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Dive into the research topics where Stephen L. Mills is active.

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Featured researches published by Stephen L. Mills.


The Journal of Comparative Neurology | 2001

Rod pathways in the mammalian retina use connexin 36.

Stephen L. Mills; Jennifer J. O'Brien; Wei Li; John O'Brien; Stephen C. Massey

Many neurons in the mammalian retina are coupled by means of gap junctions. Here, we show that, in rabbit retina, an antibody to connexin 36 heavily labels processes of AII amacrine cells, a critical interneuron in the rod pathway. Image analysis indicates that Cx36 is primarily located at dendritic crossings between overlapping AII amacrine cells. This finding suggests that Cx36 participates in homotypic gap junctions between pairs of AII amacrine cells. Cx36 was also found at AII/cone bipolar contacts, previously shown to be gap junction sites. This finding suggests that Cx36 participates at gap junctions that may be heterotypic. These results place an identified neuronal connexin in the context of a well‐defined retinal circuit. The absence of Cx36 in many other neurons known to be coupled suggests the presence of additional unidentified connexins in mammalian neurons. Conversely, Cx36 labeling in other regions of the retina is not associated with AII amacrine cells, indicating some other cell types use Cx36. J. Comp. Neurol. 436:336–350, 2001.


The Journal of Comparative Neurology | 1996

A calbindin-immunoreactive cone bipolar cell type in the rabbit retina

Stephen C. Massey; Stephen L. Mills

We have studied the distribution of the calcium‐binding protein calbindin in the adult rabbit retina by using a commercially available antibody and immunocytochemical methods. The most heavily labeled cells are A‐type horizontal cells, but B‐type horizontal cells are also lightly labeled by this antibody. Among the horizontal cells, there is a mosaic of small, well‐labeled somata, which we have identified as a subset of ON cone bipolar cells. In addition, some wide‐field amacrine cells and a few large ganglion cells are also labeled for calbindin. The calbindin bipolar cells form a regular mosaic with a peak density of approximately 1,700 cells/mm2, falling to 550 cells/mm2 in the periphery. They account for about one‐twelfth of cone bipolar cells, and they are narrowly stratified deep in sublamina 4 of the inner plexiform layer immediately above the rod bipolar terminals. Double‐label experiments using an antibody to protein kinase C (PKC) indicate that the calbindin bipolar cells are completely distinct from the population of rod bipolar cells. Rod bipolar cells outnumber the calbindin cone bipolar cells by a factor of four to five. Further double‐label experiments show that the calbindin bipolar cells are also labeled for recovering. The calbindin bipolar cells are well coupled to AII amacrine cells, and they account for roughly 23% of the AII coupled bipolar cells. This suggests that there are three to four additional ON cone bipolar cell types that are coupled to AII amacrine cells. The calbindin cone bipolar cell described in this paper shares many characteristics with a reconstructed cone bipolar cell that forms the most gap junctions with AII amacrine cells (Strettoi et al. [1994] J. Comp. Neurol. 347:139–149). We conclude that these different methodologies provide complementary descriptions of the same cone bipolar cell type. The calbindin antibody defines a subset of cone bipolar cells in the rabbit retina. The cells in this subset are almost certainly the deepest of the cone bipolar cells. The tight stratification of the calbindin cone bipolar cell suggests that the inner plexiform layer is stratified according to depth, with narrow functional divisions within the broad partition of sublamina b, where ON signals are processed. The strength of coupling between the calbindin cone bipolar cells and AII amacrine cells suggests this pathway plays a major role under scotopic conditions.


The Journal of Neuroscience | 2009

ON Inputs to the OFF Layer: Bipolar Cells That Break the Stratification Rules of the Retina

Hideo Hoshi; Wei Li Liu; Stephen C. Massey; Stephen L. Mills

The vertebrate retina is a distinctly laminar structure. Functionally, the inner plexiform layer, in which bipolar cells synapse onto amacrine and ganglion cells, is subdivided into two sublaminae. Cells that depolarize at light offset ramify in sublamina a; those that depolarize at light onset ramify in sublamina b. The separation of ON and OFF pathways appears to be a fundamental principle of retinal organization that is reflected throughout the entire visual system. We show three clear exceptions to this rule, in which the axons of calbindin-positive ON cone bipolar cells make ribbon synapses as they pass through the OFF layers with three separate cell types: (1) dopaminergic amacrine cells, (2) intrinsically photosensitive ganglion cells, and (3) bistratified diving ganglion cells. The postsynaptic location of the AMPA receptor GluR4 at these sites suggests that ON bipolar cells can make functional synapses as their axons pass through the OFF layers of the inner plexiform layer. These findings resolve a long-standing question regarding the anomalous ON inputs to dopaminergic amacrine cells and suggest that certain ON bipolar cell axons can break the stratification rules of the inner plexiform layer by providing significant synaptic output before their terminal specializations. These outputs are not only to dopaminergic amacrine cells but also to at least two ON ganglion cell types that have dendrites that arborize in sublamina a.


