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1.
J Neurophysiol ; 113(1): 203-17, 2015 Jan 01.
Article in English | MEDLINE | ID: mdl-25298382

ABSTRACT

The purpose of this study was to evaluate the expression patterns of nicotinic and muscarinic ACh receptors (nAChRs and mAChRs, respectively) in relation to one another and to understand their effects on rabbit retinal ganglion cell response properties. Double-label immunohistochemistry revealed labeled inner-retinal cell bodies and complex patterns of nAChR and mAChR expression in the inner plexiform layer. Specifically, the expression patterns of m1, m4, and m5 muscarinic receptors overlapped with those of non-α7 and α7 nicotinic receptors in presumptive amacrine and ganglion cells. There was no apparent overlap in the expression patterns of m2 muscarinic receptors with α7 nicotinic receptors or of m3 with non-α7 nicotinic receptors. Patch-clamp recordings demonstrated cell type-specific effects of nicotinic and muscarinic receptor blockade. Muscarinic receptor blockade enhanced the center responses of brisk-sustained/G4 On and G4 Off ganglion cells, whereas nicotinic receptor blockade suppressed the center responses of G4 On-cells near the visual streak but enhanced the center responses of nonstreak G4 On-cells. Blockade of muscarinic or nicotinic receptors suppressed the center responses of brisk-sustained Off-cells and the center light responses of subsets of brisk-transient/G11 On- and Off-cells. Only nicotinic blockade affected the center responses of G10 On-cells and G5 Off-cells. These data indicate that physiologically and morphologically identified ganglion cell types have specific patterns of AChR expression. The cholinergic receptor signatures of these cells may have implications for understanding visual defects in disease states that result from decreased ACh availability.


Subject(s)
Receptors, Muscarinic/metabolism , Receptors, Nicotinic/metabolism , Retinal Ganglion Cells/physiology , Animals , Bungarotoxins , Immunohistochemistry , Light , Microscopy, Confocal , Muscarinic Antagonists/pharmacology , Nicotinic Antagonists/pharmacology , Optical Imaging , Patch-Clamp Techniques , Photic Stimulation , Rabbits , Retinal Ganglion Cells/drug effects , Tissue Culture Techniques
2.
Mol Vis ; 20: 1328-56, 2014.
Article in English | MEDLINE | ID: mdl-25352741

ABSTRACT

PURPOSE: The α7 nicotinic acetylcholine receptor (nAChR) is widely expressed in the nervous system, including in the inner retinal neurons in all species studied to date. Although reductions in the expression of α7 nAChRs are thought to contribute to the memory and visual deficits reported in Alzheimer's disease (AD) and schizophrenia , the α7 nAChR knockout (KO) mouse is viable and has only slight visual dysfunction. The absence of a major phenotypic abnormality may be attributable to developmental mechanisms that serve to compensate for α7 nAChR loss. We hypothesized that the upregulation of genes encoding other nAChR subunits or muscarinic acetylcholine receptor (mAChR) subtypes during development partially accounts for the absence of major deficiencies in the α7 nAChR KO mouse. The purpose of this study was to determine whether the deletion of the α7 nAChR subunit in a mouse model resulted in changes in the regulation of other cholinergic receptors or other ion channels in an α7 nAChR KO mouse when compared to a wild-type (WT) mouse. METHODS: To examine gene expression changes, we employed a quantitative real-time polymerase chain reaction (qPCR) using whole retina RNA extracts as well as RNA extracted from selected regions of the retina. These extracts were collected using laser capture microdissection (LCM). The presence of acetylcholine receptor (AChR) subunit and subtype proteins was determined via western blotting. To determine any differences in the number and distribution of choline acetyltransferase (ChAT) amacrine cells, we employed wholemount and vertical immunohistochemistry (IHC) and cell counting. Additionally, in both WT and α7 nAChR KO mouse retinas, the distribution of the nAChR subunit and mAChR subtype proteins were determined via IHC for those KO mice that experienced mRNA changes. RESULTS: In the whole retina, there was a statistically significant upregulation of α2, α9, α10, ß4, nAChR subunit, and m1 and m4 mAChR subtype transcripts in the α7 nAChR KO mice. However, the retinal layers showed complex patterns of transcript expression. In the ganglion cell layer (GCL), m2 and m4 mAChR subtype transcripts were significantly upregulated, while ß3 and ß4 nAChR subunit transcripts were significantly downregulated. In the inner portion of the inner nuclear layer (iINL), α2, α9, ß4, nAChR subunit, and m3 and m4 mAChR subtype transcripts were significantly downregulated. In the outer portion of the inner nuclear layer (oINL), ß2, ß4, and m4 AChR subunit transcripts were significantly upregulated. Western blot experiments confirmed the protein expression of α3-α5 and α9-containing nAChR subunits and m1-m2 mAChR subtypes in mouse retinas. IHC results supported many of the mRNA changes observed. Finally, this is the first report of α9 and α10 nAChR subunit expressions in the retina of any species. CONCLUSIONS: Rather than a simple upregulation of a single AChR subunit or subtype, the absence of the α7 nAChR in the KO mice was associated with complex layer-specific changes in the expression of AChR subunits and subtypes.


