod photoreceptors instead of acting as a specific inhibitor of ICl(Ca).

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. Edman degradation and liquid chromatography of tryptic peptides combined with tandem mass spectrometry (LC-MS/MS) identified the antigen. RESULTS: Sequencing of peptides derived from the immunoprecipitated 7G6 antigen identified it as cone arrestin. The identity was confirmed by Western blot analysis with recombinant human cone arrestin and competition with the antibody in immunocytochemistry. Subcellular localization of cone arrestin in dark-adapted and bleached bovine retinas showed that cone arrestin accumulated in cone outer segments of light-adapted retina but was more concentrated in the inner segments of dark-adapted retina. By expression of truncated human cone arrestin mutants systematically deleting areas divergent from bovine and primate cone arrestins, the epitope of 7G6 was identified as a divergent loop exposed at the surface within the N-domain of cone arrestin. CONCLUSIONS: Several independent methods established that the 7G6 antigen is cone arrestin. The 7G6 epitope is contained in a divergent loop, the sequence of which is conserved in bovine and primates but not other vertebrate species consistent with the specificity of the antibody. The light-dependent translocation of cone arrestin suggests a role in light-dark adaptation of cones. Because of the location of its gene on the X-chromosome, cone arrestin is a candidate gene for X-linked cone dystrophies.

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)). External Cs(+) suppresses the inward-rectifying effect of external K(+). The reversal potential of the current, estimated by a novel method from a family of impulse responses below resting potential, indicates a channel that is permeable predominantly to K(+). Its permeability to Na(+), estimated from Goldman-Hodgkin-Katz voltage equation, was negligible. Whereas the activation of the delayed-rectifier K(+) current causes bandpass behavior (i.e., undershoots in the impulse responses) in bipolar cells, activation of the K(IR) current does not. In contrast, a slow hyperpolarization-activated current (I(h)) in rod photoreceptors leads to pronounced, slow undershoots near resting potential. Differences in the kinetics and ion selectivity of hyperpolarization-activated currents in bipolar cells (K(IR)) and in rod photoreceptors (I(h)) confer different dynamical behavior onto the two types of neurons.

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. This adaptive behavior involves two K(+) currents. The delayed-rectifier accounts for the observed gain reduction, speed increase, and bandpass behavior. The A-channel further shortens the impulse responses but suppresses bandpass features. Computer simulations of model neurons with a delayed-rectifier and varying A-channel conductances reveal that impulse responses largely reflect the flux of electrical charge through the two K(+) channels. The A-channel broadens the frequency response and preempts the action of the delayed-rectifier, thereby reducing the associated bandpass features. Admixtures of the two K(+) channels produce the observed variety of dynamics of retinal bipolar cells.