bluuebunny/crossref_metadata_2025 huggingface dataset:
Proliferation and Differentiation of Progenitor Cells Throughout the Intact Adult Rat Spinal Cord
Authors: Horner Philip J. , Power Ann E. , Kempermann Gerd , Kuhn H. Georg , Palmer Theo D. , Winkler Jürgen , Thal Leon J. , Gage Fred H.
Published: April 2018

The existence of multipotent progenitor populations in the adult forebrain has been widely studied. To extend this knowledge to the adult spinal cord we have examined the proliferation, distribution, and phenotypic fate of dividing cells in the adult rat spinal cord. Bromodeoxyuridine (BrdU) was used to label dividing cells in 13- to 14-week-old, intact Fischer rats. Single daily injections of BrdU were administered over a 12 d period. Animals were killed either 1 d or 4 weeks after the last injection of BrdU. We observed frequent cell division throughout the adult rodent spinal cord, particularly in white matter tracts (5–7% of all nuclei). The majority of BrdU-labeled cells colocalized with markers of immature glial cells. At 4 weeks, 10% of dividing cells expressed mature astrocyte and oligodendroglial markers. These data predict that 0.75% of all astrocytes and 0.82% of all oligodendrocytes are derived from a dividing population over a 4 week period. To determine the migratory nature of dividing cells, a single BrdU injection was given to animals that were killed 1 hr after the injection. In these tissues, the distribution and incidence of BrdU labeling matched those of the 4 week post injection (pi) groups, suggesting that proliferating cells dividein siturather than migrate from the ependymal zone. These data suggest a higher level of cellular plasticity for the intact spinal cord than has previously been observed and that glial progenitors exist in the outer circumference of the spinal cord that can give rise to both astrocytes and oligodendrocytes.
Late Retinal Progenitor Cells Show Intrinsic Limitations in the Production of Cell Types and the Kinetics of Opsin Synthesis
Authors: Belliveau Michael J. , Young Tracy L. , Cepko Constance L.
Published: April 2018

The seven major cell classes of the vertebrate neural retina arise from a pool of multipotent progenitor cells. Several studies suggest a model of retinal development in which both the environment and the progenitor cells themselves change over time (Cepko et al., 1996). To test this model, we used a reaggregate culture system in which a labeled population of progenitor cells from the postnatal rat retina were cultured with an excess of embryonic retinal cells. The labeled cells were then assayed for their cell fate choices and their kinetics of rod differentiation, as measured by opsin synthesis. The kinetics of opsin synthesis remained unchanged, but fewer postnatal cells adopted the rod cell fate when cultured with embryonic cells. There was an increase in the percentage of bipolar cells produced by postnatal progenitor cells, indicating a possible respecification of fate. The increase in bipolar cells could occur even after progenitor cells had completed their terminal mitoses. These alterations in cell fates appeared to be caused at least in part by a secreted factor released by the embryonic cells that requires the LIFRβ/gp130 complex for signaling. Finally, although surrounded by 20-fold more embryonic cells, the postnatal cells did not choose to adopt any fates normally produced only by embryonic cells.
Acute Changes in Maternal Thyroid Hormone Induce Rapid and Transient Changes in Gene Expression in Fetal Rat Brain
Authors: Dowling Amy L. S. , Martz Gabriel U. , Leonard Jack L. , Zoeller R. Thomas
Published: April 2018

Despite clinical evidence that thyroid hormone is essential for brain development before birth, effects of thyroid hormone on the fetal brain have been largely unexplored. One mechanism of thyroid hormone action is regulation of gene expression, because thyroid hormone receptors (TRs) are ligand-activated transcription factors. We used differential display to identify genes affected by acute T4administration to the dam before the onset of fetal thyroid function. Eight of the 11 genes that we identified were selectively expressed in brain areas known to contain TRs, indicating that these genes were directly regulated by thyroid hormone. Usingin situhybridization, we confirmed that the cortical expression of both neuroendocrine-specific protein (NSP) and Oct-1 was affected by changes in maternal thyroid status. Additionally, we demonstrated that both NSP and Oct-1 were expressed in the adult brain and that their responsiveness to thyroid hormone was retained. These data are the first to identify thyroid hormone-responsive genes in the fetal brain.
Parallel Cone Bipolar Pathways to a Ganglion Cell Use Different Rates and Amplitudes of Quantal Excitation
Authors: Freed Michael A.
Published: April 2018

The cone signal reaches the cat's On-β (X) ganglion cell via several parallel circuits (bipolar cell types b1, b2, and b3). These circuits might convey different regions of the cone's temporal bandwidth. To test this, I presented a step of light that elicited a transient depolarization followed by a sustained depolarization. The contribution of bipolar cells to these response components was isolated by blocking action potentials with tetrodotoxin and by blocking inhibitory synaptic potentials with bicuculline and strychnine. Stationary fluctuation analysis of the sustained depolarization gave the rate of quantal bombardment: ∼5100 quanta sec−1for small central cells and ∼45,000 quanta sec−1for large peripheral cells. Normalizing these rates for the vastly different numbers of bipolar synapses (150–370 per small cell vs 2000 per large cell), quantal rate was constant across the retina, ∼22 quanta synapse−1sec−1. Nonstationary fluctuation analysis gave the mean quantal EPSP amplitude: ∼240 μV for the transient depolarization and 30 μV for the sustained depolarization. The b1 bipolar cell is known from noise analysis of the On-α ganglion cell to have a near-maximal sustained release of only approximately two quanta synapse−1sec−1. This implies that the other bipolar types (b2 and b3) contribute many more quanta to the sustained depolarization (≥46 synapse−1sec−1). Type b1 probably contributes large quanta to the transient depolarization. Thus, bipolar cell types b1 and b2/b3 apparently constitute parallel circuits that convey, respectively, high and low frequencies.
Granule Cells and Cerebellar Boundaries: Analysis ofUnc5h3Mutant Chimeras
Authors: Goldowitz Dan , Hamre Kristin M. , Przyborski Stefan A. , Ackerman Susan L.
Published: April 2018