Subject(s)
Protein Subunits/genetics , RNA, Messenger/genetics , Receptors, Muscarinic/genetics , Retina/metabolism , alpha7 Nicotinic Acetylcholine Receptor/genetics , Animals , Gene Expression Profiling , Gene Expression Regulation , Laser Capture Microdissection , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Sequence Annotation , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Subunits/metabolism , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction , Receptors, Muscarinic/metabolism , alpha7 Nicotinic Acetylcholine Receptor/deficiency
3.
Invest Ophthalmol Vis Sci ; 52(13): 9445-51, 2011 Dec 09.
Article in English | MEDLINE | ID: mdl-22064991

ABSTRACT

PURPOSE: To examine the effects of nicotine on responses from the human retina measured electrophysiologically. METHODS: Electroretinogram (ERG) responses were obtained from ten healthy, visually normal adults who were nonsmokers. Nicotine (2 and 4 mg) and a placebo were administered in the form of gum 30 minutes before testing in two separate experiments. ERG responses were collected and analyzed using a full-field ERG system. Responses were recorded from one eye of each subject using a bipolar contact-lens electrode. Intensity-response curves were obtained under both dark- and light-adapted conditions. In experiment 1, both dark- and light-adapted tests were completed sequentially. In experiment 2, only light-adapted testing was performed. Intensity-response functions were analyzed using the Naka-Rushton equation. RESULTS: In experiment 1, compared with placebo, dark-adapted b-wave amplitude responses decreased significantly after chewing gum containing both 2 and 4 mg of nicotine. Under light-adapted conditions, the peak b-wave amplitude was significantly decreased after chewing gum containing 4 mg of nicotine. In experiment 2, light-adapted b-wave amplitudes were increased after 4 mg nicotine. Oscillatory potentials were measured but no significant effects under nicotine were observed. CONCLUSIONS: To the knowledge of the authors, this is the first demonstration that nicotine by itself affects responses in the human retina. These data support reports of the expression of nicotinic acetylcholine receptors in rabbit and nonhuman primate retina.


Subject(s)
Dark Adaptation/physiology , Electroretinography/drug effects , Nicotine/administration & dosage , Retina/drug effects , Administration, Oral , Adult , Dark Adaptation/drug effects , Dose-Response Relationship, Drug , Female , Ganglionic Stimulants/administration & dosage , Humans , Male , Photic Stimulation , Reference Values , Retina/physiology , Smoking/adverse effects , Smoking/physiopathology , Young Adult
4.
Am J Physiol Renal Physiol ; 299(6): F1507-14, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20926633

ABSTRACT

Recently, the use of overexpression of telomerase reverse transcriptase (TERT) has led to the generation of immortalized human cell lines. However, this cell immortalization approach has not been reported in well-differentiated mouse cells, such as renal epithelial cells. We sought to establish and then characterize a mouse collecting duct cell line, using ectopic expression of mTERT. Isolated primary cortical collecting duct (CCD) cell lines were transduced with mouse (m)TERT, using a lentiviral vector. mTERT-negative cells did not survive blasticidin selection, whereas mTERT-immortalized cells proliferated in selection media for over 40 subpassages. mTERT messenger RNA and telomerase activity was elevated in these cells, compared with an SV40-immortalized cell line. Flow cytometry with Dolichos biflorus agglutinin was used to select the CCD principal cells, and we designated this cell line mTERT-CCD. Cells were well differentiated and exhibited morphological characteristics typically found in renal epithelial cells, such as tight junction formation, microvilli, and primary cilia. Further characterization using standard immunofluorescence revealed abundant expression of aquaporin-2 and the vasopressin type 2 receptor. mTERT-CCD cells exhibited cAMP-stimulated/benzamil-inhibited whole cell currents. Whole cell patch-clamp currents were also enhanced after a 6-day treatment with aldosterone. In conclusion, we have successfully used mTERT to immortalize mouse collecting duct cells that retain the basic in vivo phenotypic characteristics of collecting duct cells. This technique should be valuable in generating cell lines from genetically engineered mouse models.


Subject(s)
Cell Culture Techniques/methods , Cell Line , Kidney Tubules, Collecting/cytology , Telomerase/genetics , Animals , Kidney Tubules, Collecting/metabolism , Mice , Reverse Transcriptase Polymerase Chain Reaction
5.
Invest Ophthalmol Vis Sci ; 51(5): 2778-89, 2010 May.
Article in English | MEDLINE | ID: mdl-20042645