Visual Neuroscience | 2007

Screening of gap junction antagonists on dye coupling in the rabbit retina

F. Pan; Stephen L. Mills; Stephen C. Massey

Many cell types in the retina are coupled via gap junctions and so there is a pressing need for a potent and reversible gap junction antagonist. We screened a series of potential gap junction antagonists by evaluating their effects on dye coupling in the network of A-type horizontal cells. We evaluated the following compounds: meclofenamic acid (MFA), mefloquine, 2-aminoethyldiphenyl borate (2-APB), 18-alpha-glycyrrhetinic acid, 18-beta-glycyrrhetinic acid (18-beta-GA), retinoic acid, flufenamic acid, niflumic acid, and carbenoxolone. The efficacy of each drug was determined by measuring the diffusion coefficient for Neurobiotin (Mills & Massey, 1998). MFA, 18-beta-GA, 2-APB and mefloquine were the most effective antagonists, completely eliminating A-type horizontal cell coupling at a concentration of 200 muM. Niflumic acid, flufenamic acid, and carbenoxolone were less potent. Additionally, carbenoxolone was difficult to wash out and also may be harmful, as the retina became opaque and swollen. MFA, 18-beta-GA, 2-APB and mefloquine also blocked coupling in B-type horizontal cells and AII amacrine cells. Because these cell types express different connexins, this suggests that the antagonists were relatively non-selective across several different types of gap junction. It should be emphasized that MFA was water-soluble and its effects on dye coupling were easily reversible. In contrast, the other gap junction antagonists, except carbenoxolone, required DMSO to make stock solutions and were difficult to wash out of the preparation at the doses required to block coupling in A-type HCs. The combination of potency, water solubility and reversibility suggest that MFA may be a useful compound to manipulate gap junction coupling.


The Journal of Comparative Neurology | 1999

Antibody to calretinin stains AII amacrine cells in the rabbit retina: Double‐label and confocal analyses

Stephen C. Massey; Stephen L. Mills

The AII or rod amacrine cell is a critical interneuron in the rod pathway of mammalian retinae. In this report, it is shown that commercially available antibodies to the calcium binding protein calretinin may be used to label the population of AII amacrine cells selectively. Calretinin‐positive amacrine cells had the morphological attributes of AII amacrine cells. Double‐labeling procedures showed that calretinin‐positive somata were surrounded by dopaminergic varicosities and that calretinin‐positive dendrites enclosed rod bipolar terminals, both as previously described for AII amacrine cells. By analyzing the surrounding kernel for each labeled pixel in the rod bipolar image, it is shown here that AII processes are adjacent to rod bipolar terminals at a level that far exceeds the random overlap present in images in which one label was rotated out of phase. Such a spatial relationship is indicative of synaptic connections, as well described for rod bipolar input to AII amacrine cells. AII amacrine cells also were double‐labeled for calretinin and parvalbumin; however, a scattergram analysis of red versus green intensity showed that the parvalbumin antibody stained additional unidentified amacrine cells. In conclusion, at the appropriate dilution, calretinin antibodies are a useful marker for AII amacrine cells in the rabbit retina. J. Comp. Neurol. 411:3–18, 1999.