Mutations in theUnc5h3gene, a receptor for the netrin 1 ligand, result in abnormal migrations of both Purkinje and granule cells to regions outside the cerebellum and of granule cells to regions within the cerebellum. Because both Purkinje and granule cells express this molecule, we sought to determine whether one or both of these cell types are the primary target of the mutation.

Chimeric mice were made between wild-type ROSA26 transgenic mouse embryos (whose cells express β-galactosidase) andUnc5h3mutant embryos. The resulting chimeric brains exhibited a range of phenotypes. Chimeras that had a limited expression of the extracerebellar phenotype (movement of cerebellar cells into the colliculus and midbrain tegmentum) and the intracerebellar phenotype (migration of granule cells into white matter) had a normal-appearing cerebellum, whereas chimeras that had more ectopic cells had attenuated anterior cerebellar lobules. Furthermore, the colonization of colliculus and midbrain tegmentum by cerebellar cells was not equivalent in all chimeras, suggesting different origins for extracerebellar ectopias in these regions.

The granule cells of the extracerebellar ectopias were almost entirely derived fromUnc5h3/Unc5h3mutant embryos, whereas the ectopic Purkinje cells were a mixture of both mutant and wild-type cells. Intracerebellar ectopias in the chimera were composed exclusively of mutant granule cells. These findings demonstrate that both inside and outside the cerebellum, the granule cell is the key cell type to demarcate the boundaries of the cerebellum.
A-Type K+Current Mediated by the Kv4 Channel Regulates the Generation of Action Potential in Developing Cerebellar Granule Cells
Authors: Shibata Riichi , Nakahira Kensuke , Shibasaki Koji , Wakazono Yoshihiko , Imoto Keiji , Ikenaka Kazuhiro
Published: April 2018

During neuronal differentiation and maturation, electrical excitability is essential for proper gene expression and the formation of synapses. The expression of ion channels is crucial for this process; in particular, voltage-gated K+channels function as the key determinants of membrane excitability. Previously, we reported that the A-type K+current (IA) and Kv4.2 K+channel subunit expression increased in cultured cerebellar granule cells with time. To examine the correlation between ion currents and the action potential, in the present study, we measured developmental changes of action potentials in cultured granule cells using the whole-cell patch-clamp method. In addition to an observed increment ofIA, we found that the Na+current also increased during development. The increase in both currents was accompanied by a change in the membrane excitability from the nonspiking type to the repetitive firing type. Next, to elucidate whether Kv4.2 is responsible for theIAand to assess the effect of Kv4 subunits on action potential waveform, we transfected a cDNA encoding a dominant-negative mutant Kv4.2 (Kv4.2dn) into cultured cells. Expression of Kv4.2dn resulted in the elimination ofIAin the granule cells. This result demonstrates that members of the Kv4 subfamily are responsible for theIAin developing granule cells. Moreover, elimination ofIAresulted in shortening of latency before the first spike generation. In contrast, expression of wild-type Kv4.2 resulted in a delay in latency. This indicates that appearance ofIAis critically required for suppression of the excitability of granule cells during their maturation.
The Role of the Hippocampus in Instrumental Conditioning
Authors: Corbit Laura H. , Balleine Bernard W.
Published: April 2018

Considerable evidence suggests that, in instrumental conditioning, rats can encode both the specific action–outcome associations to which they are exposed and the degree to which an action is causal in producing its associated outcome. Three experiments assessed the involvement of the hippocampus in encoding these aspects of instrumental learning. In each study, rats with electrolytic lesions of the dorsal hippocampus and sham-lesioned controls were trained while hungry to press two levers, each of which delivered a unique food outcome. Experiments 1A and 1B used an outcome devaluation procedure to assess the effects of the lesion on encoding the action–outcome relationship. After training, one of the two outcomes was devalued using a specific satiety procedure, after which performance on the two levers was assessed in a choice extinction test. The lesion had no detectable effect on either the acquisition of instrumental performance or on the rats' sensitivity to outcome devaluation; lesion and sham groups both reduced responding on the lever associated with the devalued outcome compared with the other lever. In experiment 2, the sensitivity of hippocampal rats to the causal efficacy of their actions was assessed by selectively degrading the contingency between one of the actions and its associated outcome. Whereas sham rats selectively reduced performance on the lever for which the action–outcome contingency had been degraded, hippocampal rats did not. These results suggest that, in instrumental conditioning, lesions of the dorsal hippocampus selectively impair the ability of rats to represent the causal relationship between an action and its consequences.
μ-Opioid Receptors Often Colocalize with the Substance P Receptor (NK1) in the Trigeminal Dorsal Horn
Authors: Aicher Sue A. , Punnoose Ann , Goldberg Alla
Published: April 2018