ABSTRACT

PURPOSE: The activation and blockade of muscarinic acetylcholine receptors (mAChRs) affects retinal ganglion cell light responses and firing rates. This study was undertaken to identify the full complement of mAChRs expressed in the rabbit retina and to assess mAChR distribution and the functional effects of mAChR activation and blockade on retinal response properties. METHODS: RT-PCR, Western blot analysis, and immunohistochemistry were used to identify the complement and distribution of mAChRs in the rabbit retina. Extracellular electrophysiology was used to determine the effects of the activation or blockade of mAChRs on ganglion cell response properties. RESULTS: RT-PCR of whole neural retina resulted in the amplification of mRNA transcripts for the m1 to m5 mAChR subtypes. Western blot and immunohistochemical analyses confirmed that all five mAChR subtypes were expressed by subpopulations of bipolar, amacrine, and ganglion cells in the rabbit retina, including subsets of cells in cholinergic and glycinergic circuits. Nonspecific muscarinic activation and blockade resulted in the class-specific modulation of maintained ganglion cell firing rates and light responses. CONCLUSIONS: The expression of mAChR subtypes on subsets of bipolar, amacrine, and ganglion cells provides a substrate for both enhancement and suppression of retinal responses via activation by cholinergic agents. Thus, the muscarinic cholinergic system in the retina may contribute to the modulation of complex stimuli. Understanding the distribution and function of mAChRs in the retina has the potential to provide important insights into the visual changes that are caused by decreased ACh in the retinas of Alzheimer's patients and the potential visual effects of anticholinergic treatments for ocular diseases.


Subject(s)
Receptors, Muscarinic/metabolism , Retina/metabolism , Retinal Ganglion Cells/physiology , Animals , Blotting, Western , Electrophysiology , Immunohistochemistry , RNA, Messenger/metabolism , Rabbits , Receptors, Muscarinic/genetics , Reverse Transcriptase Polymerase Chain Reaction
6.
Invest Ophthalmol Vis Sci ; 50(3): 1408-15, 2009 Mar.
Article in English | MEDLINE | ID: mdl-18952912

ABSTRACT

PURPOSE: The purpose of this study was to detect and establish the cellular localizations of nicotinic acetylcholine receptor (nAChR) subunits in Rhesus monkey retina. METHODS: Retinas were dissected from the eyes of monkeys killed after unrelated experiments. RNA was extracted and analyzed by RT-PCR, using primers designed against human sequences of alpha3-alpha7, alpha9, and beta2-beta4 nAChR subunits. The RT-PCR products were separated by gel electrophoresis and sequenced. Frozen sections of postmortem fixed monkey eyes were immunolabeled with well-characterized and specific monoclonal antibodies against the alpha3, alpha4, alpha6, alpha7, beta2, or beta4 nAChR subunits and visualized with fluorescence labeling. RESULTS: Products of the predicted size for the alpha3-alpha7, alpha9, and beta2-beta4 nAChR subunits were detected by RT-PCR in Rhesus monkey retina. Homology between transcripts from monkey retina and human nucleotide sequences ranged from 93 to 99%. Immunohistochemical studies demonstrated that neurons in various cell layers of monkey retina expressed alpha3, alpha4, alpha7, or beta2 nAChR subunits and cells with the morphology of microglia were immunoreactive for the alpha6 or beta4 nAChR subunits. CONCLUSIONS: nAChR subunits are expressed in the monkey retina and localize to diverse retinal neurons as well as putative microglia. Besides mediating visual processing, retinal nAChRs may influence refractive development and ocular pathologies such as neovascularization.


Subject(s)
Receptors, Nicotinic/metabolism , Retina/metabolism , Animals , DNA Primers/chemistry , Electrophoresis, Polyacrylamide Gel , Fluorescent Antibody Technique, Indirect , Macaca mulatta , Microscopy, Fluorescence , Protein Subunits/genetics , Protein Subunits/metabolism , RNA, Messenger/metabolism , Receptors, Nicotinic/genetics , Retinal Neurons/metabolism , Reverse Transcriptase Polymerase Chain Reaction
7.
J Comp Neurol ; 507(6): 1952-63, 2008 Apr 20.
Article in English | MEDLINE | ID: mdl-18273886

ABSTRACT

Although acetylcholine is one of the most widely studied neurotransmitters in the retina, many questions remain about its downstream signaling mechanisms. In this study we initially characterized the cholinergic neurotransmitter system in the salamander retina by localizing a variety of cholinergic markers. We then examined the link between both muscarinic and nicotinic receptor activation and nitric oxide production by using immunocytochemistry for cyclic guanosine monophosphate (cGMP) as an indicator. We found a large increase in cGMP-like immunoreactivity (cGMP-LI) in the inner retina in response to muscarinic (but not nicotinic) receptor activation. Based on the amplification of mRNA transcripts, receptor immunocytochemistry, and the use of selective antagonists, we identified these receptors as M2 muscarinic receptors. Using double-labeling techniques, we established that these increases in cGMP-LI were seen in GABAergic but not cholinergic amacrine cells, and that the increases were blocked by inhibitors of nitric oxide production. The creation of nitric oxide in response to cholinergic receptor activation may provide a mechanism for modulating the well-known mutual interactions of acetylcholine-glycine-GABA in the inner retina. As GABA and glycine are the primary inhibitory neurotransmitters in the retina, signaling pathways that modulate their levels or release will have major implications for the processing of complex stimuli by the retina.


