bcl-2 transgene expression can protect neurons against developmental and induced cell death.

The bcl-2 protooncogene, which protects various cell types from apoptotic cell death, is expressed in the developing and adult nervous system. To explore its role in regulation of neuronal cell death, we generated transgenic mice expressing Bcl-2 under the control of the neuron-specific enolase promoter, which forced expression uniquely in neurons. Sensory neurons isolated from dorsal root ganglia of newborn mice normally require nerve growth factor for their survival in culture, but those from the bcl-2 transgenic mice showed enhanced survival in its absence. Furthermore, apoptotic death of motor neurons after axotomy of the sciatic nerve was inhibited in these mice. The number of neurons in two neuronal populations from the central and peripheral nervous system was increased by 30%, indicating that Bcl-2 expression can protect neurons from cell death during development. The generation of these transgenic mice suggests that Bcl-2 may play an important role in survival of neurons both during development and throughout adult life.

A progesterone metabolite stimulates the release of gonadotropin-releasing hormone from GT1-1 hypothalamic neurons via the gamma-aminobutyric acid type A receptor.

The reduced progesterone metabolite tetrahydroprogesterone (3 alpha-hydroxy-5 alpha-pregnan-20-one; 3 alpha,5 alpha-THP) is a positive modulator of the gamma-aminobutyric acid type A (GABAA) receptor. Experiments performed in vitro with hypothalamic fragments have previously shown that GABA could modulate the release of gonadotropin-releasing hormone (GnRH). Using GT1-1 immortalized GnRH neurons, we investigated the role of GABAA receptor ligands, including 3 alpha,5 alpha-THP, on the release of GnRH. We first characterized the GABAA receptors expressed by these neurons. [3H]Muscimol, but not [3H]flunitrazepam, bound with high affinity to GT1-1 cell membranes (Kd = 10.9 +/- 0.3 nM; Bmax = 979 +/- 12 fmol/mg of protein), and [3H]muscimol binding was enhanced by 3 alpha,5 alpha-THP. mRNAs encoding the alpha 1 and beta 3 subunits of the GABAA receptor were detected by the reverse transcriptase polymerase chain reaction. In agreement with binding data, the benzodiazepine-binding gamma subunit mRNA was absent. GnRH release studies showed a dose-related stimulating action of muscimol. 3 alpha,5 alpha-THP not only modulated muscimol-induced secretion but also stimulated GnRH release when administered alone. Bicuculline and picrotoxin blocked the effects of 3 alpha,5 alpha-THP and muscimol. Finally, we observed that GT1-1 neurons convert progesterone to 3 alpha,5 alpha-THP. We propose that progesterone may increase the release of GnRH by a membrane mechanism, via its reduced metabolite 3 alpha,5 alpha-THP acting at the GABAA receptor.

Electrical and synaptic properties of embryonic luteinizing hormone-releasing hormone neurons in explant cultures.

Voltage- and ligand-activated channels in embryonic neurons containing luteinizing hormone-releasing hormone (LHRH) were studied by patch-pipette, whole-cell current and voltage clamp techniques. LHRH neurons were maintained in explant cultures derived from olfactory pit regions of embryonic mice. Cells were marked intracellularly with Lucifer yellow following recording. Sixty-two cells were unequivocally identified as LHRH neurons by Lucifer yellow and LHRH immunocytochemistry. The cultured LHRH neurons had resting potentials around -50 mV, exhibited spontaneous discharges generated by intrinsic and/or synaptic activities and contained a time-dependent inward rectifier (Iir). Voltage clamp analysis of ionic currents in the LHRH neuron soma revealed a tetrodotoxin-sensitive Na+ current (INa) and two major types of K+ currents, a transient current (IA), a delayed rectifier current (IK) and low- and high-voltage-activated Ca2+ currents. Spontaneous depolarizing synaptic potentials and depolarizations induced by direct application of gamma-aminobutyrate were both inhibited by picrotoxin or bicuculline, demonstrating the presence of functional gamma-aminobutyrate type A synapses on these neurons. Responses to glutamate were found in LHRH neurons in older cultures. Thus, embryonic LHRH neurons not yet positioned in their postnatal environment in the forebrain contained a highly differentiated repertoire of voltage- and ligand-gated channels.