Substance P (SP) is a peptide that is present in unmyelinated primary afferents to the dorsal horn and is released in response to painful or noxious stimuli. Opiates active at the μ-opiate receptor (MOR) produce antinociception, in part, through modulation of responses to SP. MOR ligands may either inhibit the release of SP or reduce the excitatory responses of second-order neurons to SP. We examined potential functional sites for interactions between SP and MOR with dual electron microscopic immmunocytochemical localization of the SP receptor (NK1) and MOR in rat trigeminal dorsal horn. We also examined the relationship between SP-containing profiles and NK1-bearing profiles. We found that 56% of SP-immunoreactive terminals contact NK1 dendrites, whereas 34% of NK1-immunoreactive dendrites receive SP afferents. This result indicates that there is not a significant mismatch between sites of SP release and available NK1 receptors, although receptive neurons may contain receptors at sites distant from the peptide release site. With regard to opioid receptors, we found that many MOR-immunoreactive dendrites also contain NK1 (32%), whereas a smaller proportion of NK1-immunoreactive dendrites contain MOR (17%). Few NK1 dendrites (2%) were contacted by MOR-immunoreactive afferents. These results provide the first direct evidence that MORs are on the same neurons as NK1 receptors, suggesting that MOR ligands directly modulate SP-induced nociceptive responses primarily at postsynaptic sites, rather than through inhibition of SP release from primary afferents. This colocalization of NK1 and MORs has significant implications for the development of pain therapies targeted at these nociceptive neurons.
Syntaxin Modulation of Calcium Channels in Cortical Synaptosomes As Revealed by Botulinum Toxin C1
Authors: Bergsman Jeremy B. , Tsien Richard W.
Published: April 2018

When the presynaptic membrane protein syntaxin is coexpressed inXenopusoocytes with N- or P/Q-type Ca2+channels, it promotes their inactivation (Bezprozvanny et al., 1995; Wiser et al., 1996, 1999; Degtiar et al., 2000) (I. B. Bezprozvanny, P. Zhong, R. H. Scheller, and R. W. Tsien, unpublished observations). These findings led to the hypothesis that syntaxin influences Ca2+channel function in presynaptic endings, in a reversal of the conventional flow of information from Ca2+channels to the release machinery. We examined this effect in isolated mammalian nerve terminals (synaptosomes). Botulinum neurotoxin type C1 (BoNtC1), which cleaves syntaxin, was applied to rat neocortical synaptosomes at concentrations that completely blocked neurotransmitter release. This treatment altered the pattern of Ca2+entry monitored with fura-2. Whereas the initial Ca2+rise induced by depolarization with K+-rich solution was unchanged, late Ca2+entry was strongly augmented by syntaxin cleavage. Similar results were obtained when Ca2+influx arose from repetitive firing induced by the K+-channel blocker 4-aminopyridine. Cleavage of vesicle-associated membrane protein with BoNtD or SNAP-25 with BoNtE failed to produce a significant change in Ca2+entry. The BoNtC1-induced alteration in Ca2+signaling was specific to voltage-gated Ca2+channels, not Ca2+extrusion or buffering, and it involved N-, P/Q- and R-type channels, the high voltage-activated channels most intimately associated with presynaptic release machinery. The modulatory effect of syntaxin was not immediately manifest when synaptosomes had been K+-predepolarized in the absence of external Ca2+, but developed with a delay after admission of Ca2+, suggesting that vesicular turnover may be necessary to make syntaxin available for its stabilizing effect on Ca2+channel inactivation.
Dopamine-Deficient Mice Are Hypersensitive to Dopamine Receptor Agonists
Authors: Kim Douglas S. , Szczypka Mark S. , Palmiter Richard D.
Published: April 2018

Dopamine-deficient (DA−/−) mice were created by targeted inactivation of the tyrosine hydroxylase gene in dopaminergic neurons. The locomotor activity response of these mutants to dopamine D1 or D2 receptor agonists andl-3,4-dihydroxyphenylalanine (l-DOPA) was 3- to 13-fold greater than the response elicited from wild-type mice. The enhanced sensitivity of DA−/− mice to agonists was independent of changes in steady-state levels of dopamine receptors and the presynaptic dopamine transporter as measured by ligand binding. The acute behavioral response of DA−/− mice to a dopamine D1 receptor agonist was correlated with c-fos induction in the striatum, a brain nucleus that receives dense dopaminergic input. Chronic replacement of dopamine to DA−/− mice by repeatedl-DOPA administration over 4 d relieved the hypersensitivity of DA−/− mutants in terms of induction of both locomotion and striatal c-fos expression. The results suggest that the chronic presence of dopaminergic neurotransmission is required to dampen the intracellular signaling response of striatal neurons.
Chronic Heroin Self-Administration Desensitizes μ Opioid Receptor-Activated G-Proteins in Specific Regions of Rat Brain
Authors: Sim-Selley Laura J. , Selley Dana E. , Vogt Leslie J. , Childers Steven R. , Martin Thomas J.
Published: April 2018