Subject(s)
Acetylcholine/metabolism , Ambystoma/metabolism , Neurons/metabolism , Nitric Oxide/metabolism , Receptors, Cholinergic/metabolism , Retina/metabolism , Amacrine Cells/metabolism , Ambystoma/anatomy & histology , Animals , Cyclic GMP/metabolism , Enzyme Inhibitors/pharmacology , Glycine/metabolism , Immunohistochemistry , Neural Inhibition/physiology , Neurons/cytology , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/metabolism , Receptors, Cholinergic/genetics , Receptors, Muscarinic/genetics , Receptors, Muscarinic/metabolism , Receptors, Nicotinic/genetics , Receptors, Nicotinic/metabolism , Retina/cytology , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/physiology , Synaptic Transmission/physiology , Vision, Ocular/physiology , Visual Pathways/cytology , Visual Pathways/metabolism , gamma-Aminobutyric Acid/metabolism
8.
Vis Neurosci ; 24(4): 635-45, 2007.
Article in English | MEDLINE | ID: mdl-17900379

ABSTRACT

A GABA- or glycine-induced increase in Cl(-) permeability can produce either a depolarization or hyperpolarization, depending on the Cl(-) equilibrium potential. It has been shown that retinal neurons express the chloride cotransporters, Na-K-2Cl (NKCC) and K-Cl (KCC), the primary molecular mechanisms that control the intracellular Cl(-) concentration. We thus studied (1) the localization of these cotransporters in the fish retina, and (2) how suppression of cotransporter activity in the fish retina affects function. Specific antibodies against NKCC and KCC2 revealed that both cotransporters were expressed in the outer and inner plexiform layers, and colocalized in many putative amacrine cells and in cells of the ganglion cell layer. However, the somata of putative horizontal cells displayed only NKCC immunoreactivity and many bipolar cells were only immunopositive for KCC2. In the outer retina, application of bumetanide, a specific inhibitor of NKCC activity, (1) increased the steady-state extracellular concentration of K+ ([K+](o)) and enhanced the light-induced decrease in the [K+](o), (2) increased the sPIII photoreceptor-dependent component of the ERG, and (3) reduced the extracellular space volume. In contrast, in the outer retina, application of furosemide, a specific inhibitor of KCC activity, decreased sPIII and the light-induced reduction in [K+](o), but had little effect on steady-state [K+](o). In the inner retina, bumetanide increased the sustained component of the light-induced increase in [K+](o). These findings thus indicate that NKCC and KCC2 control the [K+](o) and extracellular space volume in the retina in addition to regulating GABA- and glycine-mediated synaptic transmission. In addition, the anatomical and electrophysiological results together suggest that all of the major neuronal types in the fish retina are influenced by chloride cotransporter activity.


Subject(s)
Goldfish/physiology , Retina/physiology , Sodium-Potassium-Chloride Symporters/physiology , Algorithms , Animals , Data Interpretation, Statistical , Electrophysiology , Electroretinography , Extracellular Space/physiology , Immunohistochemistry , Photic Stimulation , Photoreceptor Cells, Vertebrate/drug effects , Photoreceptor Cells, Vertebrate/physiology , Quaternary Ammonium Compounds/pharmacology , Sodium-Potassium-Chloride Symporters/genetics
9.
Vis Neurosci ; 24(4): 523-33, 2007.
Article in English | MEDLINE | ID: mdl-17686198

ABSTRACT

Acetylcholine (ACh) enhances the preferred direction responses of directionally selective ganglion cells (DS GCs; Ariel & Daw, 1982; Ariel & Adolph, 1985) through the activation of nicotinic acetylcholine receptors (nAChRs; Ariel & Daw, 1982; Massey et al., 1997; Kittila & Massey, 1997). DS GCs appear to express at least two types of nAChRs, those that are sensitive to the partially subtype-specific antagonist methyllycaconitine (MLA), and those that are MLA-insensitive (Reed et al., 2002). Our purpose was to confirm the expression of alpha7 nAChRs by DS GCs and to assess the contributions of other nAChR subtypes to DS GC responses. Using choline as a nAChR partially subtype-specific agonist, we found that the majority of DS GCs demonstrated responses to choline while under synaptic blockade. The blockade or reduction of choline-induced responses by bath application of nanomolar (nM) concentrations of MLA provided direct evidence that the choline responses were mediated by alpha7 nAChRs. Because choline is a partial agonist for alpha3beta4 nAChRs (Alkondon et al., 1997), the residual choline responses are consistent with mediation by alpha3beta4 nAChRs. Additionally, a subset of DS GCs responded to nicotine but not to choline, indicating the expression of a third nAChR subtype. The pharmacological results were supported by single cell reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry experiments. The expression of alpha7 and specific non-alpha7 nAChR subtypes was correlated with the preferred direction. This indicates the possibility of differential responses to ACh depending on the direction of movement. This is the first description of differential expression of multiple nAChR subtypes by DS GCs.


