1Department of Vision Sciences and 2Department of Psychology, University of Alabama at Birmingham, Birmingham, Alabama
Submitted 22 December 2004 ; accepted in final form 26 April 2005
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ABSTRACT |
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cholinergic receptors; choline; neurotransmitters
In the mammalian retina, the release of acetylcholine (ACh) by starburst amacrine cells (SACs) (49) can activate nicotinic or muscarinic acetylcholine receptors (AChRs) that can affect the response properties of many retinal ganglion cells (GCs) (14, 30, 44, 47, 48, 59). Recently, a novel form of choline aceteyltransferase (ChAT) that is expressed by GCs, designated as peripheral ChAT (pChAT), has been described in mammalian retina (70). Because pChAT can be isolated only when axonal transport is blocked before enucleation, it is not clear whether pChAT is involved only in the synthesis of ACh for release at retinorecipient sites or for release in the retina proper. While ACh release that is directly instrumental in retinal function is thought to be released only from the SACs, it is possible that subsets of GCs also release ACh.
While ACh is known to gate a cation channel in dissociated GCs (44, 34), the effects of ACh are more complex in the intact retina. For example, ACh enhances the electroretinogram b-wave, an effect that is reduced by both nicotinic and muscarinic antagonists (33). Nicotine application increases intracellular free Ca2+ in most GCs (14), but ACh has also been reported to either enhance (59) or suppress GC light responses and maintained firing rates (30). ACh also contributes to the excitatory response of directionally selective (DS) GCs to a bar of light moving in the preferred direction (7, 38), is necessary for the excitatory response to a grating moving in the preferred direction (23), and contributes to responses to complex moving stimuli (57). Interestingly, when concentric GCs are stimulated in both the receptive field center and in the surround, the acetylcholinesterase (AChE) inhibitor physostygmine causes an enhanced center response, suggesting that ACh may be involved in modulation of center-surround response or GC excitability (59).
Many GC types respond to nicotine application during synaptic blockade, demonstrating that GCs directly express nAChRs (47, 56, 61). In addition, nicotinic antagonists decrease the effects of ACh and AChE and block cholinergic agonist-induced spiking of synaptically isolated GCs (29, 45, 47).
Heteromeric nAChR subunits have been localized using immunohistochemistry (15, 35, 66, 67, 69) and in situ hybridization (16, 17, 26) in the retinas of many species. The heteromeric nAChRs in rabbit retina include 2-containing nAChRs identified using immunohistochemistry and biochemistry (35). Although immunohistochemical evidence of the expression of
2-containing nAChRs (61) has been correlated with nAChR function, this correlation does not rule out a role for other nAChR subtypes composed of as yet unidentified subunit combinations.
Previous ligand-binding experiments (28, 55) demonstrated -bungarotoxin (
-Bgt) binding in the inner retina, presumably indicating
7 homomers. However, the presence of functional
7 nAChRs in the retinas of adult mammals has been controversial (e.g., Ref. 14). More recently, Wehrwein et al. (68) demonstrated that
7 nAChRs mediate protection from glutamate excitotoxicity in porcine retina. In addition, Reed et al. (56) have shown, through sensitivity to the partially subtype-specific antagonist methyllycaconitine (MLA), that
7-like nAChRs contribute to the response properties of DS GCs in rabbit retina. However, there has been no definitive demonstration of functional
7 nAChRs in retinal neurons.
We hypothesized that the MLA-sensitive responses described by Reed et al. (56) are mediated by 7 nAChRs and that these receptors are expressed by multiple GC types. Because there is evidence that
2-containing nAChRs are also widely expressed in rabbit retina (35, 61), we hypothesized that subsets of GCs express multiple nAChR subtypes. Interestingly,
7 nAChR activation has been shown to have cytoprotective effects in a variety of experimental assays (12, 31, 32, 37), including the previously mentioned model of glutamate excitotoxicity in porcine retinal ganglion cells (68). Thus the expression of functional
7 nAChRs by GCs could have clinical relevance for the exploration of alternative methods for preventing retinal injury caused by excitotoxicity or growth factor deprivation.