Quantitative measurement of intraorganelle pH in the endosomal-lysosomal pathway in neurons by using ratiometric imaging with pyranine.

Organelle acidification is an essential element of the endosomal-lysosomal pathway, but our understanding of the mechanisms underlying progression through this pathway has been hindered by the absence of adequate methods for quantifying intraorganelle pH. To address this problem in neurons, we developed a direct quantitative method for accurately determining the pH of endocytic organelles in live cells. In this report, we demonstrate that the ratiometric fluorescent pH indicator 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) is the most advantageous available probe for such pH measurements. To measure intraorganelle pH, cells were labeled by endocytic uptake of HPTS, the ratio of fluorescence emission intensities at excitation wavelengths of 450 nm and 405 nm (F450/405) was calculated for each organelle, and ratios were converted to pH values by using standard curves for F450/405 vs. pH. Proper calibration is critical for accurate measurement of pH values: standard curves generated in vitro yielded artifactually low organelle pH values. Calibration was unaffected by the use of culture medium buffered with various buffers or different cell types. By using this technique, we show that both acidic and neutral endocytically derived organelles exist in the axons of sympathetic neurons in different steady-state proportions than in the cell body. Furthermore, we demonstrate that these axonal organelles have a bimodal pH distribution, indicating a rapid acidification step in their maturation that reduces the average pH of a fraction of the organelles by 2 pH units while leaving few organelles of intermediate pH at steady state. Finally, we demonstrate a spatial gradient or organelle pH along axons, with the relative frequency of acidic organelles increasing with proximity to the cell body.

Characterization of subtype-specific antibodies to the human D5 dopamine receptor: studies in primate brain and transfected mammalian cells.

To achieve a better understanding of how D5 dopamine receptors mediate the actions of dopamine in brain, we have developed antibodies specific for the D5 receptor. D5 antibodies reacted with recombinant baculovirus-infected Sf9 cells expressing the D5 receptor but not with the D1 receptor or a variety of other catecholaminergic and muscarinic receptors. Epitope-tagged D5 receptors expressed in mammalian cells were reactive with both D5 antibodies and an epitope-specific probe. A mixture of N-linked glycosylated polypeptides and higher molecular-mass species was detected on immunoblots of membrane fractions of D5-transfected cells and also of primate brain. D5 receptor antibodies intensely labeled pyramidal neurons in the prefrontal cortex, whereas spiny medium-sized neurons and aspiny large interneurons of the caudate nucleus were relatively lightly labeled. Antibodies to the D5 dopamine receptor should prove important in experimentally determining specific roles for the D5 and D1 receptors in cortical processes and diseases.

A receptor guanylyl cyclase expressed specifically in olfactory sensory neurons.

We have cloned an additional member (GC-D) of the membrane receptor guanylyl cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] family that is specifically expressed in a subpopulation of olfactory sensory neurons. The extracellular, putative ligand-binding domain of the olfactory cyclase is similar in primary structure to two guanylyl cyclases expressed in the retina but diverges considerably from other known guanylyl cyclases. The expression of GC-D RNA is restricted to a small, randomly dispersed population of neurons that is within a single topographic zone in the olfactory neuroepithelium and resembles the pattern of the more diverse seven-transmembrane-domain odorant receptors. These observations suggest that GC-D may function directly in odor recognition or in modulating the sensitivity of a subpopulation of sensory neurons to specific odors.

Lidocaine effects on acetylcholine-elicited currents from mouse superior cervical ganglion neurons

Lidocaine is a commonly used local anaesthetic that, besides blocking voltage-dependent Na+ channels, has multiple inhibitory effects on muscle-type nicotinic acetylcholine (ACh) receptors (nAChRs). In the present study, we have investigated the effects of lidocaine on ACh-elicited currents (IAChs) from cultured mouse superior cervical ganglion (SCG) neurons, which mainly express heteromeric α3β4 nAChRs. Neurons were voltage-clamped by using the perforated-patch method and IAChs were elicited by fast application of ACh (100-300 μM), either alone or in presence of lidocaine at different concentrations. IAChs were reversibly blocked by lidocaine in a concentration-dependent way (IC50 = 41 μM; nH close to 1) and the inhibition was, at least partially, voltage-dependent, indicating an open-channel blockade. Besides, lidocaine blocked resting (closed) nAChRs, as evidenced by the increased inhibition caused by a 12 s lidocaine application just before its co-application with the agonist, and also enhanced IAChs desensitisation, at concentrations close to the IC50. These results indicate that lidocaine has diverse inhibitory actions on neuronal heteromeric nAChRs resembling those previously reported for Torpedo (muscle-type) nAChRs ( Alberola-Die et al., 2011). The similarity of lidocaine actions on different subtypes of heteromeric nAChRs differs with the specific effects of other compounds, restricted to particular subtypes of nAChRs.