Subject(s)
Receptors, Nicotinic/biosynthesis , Retinal Ganglion Cells/metabolism , Animals , Choline/pharmacology , Cobalt/pharmacology , Electrophysiology , Immunohistochemistry , Microelectrodes , Microscopy, Confocal , Nicotine/pharmacology , Nicotinic Agonists/pharmacology , Nootropic Agents/pharmacology , Photic Stimulation , Rabbits , Receptors, Nicotinic/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transcription, Genetic
10.
Invest Ophthalmol Vis Sci ; 48(4): 1504-10, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17389478

ABSTRACT

PURPOSE: To determine the expression patterns of alpha(1)- and beta(1)-adrenergic receptors in the mouse exorbital lacrimal gland (LG). An alpha- and beta-receptor agonist and antagonist were used to elucidate the receptors' relevance to protein secretion. METHODS: Mouse LGs were processed for single- and double-labeled indirect immunofluorescence studies and examined with confocal scanning microscopy. Protein secretion was measured from gland fragments in response to adrenergic agonists. RESULTS: Extensive alpha(1)-immunoreactivity (IR) was found on the surface and cytoplasm of acinar cells and much more alpha(1)-IR in the interstitial areas. In contrast, more beta(1)-IR was found in the LG, and most beta(1)-IR appeared to concentrate in the cytoplasm of acinar cells, with almost no beta(1)-IR in the interstitial areas. The protein secretion in response to phenylephrine and isoproterenol showed that direct stimulation of either the alpha(1)- or beta(1)-receptor could induce significant protein secretion from LGs. The specificity of this stimulation was further indicated by the effects of adrenergic antagonists. No synergism was observed between alpha(1)- and beta-receptor-mediated protein secretions. CONCLUSIONS: The results support the notion that there is extensive adrenergic control in the mouse LG. The adrenergic receptors may be a better choice of markers, compared with tyrosine hydroxylase and dopamine beta-hydroxylase, to reflect the extent of adrenergic control because circulating norepinephrine in the bloodstream should be taken into consideration. Both confocal microscopy observations and protein secretion data suggest the presence of alpha(1)- and beta(1)-mediated pathways in the mouse LG.


Subject(s)
Lacrimal Apparatus/physiology , Receptors, Adrenergic, alpha-1/metabolism , Receptors, Adrenergic, beta-1/metabolism , Adrenergic alpha-Agonists/pharmacology , Adrenergic alpha-Antagonists/pharmacology , Adrenergic beta-Agonists/pharmacology , Adrenergic beta-Antagonists/pharmacology , Animals , Dose-Response Relationship, Drug , Eye Proteins/metabolism , Female , Fluorescent Antibody Technique, Indirect , Lacrimal Apparatus/drug effects , Mice , Mice, Inbred C57BL , Microscopy, Confocal
11.
J Histochem Cytochem ; 55(5): 461-76, 2007 May.
Article in English | MEDLINE | ID: mdl-17189521

ABSTRACT

Cholinergic agents affect the light responses of many ganglion cells (GCs) in the mammalian retina by activating nicotinic acetylcholine receptors (nAChRs). Whereas retinal neurons that express beta2 subunit-containing nAChRs have been characterized in the rabbit retina, expression patterns of other nAChR subtypes remain unclear. Therefore, we evaluated the expression of alpha7 nAChRs in retinal neurons by means of single-, double-, and triple-label immunohistochemistry. Our data demonstrate that, in the rabbit retina, several types of bipolar cells, amacrine cells, and cells in the GC layer express alpha7 nAChRs. At least three different populations of cone bipolar cells exhibited alpha7 labeling, whereas glycine-immunoreactive amacrine cells comprised the majority of alpha7-positive amacrine cells. Some GABAergic amacrine cells also displayed alpha7 immunoreactivity; alpha7 labeling was never detected in rod bipolar cells or rod amacrine cells (AII amacrine cells). Our data suggest that activation of alpha7 nAChRs by acetylcholine (ACh) or choline may affect glutamate release from several types of cone bipolar cells, modulating GC responses. ACh-induced excitation of inhibitory amacrine cells might cause either inhibition or disinhibition of other amacrine and GC circuits. Finally, ACh may act on alpha7 nAChRs expressed by GCs themselves.


Subject(s)
Amacrine Cells/metabolism , Receptors, Nicotinic/biosynthesis , Retinal Bipolar Cells/metabolism , Retinal Ganglion Cells/metabolism , Animals , Bungarotoxins/metabolism , Immunohistochemistry , Rabbits , Subcellular Fractions/metabolism , alpha7 Nicotinic Acetylcholine Receptor
12.
Vis Neurosci ; 22(2): 143-51, 2005.
Article in English | MEDLINE | ID: mdl-15935107

ABSTRACT

Retinal neurons and Muller cells express amiloride-sensitive Na+ channels (ASSCs). Although all major subunits of these channels are expressed, their physiological role is relatively unknown in this system. In the present study, we used the electroretinogram (ERG) recorded from anesthetized rabbits and isolated rat and rabbit retina preparations to investigate the physiological significance of ASSCs in the retina. Based upon our previous study showing expression of alpha-ENaC and functional amiloride-sensitive currents in rabbit Muller cells, we expected changes in Muller cell components of the ERG. However, we observed changes in other components of the ERG as well. The presence of amiloride elicited changes in all major components of the ERG; the a-wave, b-wave, and d-wave (off response) were enhanced, while there was a reduction in the amplitude of the Muller cell response (slow PIII). These results suggest that ASSCs play an important role in retinal function including neuronal and Muller cell physiology.