In previous studies from our laboratory, chronic noncontingent morphine administration decreased μ opioid receptor-activated G-proteins in specific brainstem nuclei. In the present study, μ opioid receptor binding and receptor-activated G-proteins were examined after chronic heroin self-administration. Rats were trained to self-administer intravenous heroin for up to 39 d, achieving heroin intake up to 366 mg · kg−1 · d−1. μ opioid-stimulated [35S]GTPγS and [3H]naloxone autoradiography were performed in adjacent brain sections. Agonist-stimulated [35S]GTPγS autoradiography also examined other G-protein-coupled receptors, including δ opioid, ORL-1, GABAB, adenosine A1, cannabinoid, and 5-HT1A. In brains from heroin self-administering rats, decreased μ opioid-stimulated [35S]GTPγS binding was observed in periaqueductal gray, locus coeruleus, lateral parabrachial nucleus, and commissural nucleus tractus solitarius, as previously observed in chronic morphine-treated animals. In addition, decreased μ opioid-stimulated [35S]GTPγS binding was found in thalamus and amygdala after heroin self-administration. Despite this decrease in μ-activated G-proteins, [3H]naloxone binding demonstrated increased μ opioid receptor binding in several brain regions after heroin self-administration, and there was a significant decrease in μ receptor G-protein efficiency as expressed as a ratio between agonist-activated G-proteins and μ receptor binding. No effects on agonist-stimulated [35S]GTPγS binding were found for any other receptor examined. The effect of chronic heroin self-administration to decrease μ-stimulated [35S]GTPγS binding varied between regions and was highest in brainstem and lowest in the cortex and striatum. These results not only provide potential neuronal mechanisms that may contribute to opioid tolerance and dependence, but also may explain why various chronic effects of opioids develop to different degrees.
Functional Interactions betweenDrosophilabHLH/PAS, Sox, and POU Transcription Factors Regulate CNS Midline Expression of theslitGene
Authors: Ma Yue , Certel Kaan , Gao Yanping , Niemitz Emily , Mosher Jack , Mukherjee Ashim , Mutsuddi Mousumi , Huseinovic Neda , Crews Stephen T. , Johnson Wayne A. , Nambu John R.
Published: April 2018

DuringDrosophilaembryogenesis the CNS midline cells have organizing activities that are required for proper elaboration of the axon scaffold and differentiation of neighboring neuroectodermal and mesodermal cells. CNS midline development is dependent on Single-minded (Sim), a basic-helix-loop-helix (bHLH)-PAS transcription factor. We show here that Fish-hook (Fish), a Sox HMG domain protein, and Drifter (Dfr), a POU domain protein, act in concert with Single-minded to control midline gene expression.single-minded,fish-hook, anddrifterare all expressed in developing midline cells, and both loss- and gain-of-function assays revealed genetic interactions between these genes. The corresponding proteins bind to DNA sites present in a 1 kb midline enhancer from theslitgene and regulate the activity of this enhancer in culturedDrosophilaSchneider line 2 cells. Fish-hook directly associates with the PAS domain of Single-minded and the POU domain of Drifter; the three proteins can together form a ternary complex in yeast. In addition, Fish can form homodimers and also associates with other bHLH-PAS and POU proteins. These results indicate that midline gene regulation involves the coordinate functions of three distinct types of transcription factors. Functional interactions between members of these protein families may be important for numerous developmental and physiological processes.
Distribution of Corticosteroid Receptors in the Rhesus Brain: Relative Absence of Glucocorticoid Receptors in the Hippocampal Formation
Authors: Sánchez M. Mar , Young Larry J. , Plotsky Paul M. , Insel Thomas R.
Published: April 2018

Chronic stress has been associated with degenerative changes in the rodent and primate hippocampus, presumably mediated in part via neuronal glucocorticoid receptors (GRs). In the rat brain, GRs are widely distributed and are particularly dense in the hippocampus. The distribution of GRs in the primate brain, however, has not been fully characterized. In this study, we usedin situhybridization histochemistry and immunohistochemistry to map the distribution of GR mRNA and GR protein, respectively, in adult rhesus monkeys (Macaca mulatta). In contrast to its well established distribution in the rat brain, GR mRNA was only weakly detected in the dentate gyrus (DG) and Cornu Ammonis (CA) of the macaque hippocampus, whereas it was abundant in the pituitary (PIT), cerebellum (CBL), hypothalamic paraventricular nucleus (PVN), and, to a lesser extent, the neocortex. Immunohistochemical staining indicated a very low density of GR-like immunoreactive cells within the macaque hippocampal formation in contrast to the high density observed within the PVN, prefrontal and entorhinal cortices, and cerebellar cortex. Relative to the low level of GR, mineralocorticoid receptor (MR) mRNA and protein expression were abundant within the DG and CA of the rhesus monkey hippocampal formation. These results indicate that, in the primate, neocortical and hypothalamic areas may be more important targets for GR-mediated effects of glucocorticoids than the hippocampus. Alternatively, it is also possible that glucocorticoid effects are mediated through the MRs present in the hippocampal formation.
Ionic Mechanisms Underlying Repetitive High-Frequency Burst Firing in Supragranular Cortical Neurons
Authors: Brumberg Joshua C. , Nowak Lionel G. , McCormick David A.
Published: April 2018

