Colocalization of hyperpolarization‐activated, cyclic nucleotide‐gated channel subunits in rat retinal ganglion cells

Stradleigh, Tyler W.; Ogata, Genki; Partida, Gloria J.; Oi, Hanako; Greenberg, Kenneth P.; Krempely, Kalen S.; Ishida, Andrew T.

doi:10.1002/cne.22638

Cited by 26 publications

(57 citation statements)

References 101 publications

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“…The specificity of anti-HCNs (N70/28, APC-056, APC-052) has been confirmed previously with Western blots revealing a single band of expected molecular weight (Stradleigh et al 2011) (Scanalytics, Inc.). In these experiments, the antibody labeling luminance was measured in IPLab with no digital enhancement by subtracting the mean of 400 pixels at four background areas from the mean of 300 pixels at the three brightest areas inside each neuron.…”

Section: Immunocytochemistrysupporting

confidence: 68%

I h and HCN Channels in Murine Spiral Ganglion Neurons: Tonotopic Variation, Local Heterogeneity, and Kinetic Model

Liu

Manis

Davis

2014

JARO

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One of the major contributors to the response profile of neurons in the auditory pathways is the I h current. Its properties such as magnitude, activation, and kinetics not only vary among different types of neurons (Banks et al., J Neurophysiol 70:1420-1432, 1993 Fu et al., J Neurophysiol 78:2235-2245 Bal and Oertel, J Neurophysiol 84:806-817, 2000; Cao and Oertel, J Neurophysiol 94:821-832, 2005; Rodrigues and Oertel, J Neurophysiol 95:76-87, 2006; Yi et al., J Neurophysiol 103:2532-2543, 2010), but they also display notable diversity in a single population of spiral ganglion neurons (Mo and Davis, J Neurophysiol 78:3019-3027, 1997), the first neural element in the auditory periphery. In this study, we found from somatic recordings that part of the heterogeneity can be attributed to variation along the tonotopic axis because I h in the apical neurons have more positive half-activation voltage levels than basal neurons. Even within a single cochlear region, however, I h current properties are not uniform. To account for this heterogeneity, we provide immunocytochemical evidence for variance in the intracellular density of the hyperpolarization-activated cyclic nucleotide-gated channel α-subunit 1 (HCN1), which mediates I h current. We also observed different combinations of HCN1 and HCN4 α-subunits from cell to cell. Lastly, based on the physiological data, we performed kinetic analysis for the I h current and generated a mathematical model to better understand varied I h on spiral ganglion function. Regardless of whether I h currents are recorded at the nerve terminals (Yi et al., J Neurophysiol 103:2532-2543, 2010 or at the somata of spiral ganglion neurons, they have comparable mean half-activation voltage and induce similar resting membrane potential changes, and thus our model may also provide insights into the impact of I h on synaptic physiology.

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Section: Immunocytochemistrysupporting

confidence: 68%

I h and HCN Channels in Murine Spiral Ganglion Neurons: Tonotopic Variation, Local Heterogeneity, and Kinetic Model

Liu

Manis

Davis

2014

JARO

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“…Signal attributed to GFP was amplified with an Alexa Fluor 488-conjugated rabbit polyclonal antibody against green fluorescent protein (GFP), raised against GFP isolated from Aequorea victoria and diluted to 2 μg/mL (Life Technologies, Cat# A-21311, RRID:AB_10058149). This antibody has been used in rodent retina previously (Xu and Tian, 2006;Stradleigh et al, 2011), and we detected no binding of this antibody in rat or rabbit retinae unless GFP expression was induced.…”

Section: Immunohistochemistrymentioning

confidence: 55%

Section: Applicabilitymentioning

confidence: 89%

“…FA-based fixatives that do not induce beading have enabled us to visualize dendritic shape, contour, branching, and arborization (Stradleigh et al, 2011), contrast axon calibers (Stradleigh et al, 2011), gauge protein colocalizations (Stradleigh et al, 2011;Partida et al, 2012), and compare the binding of antibodies in dark-and light-adapted retinae (Ogata et al, 2012). Moreover, we have found that replacing NaCl by sucrose suppresses beading even if the fixative pH is shifted to alter fixation speed and efficacy (Berod et al, 1981;Eldred et al, 1983;Stradleigh et al, 2011;Ogata et al, 2012). The improved preservation of cells by the protectant fixatives we have described here is of particular interest because immunostaining with some antibodies (e.g., monoclonal and polyclonal antibodies directed against D1 dopamine receptors, HCN channel subunits, cAMP, and phosphorylated CaMKII) is compatible with FA-based fixatives (Pow et al, 1995;Hayashida et al, 2009;Stradleigh et al, 2011;Ogata et al, 2012) but not with a variety of other fixatives (e.g., Clarke's fluid, glyoxal, ice cold acetone; unpublished observations).…”