Subject(s)
Amiloride/pharmacology , Retina/physiology , Sodium Channel Blockers/pharmacology , Sodium Channels/physiology , Animals , Dark Adaptation , Electroretinography/drug effects , Epithelial Sodium Channels , Fluorescent Antibody Technique, Indirect , Neurons/physiology , Rabbits , Rats , Reverse Transcriptase Polymerase Chain Reaction , Sodium Channels/drug effects
13.
Am J Physiol Cell Physiol ; 289(3): C644-55, 2005 Sep.
Article in English | MEDLINE | ID: mdl-15872006

ABSTRACT

It is well known that cholinergic agents affect ganglion cell (GC) firing rates and light responses in the retinas of many species, but the specific receptor subtypes involved in mediating these effects have been only partially characterized. We sought to determine whether functional alpha(7) nicotinic acetylcholine receptors (nAChRs) contribute to the responses of specific retinal GC classes in rabbit retina. We used electrophysiology, pharmacology, immunohistochemistry, and reverse transcriptase-polymerase chain reaction to determine the pharmacological properties and expression of nAChR subtypes by specific rabbit retinal GC classes. Choline was used as an alpha(7) nAChR agonist. Methyllycaconitine (MLA) was used as a competitive alpha(7) nAChR antagonist. The application of choline before synaptic blockade resulted in changes in retinal GC activity, including increases or decreases in maintained firing and/or enhancement or suppression of light responses. Many physiologically identified GC types, including sustained off, sustained on, transient off, and transient on cells, demonstrated responses to choline application while under synaptic blockade. The choline-induced responses could be blocked with MLA, confirming alpha(7) nAChR activation. Individual choline-responsive GCs displayed mRNA transcripts consistent with the expression of functional alpha(7) nAChRs. Other GCs demonstrated physiological responses and mRNA expression consistent with the expression of both alpha(7) and non-alpha(7) nAChRs. Thus mRNA is present for multiple nAChR subunits in whole retina extracts, and functional alpha(7) nAChRs are capable of modulating the responses of GCs in adult rabbit retina. We also demonstrate through physiological responses that subsets of GCs express more than one nAChR subtype.


Subject(s)
Receptors, Nicotinic/genetics , Receptors, Nicotinic/physiology , Retinal Ganglion Cells/physiology , Animals , Choline/pharmacology , Electrophysiology , Evoked Potentials/drug effects , Evoked Potentials/physiology , Ganglionic Blockers/pharmacology , Ganglionic Stimulants/pharmacology , Hexamethonium/pharmacology , Nicotine/pharmacology , Nootropic Agents/pharmacology , RNA, Messenger/analysis , Rabbits , Reverse Transcriptase Polymerase Chain Reaction , alpha7 Nicotinic Acetylcholine Receptor
14.
Proc Natl Acad Sci U S A ; 100(26): 16047-52, 2003 Dec 23.
Article in English | MEDLINE | ID: mdl-14665697

ABSTRACT

The ability of directionally selective (DS) retinal ganglion cells to respond selectively to stimulus motion in one direction is a classic unresolved example of computation in a local neural circuit. Recent evidence indicates that DS responses occur first in the retina in the dendrites of starburst amacrine cells (interneurons presynaptic to the ganglion cells). We report that the directional responses of starburst-cell dendrites and DS ganglion cells are highly sensitive to the polarity of the transmembrane chloride gradient. Reducing the transmembrane chloride gradient by ion substitution or by blocking the K-Cl cotransporter resulted in the starburst cells responding equally to light moving in opposite directions. Conversely, increasing the chloride gradient by blocking the Na-K-Cl cotransporter eliminated responses to light moving in either direction. Moreover, in each case, blocking the chloride cotransporters or reducing the transmembrane chloride gradient eliminated the directional responses of DS ganglion cells in a manner opposite that of the starburst cells. These results indicate that chloride cotransporters play a key role in the generation of direction selectivity and that the directional responses of starburst cells and DS ganglion cells are exquisitely sensitive to the chloride equilibrium potential. The findings further suggest that the directional responses of DS ganglion cells are mediated in part by the directional release of gamma-aminobutyric acid from starburst dendrites and that the asymmetric distribution of the two cotransporters along starburst-cell dendrites mediates direction selectivity. A model of direction selectivity in the retina that incorporates these and other findings is discussed.