Neocortical neurons in awake, behaving animals can generate high-frequency (>300 Hz) bursts of action potentials, either in single bursts or in a repetitive manner. Intracellular recordings of layer II/III pyramidal neurons were obtained from adult ferret visual cortical slices maintainedin vitroto investigate the ionic mechanisms by which a subgroup of these cells generates repetitive, high-frequency burst discharges, a pattern referred to as “chattering.” The generation of each but the first action potential in a burst was dependent on the critical interplay between the afterhyperpolarizations (AHPs) and afterdepolarizations (ADPs) that followed each action potential. The spike-afterdepolarization and the generation of action potential bursts were dependent on Na+, but not Ca2+, currents. Neither blocking of the transmembrane flow of Ca2+nor the intracellular chelation of free Ca2+with BAPTA inhibited the generation of intrinsic bursts. In contrast, decreasing the extracellular Na+concentration or pharmacologically blocking Na+currents with tetrodotoxin, QX-314, or phenytoin inhibited bursting before inhibiting action potential generation. Additionally, a subset of layer II/III pyramidal neurons could be induced to switch from repetitive single spiking to a burst-firing mode by constant depolarizing current injection, by raising extracellular K+concentrations, or by potentiation of the persistent Na+current with the Na+channel toxin ATX II. These results indicate that cortical neurons may dynamically regulate their pattern of action potential generation through control of Na+and K+currents. The generation of high-frequency burst discharges may strongly influence the response of postsynaptic neurons and the operation of local cortical networks.
Long-Term Specification of AMPA Receptor Properties after Synapse Formation
Authors: Lawrence J. Josh , Trussell Laurence O.
Published: April 2018

AMPA receptors expressed at auditory nerve synapses in the mammalian and avian cochlear nuclei display exceptionally rapid channel gating, an adaptation well suited for acoustic processing. We examined whether cellular interactions during development might determine the subunit composition of these receptors. After synapse formation in the avian nucleus magnocellularis (nMag)in vivo, the rate of receptor desensitization increased threefold, sensitivity to channel block by polyamines increased, and sensitivity to cyclothiazide, an inhibitor of desensitization, increased, indicating a reduction in glutamate receptor subunit 2 and of flip splice variants. This phenotypic switch was prevented, but not reversed, by isolating nMag neurons in a cell-culture environment. We propose that the switch in receptor kinetics is an outcome of cellular interactions during a critical period that result in the long-term determination of receptor phenotype.
Immunoisolation of GABA-Specific Synaptic Vesicles Defines a Functionally Distinct Subset of Synaptic Vesicles
Authors: Takamori Shigeo , Riedel Dietmar , Jahn Reinhard
Published: April 2018

Synaptic vesicles from mammalian brain are among the best characterized trafficking organelles. However, so far it has not been possible to characterize vesicle subpopulations that are specific for a given neurotransmitter. Taking advantage of the recent molecular characterization of vesicular neurotransmitter transporters, we have used an antibody specific for the vesicular GABA transporter (VGAT) to isolate GABA-specific synaptic vesicles. The isolated vesicles are of exceptional purity as judged by electron microscopy. Immunoblotting revealed that isolated vesicles contain most of the major synaptic vesicle proteins in addition to VGAT and are devoid of vesicular monoamine and acetylcholine transporters. The vesicles are 10-fold enriched in GABA uptake activity when compared with the starting vesicle fraction. Furthermore, glutamate uptake activity and glutamate-induced but not chloride-induced acidification are selectively lost during immunoisolation. We conclude that the population of GABA-containing synaptic vesicles is separable and distinct from vesicle populations transporting other neurotransmitters.
Slit2 Is a Repellent for Retinal Ganglion Cell Axons
Authors: Niclou Simone P. , Jia Li , Raper Jonathan A.
Published: April 2018

We set out to isolate inhibitory guidance cues that affect retinal ganglion cell (RGC) axonsin vitroand that could potentially be involved in RGC pathfinding decisions. Here we describe the biochemical purification of an RGC growth cone collapsing factor from bovine brain membranes and its identification as Slit2. Recombinant human Slit2 collapses and repels RGC growth cones from all quadrants of the chick retina. In the developing mouse visual system, slit2 is expressed in the eye, in the optic stalk, and in the ventral diencephalon. Slit2 expression is strong in anterior ventral diencephalic structures but is absent from the ventral midline where the optic chiasm forms. The putative receptors for Slits, robo1 and robo2, are expressed in the inner retinal layer in which RGCs are located. A comparison of the expression patterns of Slit2 and retinal axon trajectories suggests that slit2 acts as a short range repellent for retinal ganglion cell axons.
Rapid Dendritic Remodeling in the Developing Retina: Dependence on Neurotransmission and Reciprocal Regulation by Rac and Rho
Authors: Wong Wai T. , Faulkner-Jones Beverly E. , Sanes Joshua R. , Wong Rachel O. L.
Published: April 2018

We demonstrate that within the intact and spontaneously active retina, dendritic processes of ganglion cells exhibit rapid and extensive movements during the period of synaptogenesis. Marked restructuring occurs in seconds, but structural changes are relatively balanced across the dendritic arbor, maintaining overall arbor size and complexity over hours. Dendritic motility is regulated by spontaneous glutamatergic transmission. Both the rate and extent of the movements are decreased by antagonists to NMDA and non-NMDA glutamate receptors but are unaffected by tetrodotoxin, a sodium channel blocker. The dendritic movements are actin dependent and are controlled by the Rho family of small GTPases. Transfection of dominant-negative and constitutively active mutants into ganglion cells showed that Rac and Rho exert reciprocal effects on motility. We suggest that the Rho family of small GTPases could integrate activity-dependent and -independent signals from afferents, thereby adjusting target motility and maximizing the chance for initial contact and subsequent synaptogenesis.
In VivoImaging of Functional Inhibitory Networks on the Mauthner Cell of Larval Zebrafish
Authors: Takahashi Masaharu , Narushima Madoka , Oda Yoichi
Published: April 2018