We present evidence below for the expression of mRNA transcripts for several nAChR subunits in whole rabbit retina, as well as direct evidence that functional 7 nAChRs are expressed by physiologically identified GC types, and that some GCs express more than one nAChR subtype. We also provide evidence that that
7 nAChRs are capable of modulating the response properties of individual retinal GCs. Portions of these results have been published previously in abstract form (62, 63).
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MATERIALS AND METHODS |
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RT-PCR
Primer design.
Primers were designed for 3,
4,
6,
7,
2,
3, and
4 nAChR subunit mRNA. Because rabbit sequences were not available, sequences available in the public domain for other species were obtained from GenBank (National Center for Biotechnology Information, Bethesda, MD; http://www.ncbi.nlm.nih.gov/). The sequences were aligned, and subunit-specific primers were chosen in regions of high cross-species homology using Primer Premier (version 5.0; Premier Biosoft, Palo Alto, CA). Some primers were adapted from those used by Léna et al. (41). Primers were obtained from Cruachem (Omaha, NE), Invitrogen (Carlsbad, CA), or Sigma Genosys (The Woodlands, TX). The primers used for amplification of mRNA extracted from whole retina are shown in Table 1.
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Rabbit-specific primers were designed from the rabbit sequences obtained for 3 and
7 primer pairs. Sequencing of the subsequent products verified the identity of the RT-PCR product created from each rabbit-specific primer pair as
3 or
7 nAChR subunits. All primers were located within the coding region of the mRNA, and the rabbit-specific primer pairs spanned at least two exons (Table 1).
RNA isolation and amplification. Intact retinas were dissected into RNAlater (Ambion, Austin, TX) immediately after enucleation. RNA was extracted using the Absolutely RNA RT-PCR MiniPrep kit (Stratagene, La Jolla, CA). Contaminating DNA bound to the fiber matrix was removed by DNase digestion and followed by a series of low- and high-salt buffer rinses.
The single-cell multiplex RT-PCR protocol was adapted from that of Léna et al. (41) and Klink et al. (39). After physiological characterization, physiologically identified cells were harvested from the retina via clean glass capillaries with tip diameters of 510 µM. Figure 1 shows the harvesting process. A clean micropipette was lowered next to the cell, after which the cell was pulled into the tip using the "fill" setting of a picoinjector (Harvard Apparatus, Hellstrom, MA). The tip of the capillary was broken into 5 µl of RNAlater (Ambion) and stored at 4°C in RNAse-free tubes.
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Reverse transcription occurred at 50°C for 30 min, followed by one cycle at 95°C for 15 min to inactivate the reverse transcriptase and activate the Taq polymerase. Denaturing, annealing, and extension consisted of 3540 cycles at 94°C for 1 min, 5260°C for 1.5 min, and 72°C for 2 min, respectively. Final extension took place at 72°C for 20 min. For second-round PCR amplification of single-cell products, 12 µl of first-round product was used as the template, and the reverse transcription cycle was omitted. Gel electrophoresis was used to separate RT-PCR and PCR products on 2% agarose gels. Ethidium bromide was used to visualize product bands. Appropriately sized products were gel purified using a QIAquick PCR purification kit (Qiagen, Valencia, CA) and sequenced (Center for AIDS Research, University of Alabama at Birmingham, Birmingham, AL). The sequences from the DNA extraction were aligned with one another using Clustal W (European Bioinformatics Institute, Cambridge, UK; http://www.ebi.ac.uk/clustalw/) and compared with known cDNA sequences through GenBank by performing NCBI BLAST query.
Electrophysiology
Extracellular recordings were made under visual control (x40 water-immersion lens objective under dim red light) with carbon fiber in glass microelectrodes. Cells recorded from the inferior retina were located 14 mm below the myelinated fiber band. Recordings were also made for some cells in the superior retina. Signals were amplified through a differential alternating current amplifier (A-M Systems, Carlsborg, WA) with the band pass set between 300 Hz and 10 kHz. Spikes passing a Schmidt trigger threshold were digitized for later offline analysis of spike frequency. Cell groups were divided into four broad categories on the basis of previously published physiological criteria (9, 10). Briefly, the receptive field for each cell was mapped, and the light response was characterized as either on or off on the basis of the response to an optimally sized spot that produced the largest centering response. Sustained vs. transient light responses were characterized on the basis of maintained firing at >25% of the peak firing rate for at least 150 ms (9).