Subject(s)
Retinal Ganglion Cells/physiology , Symporters/physiology , Animals , Cations/metabolism , Chlorides/metabolism , Dendrites/physiology , Dendrites/radiation effects , Light , Rabbits , Receptors, GABA/physiology , Retina/physiology , Retinal Ganglion Cells/radiation effects , gamma-Aminobutyric Acid/physiology
15.
Vis Neurosci ; 20(2): 165-76, 2003.
Article in English | MEDLINE | ID: mdl-12916738

ABSTRACT

Acetylcholine is one of the primary excitatory neurotransmitters/neuromodulators in the retina, but little is known about the downstream signaling pathways it can activate. The present study immunocytochemically examines the potential sources of acetylcholine and the location of the nicotinic cholinergic receptors in the turtle retina. It also examines how activation of these receptors can influence the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signal-transduction pathways. Photoreceptors, amacrine cells, and potentially ganglion cells contain choline acetyltransferase-like immunoreactivity (LI). Nicotinic acetylcholine receptors are immunocytochemically localized on photoreceptors, horizontal, bipolar, and ganglion cells. Nitric oxide imaging indicates that stimulation with nicotine increases NO production primarily in photoreceptors, horizontal, Muller, bipolar, and ganglion cells. In turn, very select populations of amacrine cells respond to this NO with increased levels of cGMP-LI. Selective inhibitors reveal that nitric oxide synthase is involved in most, but not all, of these increases in cGMP-LI. These results show that acetylcholine can activate the NO/cGMP signal-transduction pathways in both the inner and outer retina. This indicates that both of the major excitatory retinal transmitters, glutamate and acetylcholine, can stimulate NO production that increases levels of cGMP-LI in overlapping populations of retinal cells.


Subject(s)
Cyclic GMP/metabolism , Nicotine/pharmacology , Nitric Oxide/metabolism , Retina/metabolism , Signal Transduction/drug effects , Animals , Choline O-Acetyltransferase/metabolism , Enzyme-Linked Immunosorbent Assay , Fluorescein , Immunohistochemistry , In Vitro Techniques , Indicators and Reagents , Nitric Oxide Synthase/metabolism , Receptors, Nicotinic/metabolism , Retina/drug effects , Turtles
16.
J Comp Neurol ; 461(1): 76-90, 2003 Jun 16.
Article in English | MEDLINE | ID: mdl-12722106

ABSTRACT

Starburst amacrine cells in the macaque retina were studied by electron microscopic immunohistochemistry. We found that these amacrine cells make a type of synapse not described previously; they are presynaptic to axon terminals of bipolar cells. We also confirmed that starburst amacrine cells are presynaptic to ganglion cell dendrites and amacrine cell processes. In order to determine the functions of these synapses, we localized acetylcholine receptors using a monoclonal antibody (mAb210) that recognizes human alpha3- and alpha5-containing nicotinic receptors and also antisera against the five known subtypes of muscarinic receptors. The majority of the mAb210-immunoreactive perikarya were amacrine cells and ganglion cells, but a subpopulation of bipolar cells was also labeled. A subset of bipolar cells and a subset of horizontal cells were labeled with antibodies to M3 muscarinic receptors. A subset of amacrine cells, including those that contain cholecystokinin, were labeled with antibodies to M2 receptors. Taken together, these results suggest that acetylcholine can modulate the activity of retinal ganglion cells by multiple pathways.


Subject(s)
Amacrine Cells/ultrastructure , Macaca mulatta/physiology , Receptors, Cholinergic/metabolism , Retina/ultrastructure , Synapses/ultrastructure , Acetylcholine/metabolism , Amacrine Cells/metabolism , Animals , Eye/innervation , Immunohistochemistry , Interneurons/physiology , Microscopy, Electron , Retina/metabolism , Retinal Ganglion Cells/physiology , Synapses/metabolism , Synaptic Transmission/physiology , Tissue Distribution , gamma-Aminobutyric Acid/metabolism
17.
Invest Ophthalmol Vis Sci ; 44(4): 1513-20, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12657587

ABSTRACT

PURPOSE: To explore the sympathetic innervation pattern and the role of sympathetic nervous system control of protein secretion in the exorbital lacrimal glands of normal mice. METHODS: Mouse lacrimal glands were processed for single- and double-label indirect immunofluorescence studies to show their innervation patterns. The sucrose-potassium phosphate-glyoxylic acid method was also used to visualize the adrenergic innervation. The effects of adrenergic and cholinergic agonists on protein secretion were evaluated. RESULTS: The mouse lacrimal gland can be divided into two different areas based on the innervation density and distribution pattern. One area, approximately 10% to 30% of the gland, exhibited much higher innervation density, both parasympathetic and sympathetic, than the rest of the gland. The adrenergic agonists norepinephrine and phenylephrine induced increases in protein secretion that were of a magnitude similar to the increases induced by the cholinergic agonist carbachol at 10(-6) to 10(-4) M. Isoproterenol, the beta-adrenergic agonist, also elicited protein secretion at 10(-5) to 10(-4) M. CONCLUSIONS: The data indicate that there is extensive sympathetic innervation of the mouse lacrimal gland and that sympathetic input can modulate protein secretion. The division of the lacrimal gland into two areas suggests that the mouse lacrimal gland is a mixed gland and that these two areas may play different roles in secreting tears of different compositions in various situations. These data appear to support the notion that differential secretion is accomplished by activating different populations of secretory cells that are differentially innervated.