Noninvasivein vivocalcium imaging was used to observe and characterize inhibitory circuitry in intact larval zebrafish. In the teleost hindbrain, the inhibitory network onto the major pair of reticulospinal neurons known as Mauthner cells (M-cells) has been described in detail. There are three sources of inhibition onto M-cells: recurrent inhibition mediated by an ipsilateral collateral of the M-cell axon, feedforward inhibition driven by sensory afferents, and reciprocal inhibition between bilaterally opposed M-cells. To visualize these inhibitions, M-cells were retrogradely loaded with the calcium indicator calcium green dextran. Recurrent inhibition attenuated the Ca2+response associated with an action potential in M-cells. Whole-cell recording revealed recurrent IPSCs, the conductance of which may underlie the shunting effect on action potentials and the attenuation of the Ca2+signal in M-cells. Blocking synaptic transmission within the recurrent network abolished both the Ca2+signal attenuation and the IPSCs. Electrical stimulation of the otic vesicle to activate VIII nerve afferents resulted in feedforward suppression of antidromically evoked test Ca2+responses in the contralateral M-cell. Orthodromic activation of M-cells produced a reciprocal reduction of the test Ca2+response in the contralateral M-cell. Thus, in the present study, we visualized the three types of inhibition and demonstrated that they are functional at 4 d after fertilization. The use of noninvasive techniques to image inhibitionin vivosuggest the plausibility of studying the hypothesis previously tested in adult goldfish that use-dependent changes in inhibitions underlie sound conditioning in escape behavior.
FM1-43 Reveals Membrane Recycling in Adult Inner Hair Cells of the Mammalian Cochlea
Authors: Griesinger Claudius B. , Richards Chistopher D. , Ashmore Jonathan F.
Published: April 2018

Neural transmission of complex sounds demands fast and sustained rates of synaptic release from the primary cochlear receptors, the inner hair cells (IHCs). The cells therefore require efficient membrane recycling. Using two-photon imaging of the membrane marker FM1-43 in the intact sensory epithelium within the cochlear bone of the adult guinea pig, we show that IHCs possess fast calcium-dependent membrane uptake at their apical pole. FM1-43 did not permeate through the stereocilial mechanotransducer channel because uptake kinetics were neither changed by the blockers dihydrostreptomycin andd-tubocurarine nor by treatment of the apical membrane with BAPTA, known to disrupt mechanotransduction. Moreover, the fluid phase marker Lucifer Yellow produced a similar labeling pattern to FM1-43, consistent with FM1-43 uptake via endocytosis. We estimate the membrane retrieval rate at ∼0.5% of the surface area of the cell per second. Labeled membrane was rapidly transported to the base of IHCs by kinesin-dependent trafficking and accumulated in structures that resembled synaptic release sites. Using confocal imaging of FM1-43 in excised strips of the organ of Corti, we show that the time constants of fluorescence decay at the basolateral pole of IHCs and apical endocytosis were increased after depolarization of IHCs with 40 mmpotassium, a stimulus that triggers calcium influx and increases synaptic release. Blocking calcium channels with either cadmium or nimodipine during depolarization abolished the rate increase of apical endocytosis. We suggest that IHCs use fast calcium-dependent apical endocytosis for activity-associated replenishment of synaptic membrane.
β-Amyloid-Induced Synthesis of the Ganglioside Gd3 Is a Requisite for Cell Cycle Reactivation and Apoptosis in Neurons
Authors: Copani Agata , Melchiorri Daniela , Caricasole Andrea , Martini Francesca , Sale Patrizio , Carnevale Roberto , Gradini Roberto , Sortino Maria Angela , Lenti Luisa , De Maria Ruggero , Nicoletti Ferdinando
Published: April 2018

We have shown that cortical neurons challenged with toxic concentrations of β-amyloid peptide (βAP) enter the S phase of the cell cycle before apoptotic death. Searching for a signaling molecule that lies at the border between cell proliferation and apoptotic death, we focused on the disialoganglioside GD3. Exposure of rat cultured cortical neurons to 25 μmβAP(25–35) induced a substantial increase in the intracellular levels of GD3 after 4 hr, a time that precedes neuronal entry into S phase. GD3 levels decreased but still remained higher than in the control cultures after 16 hr of exposure to βAP(25–35). Confocal microscopy analysis showed that the GD3 synthesized in response to βAP colocalized with nuclear chromatin. The increase in GD3 was associated with a reduction of sphingomyelin (the main source of the ganglioside precursor ceramide) and with the induction of α-2,8-sialyltransferase (GD3 synthase), the enzyme that forms GD3 from the monosialoganglioside GM3. A causal relationship between GD3, cell-cycle activation, and apoptosis was demonstrated by treating the cultures with antisense oligonucleotides directed against GD3 synthase. This treatment, which reduced βAP(25–35)-stimulated GD3 formation by ∼50%, abolished the neuronal entry into the S phase and was protective against βAP(25–35)-induced apoptosis.
Signaling Cascade Regulating Long-Term Potentiation of GABAAReceptor Responsiveness in Cerebellar Purkinje Neurons
Authors: Kawaguchi Shin-ya , Hirano Tomoo
Published: April 2018

Synaptic plasticity, a cellular basis of learning and memory, has been studied extensively at excitatory synapses. Although synaptic plasticity has also been reported at inhibitory synapses, the molecular mechanism remains elusive. Here we attempted to clarify the overall signaling cascades regulating the induction of inhibitory synaptic plasticity in the cerebellum.