The Journal of Comparative Neurology | 1999

AII Amacrine Cells Limit Scotopic Acuity in Central Macaque Retina: A Confocal Analysis of Calretinin Labeling

Stephen L. Mills; Stephen C. Massey

We have used calretinin antibodies to label selectively the mosaic of AII amacrine cells in the macaque retina. Confocal analysis of double‐labeled material indicated that AII dendrites spiral down around descending rod bipolar axons before enveloping the synaptic terminals. Processes from a previously observed dopaminergic plexus in the inner nuclear layer were observed to contact the somata of calretinin‐positive AII somata. Intracellular neurobiotin injection revealed that AII amacrine cells are tracer coupled to other AII amacrine cells and to some unidentified cone bipolar cells. An analysis of the retinal distribution of macaque AII amacrine cells, including an area in and around the fovea, showed a peak density of approximately 5,000 cells/mm2 at an eccentricity of 1.5 mm. Staining of AII amacrine cells in central retina with antibodies to calretinin was confirmed by confocal microscopy. These results indicate that calretinin antibodies can be used to label the AII amacrine cell population selectively and that primate AII amacrine cells share many of the features of previously described mammalian AII amacrine cells. The peak AII cell density closely matched the peak sampling rate of scotopic visual acuity. Calculations suggest that, in central macaque retina, where midget ganglion cells are more numerous, AII amacrine cells form the limit of scotopic visual acuity (Wässle et al. [1995] J. Comp. Neurol. 361:537–551). As the ganglion cell density falls rapidly away from the fovea, there is a cross‐over point at around 15° eccentricity that matches the inflection point in a psychophysically derived plot of scotopic visual acuity versus eccentricity (Lennie and Fairchild [1994] Vision Res. 34:477–482). The correspondence between the anatomic and psychophysical data supports our interpretation that the anatomic sampling rate of AII amacrine cells limits central scotopic acuity. J. Comp. Neurol. 411:19–34, 1999.


Visual Neuroscience | 1994

Distribution and coverage of A- and B-type horizontal cells stained with Neurobiotin in the rabbit retina

Stephen L. Mills; Stephen C. Massey

Both A- and B-type horizontal cells in the rabbit retina were labeled by brief in vitro incubations of the isolated retina in the blue fluorescent dye 4,6-diamino-2-phenylindole. Intracellular injection of Lucifer Yellow into the somata revealed the morphology of the individual cells. Dye-coupling with Lucifer Yellow was seen only between A-type horizontal cells. By contrast, injection of the tracer Neurobiotin showed dye-coupling between both A- and B-type horizontal cells. There also appeared to be coupling between the axon terminals of B-type horizontal cells. The extensive dye-coupling seen following injection of Neurobiotin into a single horizontal cell soma can be used to obtain population counts of each cell type. Staining of large numbers of each cell type across the retina showed that each type increased in number and declined in dendritic diameter as the visual streak was approached, such that relatively constant coverage across the retina was maintained. In the visual streak, A-type horizontal cells numbered 555 cells/mm2 and averaged 120 microns in diameter, compared to 1375 cells/mm2 and 100 microns for B-type horizontal cells. In the periphery, the A- and B-types numbered 250 cells/mm2 and 400 cells/mm2, respectively. The average diameters of the dendritic trees at these locations were 225 microns for the A-type and 175 microns for the B-type. Coverage across the retina averaged almost six for A-type horizontal cells and 8-10 for the B-type. A-type horizontal cells in the visual streak whose elliptical dendritic fields were shown by Bloomfield (1992) to correlate physiologically with orientation bias were shown to be dye-coupled to cells with symmetrical dendritic fields.


The Journal of Comparative Neurology | 2001

Coupling from AII amacrine cells to ON cone bipolar cells is bidirectional

E. Brady Trexler; Wei Li; Stephen L. Mills; Stephen C. Massey

The AII amacrine cell is a critical interneuron in the rod pathway of the mammalian retina. Rod signals pass into cone pathways by means of gap junctions between AII amacrine cells and ON cone bipolar cells. Filling AII amacrine cells with Neurobiotin produces labeling of cone bipolar cells by means of these gap junctions. However, tracer injections into bipolar cells do not produce labeling of the AII network (Vaney [1997] Invest Ophthalmol Vis Sci. 38:267–273), which suggests that the AII/bipolar gap junctions allow the passage of tracer in only one direction. This mechanism stands in contrast to physiological results, which indicate that light adapted signals can pass from ON cone bipolar cells into the AII network (Xin and Bloomfield [1999] Vis Neurosci. 16:653–665). Here, we report that a variety of ON and OFF bipolar cells are sometimes anomalously coupled to the A‐type horizontal cell network. These relatively rare examples do not result from dye injection errors, but seem to represent minor developmental errors. However, this provides a method to obtain Neurobiotin‐filled cone bipolar cells without the necessity of impaling them with a microelectrode. Under these conditions, Neurobiotin spreads from ON cone bipolar cells into neighboring AII amacrine cells. The dye‐coupled AII amacrine cells, positively identified by double labeling with an antibody against calretinin, were centered around anomalously coupled ON bipolar cells. These results indicate that AII/bipolar cell gap junctions allow tracer coupling in both directions, consistent with previous physiological results. The previous failure to detect the passage of neuronal tracer from injected bipolar cells to AII amacrine cells may reflect electrode damage or perhaps the asymmetrical voltage sensitivity of a heterotypic gap junction. J. Comp. Neurol. 437:408–422, 2001.