Subject(s)
Lacrimal Apparatus/innervation , Parasympathetic Nervous System/anatomy & histology , Sympathetic Nervous System/anatomy & histology , Adrenergic Agonists/pharmacology , Animals , Cholinergic Agonists/pharmacology , Eye Proteins/metabolism , Female , Fluorescent Antibody Technique, Indirect , Lacrimal Apparatus/drug effects , Lacrimal Apparatus/metabolism , Mice , Mice, Inbred C57BL , Parasympathetic Nervous System/physiology , Sympathetic Nervous System/physiology
18.
J Comp Neurol ; 456(2): 167-75, 2003 Feb 03.
Article in English | MEDLINE | ID: mdl-12509873

ABSTRACT

The light-evoked release of acetylcholine (ACh) affects the responses of many retinal ganglion cells, in part via nicotinic acetylcholine receptors (nAChRs). nAChRs that contain beta2alpha3 neuronal nicotinic acetylcholine receptors have been identified and localized in the rabbit retina; these nAChRs are recognized by the monoclonal antibody mAb210. We have examined the expression of beta2alpha3 nAChRs by glycinergic amacrine cells in the rabbit retina and have identified different subpopulations of nicotinic cholinoceptive glycinergic cells using double and triple immunohistochemistry with quantitative analysis. Here we demonstrate that about 70% of the cholinoceptive amacrine cells in rabbit retina are glycinergic cells. At least three nonoverlapping subpopulations of mAb210 glycine-immunoreactive cells can be distinguished with antibodies against calretinin, calbindin, and gamma-aminobutyric acid (GABA)(A) receptors. The cholinergic cells in rabbit retina are thought to synapse only on other cholinergic cells and ganglion cells. Thus, the expression of beta2alpha3 nAChRs on diverse populations of glycinergic cells is puzzling. To explore this finding, the subcellular localization of beta2alpha3 was studied at the electron microscopic level. mAb210 immunoreactivity was localized on the dendrites of amacrines and ganglion cells throughout the inner plexiform layer, and much of the labeling was not associated with recognizable synapses. Thus, our findings indicate that ACh in the mammalian retina may modulate glycinergic circuits via extrasynaptic beta2alpha3 nAChRs.


Subject(s)
Neurons/cytology , Receptors, Nicotinic/biosynthesis , Retina/cytology , Amino Acid Transport Systems, Neutral/biosynthesis , Animals , Biomarkers/analysis , Choline O-Acetyltransferase/biosynthesis , Glycine/biosynthesis , Glycine Plasma Membrane Transport Proteins , Immunohistochemistry , Microscopy, Immunoelectron , Neurons/metabolism , Neurons/ultrastructure , Rabbits , Retina/metabolism , Retina/ultrastructure , gamma-Aminobutyric Acid/biosynthesis
19.
Vis Neurosci ; 20(6): 651-62, 2003.
Article in English | MEDLINE | ID: mdl-15088718

ABSTRACT

Acetylcholine (ACh) affects the response properties of many retinal ganglion cells (GCs) through the activation of nicotinic acetylcholine receptors (nAChRs). To date there have been few studies directly correlating the expression of specific nAChR subtypes with the physiological and morphological characteristics of specific retinal GCs. This study was designed to correlate responses to nicotine application with immunohistochemical evidence of nAChR expression in physiologically and morphologically identified ganglion cells. Extracellular recordings were used to physiologically identify rabbit retinal GCs, based on responses to light stimulation. Cells were then tested for responses to nicotine application and/or for expression of nAChRs, as judged by immunoreactivity to mAb210, an nAChR antibody. The morphologies of many physiologically identified cells were also determined by dye injection. More than three-fourths of ganglion cells tested responded to nicotine application under cobalt-induced synaptic blockade. The nicotine sensitivity was consistent with nAChR immunoreactivity and was also correlated with specific morphological subgroups of GCs. Overall, approximately two-thirds of all physiologically identified GCs that were processed for immunohistochemistry displayed immunoreactivity. In total, 18 of 22 physiologically identified cells demonstrated both sensitivity to nicotine application under synaptic blockade and mAb210 immunoreactivity (mAb210-IR). Thus, mAb210-IR is likely to represent functional nAChRs that can modulate retinal information processing and visual functioning via direct excitation of a number of GC classes.


Subject(s)
Nicotine/pharmacology , Nicotinic Agonists/pharmacology , Receptors, Nicotinic/metabolism , Retinal Ganglion Cells/drug effects , Animals , Dark Adaptation , Electrophysiology , Fluorescent Antibody Technique, Indirect , Microscopy, Confocal , Rabbits , Retinal Ganglion Cells/metabolism
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