Loss of Outer Retinal Neurons and Circuitry Alterations in the DBA/2J Mouse

Purpose. The DBA/2J mouse line develops essential iris atrophy, pigment dispersion, and glaucomatous age-related changes, including an increase of IOP, optic nerve atrophy, and retinal ganglion cell (RGC) death. The aim of this study was to evaluate possible morphological changes in the outer retina of the DBA/2J mouse concomitant with disease progression and aging, based on the reduction of both the a- and b-waves and photopic flicker ERGs in this mouse line. Methods. Vertically sectioned DBA/2J mice retinas were evaluated at 3, 8, and 16 months of age using photoreceptor, horizontal, and bipolar cell markers. Sixteen-month-old C57BL/6 mice retinas were used as controls. Results. The DBA/2J mice had outer retinal degeneration at all ages, with the most severe degeneration in the oldest retinas. At 3 months of age, the number of photoreceptor cells and the thickness of the OPL were reduced. In addition, there was a loss of horizontal and ON-bipolar cell processes. At 8 months of age, RGC degeneration occurred in patches, and in the outer retina overlying these patches, cone morphology was impaired with a reduction in size as well as loss of outer segments and growth of horizontal and bipolar cell processes into the outer nuclear layer. At 16 months of age, connectivity between photoreceptors and horizontal and bipolar cell processes overlying these patches was lost. Conclusions. Retinal degeneration in DBA/2J mice includes photoreceptor death, loss of bipolar and horizontal cell processes, and loss of synaptic contacts in an aging-dependent manner.

Regulation of dendrite growth, placement, and patterning in Drosophila melanogaster class 4 dendrite arborization neurons

Thesis (Ph.D.)--University of Washington, 2016-06

The Epstein-Barr virus nuclear antigen-6 protein co-localizes with EBNA-3 and survival of motor neurons protein

The Epstein-Barr virus nuclear antigen (EBNA)-6 protein is essential for Epstein-Barr virus (EBV)-induced immortalization of primary human B-lymphocytes in vitro. In this study, fusion proteins of EBNA-6 with green fluorescent protein (GFP) have been used to characterize its nuclear localization and organization within the nucleus. EBNA-6 associates with nuclear structures and in immunofluorescence demonstrate a punctate staining pattern. Herein, we show that the association of EBNA-6 with these nuclear structures was maintained throughout the cell cycle and with the use of GFP-E6 deletion mutants, that the region amino acids 733-808 of EBNA-6 contains a domain that can influence the association of EBNA-6 with these nuclear structures. Co-immunofluorescence and confocal analyses demonstrated that EBNA-6 and EBNA-3 co-localize in the nucleus of cells. Expression of EBNA-6, but not EBNA-3, caused a redistribution of nuclear survival of motor neurons protein (SMN) to the EBNA-6 containing nuclear structures resulting in co-localization of SMN with EBNA-6. (C) 2003 Elsevier Inc. All rights reserved.