The Journal of Neuroscience | 2004

Cone Photoreceptors in Bass Retina Use Two Connexins to Mediate Electrical Coupling

John O'Brien; H. Bao Nguyen; Stephen L. Mills

Electrical coupling via gap junctions is a common property of CNS neurons. In retinal photoreceptors, coupling plays important roles in noise filtering, intensity coding, and spatial processing. In many vertebrates, coupling is regulated during the course of light adaptation. To understand the mechanisms of this regulation, we studied photoreceptor gap junction proteins. We found that two connexins were expressed in bass cone photoreceptors. Connexin 35 (Cx35) mRNA was present in many cell types, including photoreceptors and amacrine, bipolar, and a few ganglion cells. Antibodies to Cx35 labeled abundant gap junctions in both the inner and outer plexiform layers. In the outer plexiform layer, numerous plaques colocalized with cone telodendria at crossing contacts and tip-to-tip contacts. Cx34.7 mRNA was found predominantly in the photoreceptor layer, primarily in cones. Cx34.7 immunolabeling was limited to small plaques immediately beneath cone pedicles and did not colocalize with Cx35. Cx34.7 plaques were associated with a dense complex of cone membrane beneath the pedicles, including apparent contacts between telodendria and cone pedicles. Tracer coupling studies of the connexins expressed in HeLa cells showed that coupling through Cx35 gap junctions was reduced by protein kinase A (PKA) activation and enhanced by PKA inhibition through a greater than fivefold activity range. Cx34.7 was too poorly expressed to study. PKA regulation suggests that coupling through Cx35 gap junctions can be controlled dynamically through dopamine receptor pathways during light adaptation. If Cx34.7 forms functional cell-cell channels between cones, it would provide a physically separate pathway for electrical coupling.


Visual Neuroscience | 2007

Dopaminergic modulation of tracer coupling in a ganglion-amacrine cell network.

Stephen L. Mills; Xiao Bo Xia; Hideo Hoshi; Sally I. Firth; Margaret E. Rice; Laura J. Frishman; David W. Marshak

Many retinal ganglion cells are coupled via gap junctions with neighboring amacrine cells and ganglion cells. We investigated the extent and dynamics of coupling in one such network, the OFF alpha ganglion cell of rabbit retina and its associated amacrine cells. We also observed the relative spread of Neurobiotin injected into a ganglion cell in the presence of modulators of gap junctional permeability. We found that gap junctions between amacrine cells were closed via stimulation of a D(1) dopamine receptor, while the gap junctions between ganglion cells were closed via stimulation of a D(2) dopamine receptor. The pairs of hemichannels making up the heterologous gap junctions between the ganglion and amacrine cells were modulated independently, so that elevations of cAMP in the ganglion cell open the ganglion cell hemichannels, while elevations of cAMP in the amacrine cell close its hemichannels. We also measured endogenous dopamine release from an eyecup preparation and found a basal release from the dark-adapted retina of approximately 2 pmol/min during the day. Maximal stimulation with light increased the rate of dopamine release from rabbit retina by 66%. The results suggest that coupling between members of the OFF alpha ganglion cell/amacrine cell network is differentially modulated with changing levels of dopamine.

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Stephen C. Massey

University of Texas at Austin

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Hideo Hoshi

University of Texas Health Science Center at Houston

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John O'Brien

University of Texas Health Science Center at Houston

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David W. Marshak

University of Texas at Austin

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Harry G. Sperling

University of Texas Health Science Center at Houston

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Wei Li

University of Texas Health Science Center at Houston

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Alejandro Vila

University of Texas at Austin

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Anthony A. Wright

University of Texas Health Science Center at Houston

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F. Pan

University of Texas Health Science Center at Houston

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Hongyan Li

University of Texas at Austin

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