Rebound potentiation (RP), a long-lasting increase in GABAAreceptor (GABAAR) responsiveness, is induced by postsynaptic depolarization of a Purkinje neuron (PN) at synapses formed with inhibitory interneurons (stellate or basket neurons). Previously, we showed that RP is suppressed by homosynaptic activation during depolarization through activation of the postsynaptic GABABreceptor (GABABR). Activation of GABABR reduces cAMP-dependent protein kinase (PKA) activity via the Gi/Go-protein. Here we examined the molecular pathway through which PKA activity affects RP induction.

We confirmed that inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) or PKA suppresses RP. We also found that inhibition of protein phosphatase 1 (PP-1) or calcineurin (PP-2B) impaired suppression of RP induction. Inhibition of either PP-1 or calcineurin abolished RP impairment by PKA inhibition, but not that by CaMKII inhibition. Antisense oligonucleotide-mediated knock down of DARPP-32, which is a substrate of PKA and calcineurin and inhibits PP-1 when phosphorylated by PKA, suppressed RP. Furthermore, activation of GABABR inhibited CaMKII activation through PKA inhibition and PP-1 activity. These results suggest that calcineurin activation accompanied by PKA inhibition in a PN causes dephosphorylation of DARPP-32, which releases PP-1 from inhibition. PP-1 in turn inhibits CaMKII activity, which is then directly involved in the RP induction.
N-Cadherin Mediates Axon-Aligned Process Growth and Cell–Cell Interaction in Rat Schwann Cells
Authors: Wanner Ina B. , Wood Patrick M.
Published: April 2018

The molecular mechanisms underlying the contact behavior of Schwann cells (SCs) and SC–axon association are poorly understood. SC–SC and SC–axon interactions were studied using purified adult rat SCs and cocultures of SCs with embryonic dorsal root ganglion neurons. After contact of SCs with axons, SCs start to extend processes in alignment with axons. This unique alignment was quantitated using a new assay. SC–axon alignment and SC–SC band formation were disrupted in medium containing low extracellular calcium, indicating the involvement of calcium-dependent adhesion molecules. N-cadherin expression was strong in developing rat sciatic nerves but weak in adult sciatic nerves. In purified adult-derived rat SCs, N-cadherin expression was increased by mitogens (neuregulins) and decreased by high cell density. High-resolution confocal images show intense N-cadherin signals in SC process tips. Subcellular N-cadherin was accumulated in bands at intercellular junctions between SCs and was clustered at axon–SC contact sites. Blocking antibodies (rabbit and guinea pig IgG directed against the first extracellular domain of N-cadherin) and cyclic pentapeptides (including the HAV motif) were used to perturb N-cadherin function. All blocking agents reduced the number of N-cadherin-positive SC–SC junctions and perturbed axon-aligned growth of SC processes. Averaging over all N-cadherin-perturbation experiments, in controls 67–86% of SCs exhibited axon-aligned process growth, whereas in treated cultures only 41% of the SCs aligned with axons. These results are evidence that in mammals N-cadherin is important for formation of SC–SC junctions and SC process growth in alignment with axons.
The Effect of Intensity and Duration on the Light-Induced Sodium and Potassium Currents in theHermissendaType B Photoreceptor
Authors: Blackwell Kim T.
Published: April 2018

Light duration and intensity influence classical conditioning inHermissendathrough their effects on the light-induced currents. Furthermore, the contribution of voltage-dependent potassium currents to the long-lasting depolarization in type B photoreceptors depends on light-induced currents active at resting potentials. Thus, the present study measures the effect of holding potential, duration, and intensity on the light-induced currents in discontinuous single-electrode voltage clamp mode. Three distinct current components are distinguished by their temporal and voltage characteristics and sensitivity to pharmacological agents. One current component is a transient sodium current,INalgt; another is a plateau sodium current,Iplateau, which persists for the duration of the light stimulus. Substitution of trimethylammonium chloride for sodium reduces both currents equally, suggesting thatIplateaurepresents partial inactivation ofINalgt. The third current component is a prolonged reduction in potassium currents,IKlgt; it is accompanied by an increase in input resistance, and it appears at potentials close to rest. An increase in light duration or intensity causes an increase in the peak conductance of bothINalgtandIKlgt. Latency ofINalgtis decreased by intensity, whereas rise time is increased by duration. An increase in light duration or intensity causes an increase in the time-to-peak and duration ofIKlgt. Characteristics of these currents suggest thatIKlgtis responsible for the long-lasting depolarization seen after light termination, and thus plays a role in classical conditioning.
Sensory Activation and Role of Inhibitory Reticulospinal Neurons that Stop Swimming in Hatchling Frog Tadpoles
Authors: Perrins Ray , Walford Alison , Roberts Alan
Published: April 2018

Activity in neuronal networks underlying locomotion and other rhythmic actions can start and stop in response to specific sensory stimuli. In vertebrate locomotion, some reticulospinal neurons such as Mauthner neurons can initiate activity, but the neurons controlling stopping are not defined. We have studied swimming in tadpoles of the frog,Xenopus, which is started by touching the skin and stops when the head contacts a solid surface. Using an immobilized tadpole preparation, the same stimuli control fictive swimming. When head contact is imitated by pressure to the head skin sensory neurons in the trigeminal ganglion are active, spinal neurons receive GABAergic inhibition, and swimming stops. Here we record intracellularly from neurons in the hindbrain that are excited by pressure or electrical stimulation to the head skin. By intracellular filling with neurobiotin, we identify these anatomically as mid-hindbrain reticulospinal neurons (MHRs). These have contralateral descending projections to the spinal cord and GABA-like immunoreactivity. They are rhythmically inhibited during fictive swimming. Individual MHRs reliably stopped ongoing swimming when brief firing was induced by intracellular current injection. The ability of individual MHRs to stop swimming was blocked by the GABAAantagonist bicuculline. Our evidence indicates that MHRs receive direct excitation from trigeminal sensory neurons and in turn release GABA to directly inhibit spinal neurons and turn off the swimming central pattern generator.
Variation in Inter-Animal Susceptibility to Noise Damage Is Associated with α9 Acetylcholine Receptor Subunit Expression Level
Authors: Luebke Anne E. , Foster Paul K.
Published: April 2018