Moving molecules: mRNA trafficking in mammalian oligodendrocytes and neurons

Numerous mRNA molecules are localized in regions of the dendrites of neurons, some moving along dendrites in response to synaptic activity. The proteins encoded by these RNAs have diverse functions, including participation in memory formation and long-term potentiation. Recent experiments have shown that a cytoplasmic RNA trafficking pathway described for oligodendrocytes also operates in neurons. Transported RNAs possess a cis-acting element that directs them to granules, which are transported along microtubules by the motor proteins kinesin and dynein. These RNA molecules are recruited to the cytoplasmic transport granules by cooperative interaction with a cognate trans-acting factor. mRNAs containing the 11-nucleotide A2RE11 or 21-nucleotide A2RE sequences bind heterogeneous nuclear ribonucleoproteins A2 and A3, which are abundant in the brain. Mutations in this cis-acting element that weaken its interaction with hnRNP A2 also interfere with RNA trafficking. Several dendritically localized mRNAs, including those encoding calcium-calmodulin-dependent protein kinase 11 a subunit and neurogranin, possess A2RE-like sequences, suggesting that they may be localized by interaction with these heterogeneous nuclear ribonucleoproteins. Calcium-calmodulin-dependent protein kinase 11 a subunit is of particular interest: Its RNA is transported in depolarized neurons, and the protein it encodes is essential for establishing long-term memory. Several other cis-acting sequences and trans-acting factors that participate in neuronal RNA localization have been discovered.

Type 1 nitrergic (ND1) cells of the rabbit retina: Comparison with other axon-bearing amacrine cells

NADPH diaphorase (NADPHd) histochemistry labels two types of nitrergic amacrine cells in the rabbit retina. Both the large ND1 cells and the small ND2 cells stratify in the middle of the inner plexiform layer, and their overlapping processes produce a dense plexus, which makes it difficult to trace the morphology of single cells. The complete morphology of the ND1 amacrine cells has been revealed by injecting Neurobiotin into large round somata in the inner nuclear layer, which resulted in the labelling of amacrine cells whose proximal morphology and stratification matched those of the ND1 cells stained by NADPHd histochemistry. The Neurobiotin-injected ND1 cells showed strong homologous tracer coupling to surrounding ND1 cells, and double-labelling experiments confirmed that these coupled cells showed NADPHd reactivity. The ND1 amacrine cells branch in stratum 3 of the inner plexiform layer, where they produce a sparsely branched dendritic tree of 400-600 mum diameter in ventral peripheral retina. In addition, each cell gives rise to several fine beaded processes, which arise either from a side branch of the dendritic tree or from the tapering of a distal dendrite. These axon-like processes branch successively within the vicinity of the dendritic field before extending, with little or no further branching, for 3-5 mm from the soma in ventral peripheral retina. Consequently, these cells may span one-third of the visual field of each eye, and their spatial extent appears to be greater than that of most other types of axon-bearing amacrine cells injected with Neurobiotin in this study. The morphology and tracer-coupling pattern of the ND1 cells are compared with those of confirmed type 1 catecholaminergic cells, a presumptive type 2 catecholaminergic cell, the type 1 polyaxonal. cells, the long-range amacrine cells, a novel type of axon-bearing cell that also branches in stratum 3, and a type of displaced amacrine cell that may correspond to the type 2 polyaxonal cell. (C) 2004 Wiley-Liss, Inc.

Involvement of Islet-2 in the Slit signaling for axonal branching and defasciculation of the sensory neurons in embryonic zebrafish

In Drosophila melanogaster, Slit acts as a repulsive cue for the growth cones of the commissural axons which express a receptor for Slit, Roundabout (Robo), thus preventing the commissural axons from crossing the midline multiple times. Experiments using explant culture have shown that vertebrate Slit homologues also act repulsively for growth cone navigation and neural migration, and promote branching and elongation of sensory axons. Here, we demonstrate that overexpression of Slit2 in vivo in transgenic zebrafish embryos severely affected the behavior of the commissural reticulospinal neurons (Mauthner neurons), promoted branching of the peripheral axons of the trigeminal sensory ganglion neurons, and induced defasciculation of the medial longitudinal fascicles. In addition, Slit2 overexpression caused defasciculation and deflection of the central axons of the trigeminal sensory ganglion neurons from the hindbrain entry point. The central projection was restored by either functional repression or mutation of Robo2, supporting its role as a receptor mediating the Slit signaling in vertebrate neurons. Furthermore, we demonstrated that Islet-2, a LIM/homeodomain-type transcription factor, is essential for Slit2 to induce axonal branching of the trigeminal sensory ganglion neurons, suggesting that factors functioning downstream of Islet-2 are essential for mediating the Slit signaling for promotion of axonal branching. (C) 2004 Elsevier Ireland Ltd. All rights reserved.