Section: Applicabilitymentioning

confidence: 99%

“…Moreover, we have found that replacing NaCl by sucrose suppresses beading even if the fixative pH is shifted to alter fixation speed and efficacy (Berod et al, 1981;Eldred et al, 1983;Stradleigh et al, 2011;Ogata et al, 2012). The improved preservation of cells by the protectant fixatives we have described here is of particular interest because immunostaining with some antibodies (e.g., monoclonal and polyclonal antibodies directed against D1 dopamine receptors, HCN channel subunits, cAMP, and phosphorylated CaMKII) is compatible with FA-based fixatives (Pow et al, 1995;Hayashida et al, 2009;Stradleigh et al, 2011;Ogata et al, 2012) but not with a variety of other fixatives (e.g., Clarke's fluid, glyoxal, ice cold acetone; unpublished observations). GFP-expressing retinal ganglion cells preserved for 24 hr in standard FA solution (formaldehyde diluted to 4% w/v in phosphate-buffered saline).…”

Section: Applicabilitymentioning

confidence: 99%

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Moniliform deformation of retinal ganglion cells by formaldehyde‐based fixatives

Stradleigh

Greenberg

Partida

et al. 2015

J of Comparative Neurology

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Protocols for characterizing cellular phenotypes commonly use chemical fixatives to preserve anatomical features, mechanically stabilize tissue, and stop physiological responses. Formaldehyde, diluted in either phosphate-buffered saline or phosphate buffer, has been widely used in studies of neurons, especially in conjunction with dyes and antibodies. However, previous studies have reported that these fixatives induce the formation of bead-like varicosities in the dendrites and axons of brain and spinal cord neurons. We report here that these formaldehyde formulations can induce bead formation in the dendrites and axons of adult rat and rabbit retinal ganglion cells, and that retinal ganglion cells differ from hippocampal, cortical, cerebellar, and spinal cord neurons in that bead formation is not blocked by glutamate receptor antagonists, a voltage-gated Na + channel toxin, extracellular Ca 2+ ion exclusion, or temperature shifts. Moreover, we describe a modification of formaldehyde-based fixatives that prevents bead formation in retinal ganglion cells visualized by green fluorescent protein expression and by immunohistochemistry.

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Dopamine and full‐field illumination activate D1 and D2‐D5‐type receptors in adult rat retinal ganglion cells

Ogata

Stradleigh

Partida

et al. 2012

J of Comparative Neurology

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Dopamine can regulate signal generation and transmission by activating multiple receptors and signaling cascades, especially in striatum, hippocampus, and cerebral cortex. Dopamine modulates an even larger variety of cellular properties in retina, yet has been reported to do so by only D1 receptor-driven cyclic adenosine monophosphate (cAMP) increases or D2 receptor-driven cAMP decreases. Here, we test the possibility that dopamine operates differently on retinal ganglion cells, because the ganglion cell layer binds D1 and D2 receptor ligands, and displays changes in signaling components other than cAMP under illumination that should release dopamine. In adult rat retinal ganglion cells, based on patch-clamp recordings, Ca2+ imaging, and immunohistochemistry, we find that 1) spike firing is inhibited by dopamine and SKF 83959 (an agonist that does not activate homomeric D1 receptors or alter cAMP levels in other systems); 2) D1 and D2 receptor antagonists (SCH 23390, eticlopride, raclopride) counteract these effects; 3) these antagonists also block light-induced rises in cAMP, light-induced activation of Ca2+/calmodulin-dependent protein kinase II, and dopamine-induced Ca2+ influx; and 4) the Ca2+ rise is markedly reduced by removing extracellular Ca2+ and by an IP3 receptor antagonist (2-APB). These results provide the first evidence that dopamine activates a receptor in adult mammalian retinal neurons that is distinct from classical D1 and D2 receptors, and that dopamine can activate mechanisms in addition to cAMP and cAMP-dependent protein kinase to modulate retinal ganglion cell excitability.

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Colocalization of hyperpolarization‐activated, cyclic nucleotide‐gated channel subunits in rat retinal ganglion cells

Cited by 26 publications

References 101 publications

I h and HCN Channels in Murine Spiral Ganglion Neurons: Tonotopic Variation, Local Heterogeneity, and Kinetic Model

I h and HCN Channels in Murine Spiral Ganglion Neurons: Tonotopic Variation, Local Heterogeneity, and Kinetic Model

Moniliform deformation of retinal ganglion cells by formaldehyde‐based fixatives

Dopamine and full‐field illumination activate D1 and D2‐D5‐type receptors in adult rat retinal ganglion cells

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