Large intersubject variabilities in acoustic injury are known to occur in both humans and animals; however, the mechanisms underlying such differences are poorly understood. The olivocochlear efferent system has been hypothesized to play a significant role in protecting the cochlea from noise overexposure. In this study, we demonstrate that a newly developed test for determining average efferent system strength can predict intersubject variations in acoustic injury. In addition, the intersubject variability in cochlear expression of the α9 subunit of the nicotinic acetylcholine receptor was found to be proportional to an animals average efferent strength. Therefore, the inter-animal variability in the α9-containing acetylcholine receptor expression may be one mechanism contributing to the inter-animal variability in acoustic injury.
The Wake-Promoting Hypocretin–Orexin Neurons Are in an Intrinsic State of Membrane Depolarization
Authors: Eggermann Emmanuel , Bayer Laurence , Serafin Mauro , Saint-Mleux Benoı̂t , Bernheim Laurent , Machard Danièle , Jones Barbara E. , Mühlethaler Michel
Published: April 2018

Wakefulness depends on the activity of hypocretin–orexin neurons because their lesion results in narcolepsy. How these neurons maintain their activity to promote wakefulness is not known. Here, by recording for the first time from hypocretin–orexin neurons and comparing their properties with those of neurons expressing melanin-concentrating hormone, we show that hypocretin–orexin neurons are in an intrinsic state of membrane depolarization that promotes their spontaneous activity. We propose that wakefulness and associated energy expenditure thus depend on that property, which allows the hypocretin–orexin neurons to maintain a tonic excitatory influence on the central arousal and peripheral sympathetic systems.
Glial Cell Inhibition of Neurons by Release of ATP
Authors: Newman Eric A.
Published: April 2018

ATP is released by neurons and functions as a neurotransmitter and modulator in the CNS. Here I show that ATP released from glial cells can also serve as a potent neuromodulator, inhibiting neurons in the retina of the rat. Activation of glial cells by focal ejection of ATP, ATPγS, dopamine, thrombin, or lysophosphatidic acid or by mechanical stimulation evoked hyperpolarizing responses and outward currents in a subset of retinal ganglion cells by increasing a Ba2+-sensitive K+conductance in the neurons. This glia-evoked inhibition reduced the firing rate of those neurons that displayed spontaneous spike activity. The inhibition was abolished by the A1adenosine receptor antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine) (10 nm) and was reduced by the ecto-ATPase inhibitor ARL-67156 (6-N,N-diethyl-d-β,γ-dibromomethyleneATP) (50 μm) and by the ectonucleotidase inhibitor AOPCP [adenosine-5′-O-(α,β-methylene)-diphosphonate] (250 μm). Selective activation of retinal glial cells demonstrated that Müller cells, but not astrocytes, mediate the inhibition. ATP release from Müller cells into the inner plexiform layer of the retina was shown using the luciferin–luciferase chemiluminescence assay. These findings demonstrate that activated glial cells can inhibit neurons in the retina by the release of ATP, which is converted to adenosine by ectoenzymes and subsequently activates neuronal adenosine receptors. The results lend support to the hypothesis that glial cells play an active role in information processing in the CNS.
Optical Imaging of Long-Term Depression in the Mouse Cerebellar CortexIn Vivo
Authors: Gao Wangcai , Dunbar Robert L. , Chen Gang , Reinert Kenneth C. , Oberdick John , Ebner Timothy J.
Published: April 2018

Conjunctive stimulation of climbing fiber and parallel fiber inputs results in long-term depression (LTD) at parallel fiber–Purkinje cell synapses. Although hypothesized to play a major role in cerebellar motor learning, there has been no characterization of the cellular and molecular mechanisms of LTD in the whole animal, let alone its spatial properties, both of which are critical to understanding the role of LTD in cerebellar function. Neutral red optical imaging of the cerebellar cortex in the anesthetized mouse was used to visualize the spatial patterns of activation. Stimulation of the parallel fibers evoked a transverse beam of optical activity, and stimulation of the contralateral inferior olive evoked parasagittal bands. Conjunctive stimulation of parallel fibers and climbing fibers induced a long-term decrease (at least 1 hr) in the optical response to subsequent parallel fiber activation confined to the region of interaction between these two inputs. Activation of climbing fibers alone failed to induce the long-term decrease. Field potential recordings confirmed that the depression is postsynaptic and restricted to the interaction site. The long-term depression in the beam was prevented by a group 1 metabotropic glutamate receptor (mGluR1) antagonist and was absent in transgenic mice selectively expressing an inhibitor of protein kinase C (PKC) in Purkinje cells. Conversely, the long-term depression occurred in the mGluR4knock-out mouse, consistent with its postsynaptic origin. In addition to providing the first visualization of parallel fiber–Purkinje cell LTD in the cerebellar cortex, this study demonstrates the spatial specificity of LTD and its dependence on mGluR1and PKCin vivo.