Pharmacology
In some experiments, agonists and/or antagonists were applied before the application of cobalt to test the effects on maintained firing rates. Agonists and/or antagonists were also applied before or concurrently with light stimuli to test the effect on light responses. The effects were defined as enhancement if spiking increased during agonist application or if the agonist/antagonist-induced spiking was additive with light-induced spiking. The effects were defined as suppression if the spiking decreased or the light response was decreased or blocked during agonist/antagonist application.
Agonists were dissolved in distilled (DI) H2O and focally applied using Picoinjector (Harvard Apparatus, Hellstrom, MA) via glass capillaries with tip diameters of 510 µM. Separate pipettes were loaded for each agonist concentration and changed between tests. In some cases, Azure B dye was included in the pipette to allow for visual confirmation of agonist application at and around the soma of the recorded cell. Negative controls included focal application of DI H2O and DI H2O containing Azure B to preclude the possibility that any effects might result solely from pressure on or movement of the cell.
Nicotine was used as a nonspecific agonist (42) and choline was used as an agonist for 7 nAChRs (51). We used concentrations of 24 µM nicotine because this concentration range activates
75% of synaptically isolated GCs in rabbit retina (61). Choline concentrations ranging from 200 µM-400 mM were tested. We initially tested cells using high choline concentrations. When consistent responses were confirmed at the higher concentrations, we then explored the relationship between dose and response in a subset of cells. The lowest consistently effective choline concentration (400 µM) was determined, and later experiments used low concentrations of choline almost exclusively. At low agonist concentrations, partial occupancy of the
7 nAChR binding sites is thought to occur, resulting in prolonged activation of the receptor without desensitization (65). Thus the hydrolysis of ACh by AChE can create a second agonist (4). Higher concentrations of choline can act as a partial agonist for
3
4 nAChRs (4, 51, 52), inhibit the ACh response of
4
4 nAChRs (71), or inhibit the binding of muscarinic AChR antagonists (19).
Antagonists were applied by bath application via gravity feed into the superfusate line. In addition to the general nAChR antagonist hexamethonium bromide (HMB), MLA, which is an alkaloid toxin from the Delphinium brownii, was used as a subunit specific nAChR antagonist. MLA acts as a competitive antagonist for 7 nAChRs with 100- to 1,000-fold higher affinity for
7 than for
3
2 or
4
2 nAChRs (6), but MLA also has been found to inhibit
6-containing nAChRs (39, 50). Thus a positive identification of
7 nAChRs was made only in cases in which nanomolar concentrations of MLA blocked activation by choline. In some cases, atropine was bath applied to assess whether activation of muscarinic AChRs contributed to the choline response. Effective concentrations of agonists and antagonists are summarized in Table 2.
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Statistical Analysis
The Wilcoxon matched-pairs signed-rank test (27) was used to determine the significance of differences in cell responses between experimental conditions. The Bonferroni correction was used to adjust the significance level for multiple comparisons. There were two families of statistical comparisons. The first included comparisons designed to test the effects of agonist on maintained firing and light responses before synaptic blockade (see Pharmacology). The a priori comparisons for the first family were 1) spontaneous rate before and after drug application and 2) light response before and after drug application in Ames medium without cobalt. The P value required for significance of the first family of comparisons was adjusted to <0.025 per comparison. The second family of comparisons was designed to test the effects of agonist and antagonist after synaptic isolation. The a priori comparisons for the second family included the effects of the following: 1) agonist application in Ames medium containing cobalt (synaptic isolation) compared with baseline under synaptic isolation, 2) agonist application under synaptic isolation against the effects of agonist application with antagonist included in the bath, and 3) agonist/antagonist application under synaptic blockade against the baseline recorded in Ames containing cobalt and antagonist. The P value required for significance of the second family of comparisons was adjusted to <0.016 per comparison. For the reporting of results, cells with similar light responses and responses to pharmacological agents were grouped, and the lowest significant P value obtained from cells in that group was reported.
Immunohistochemistry
After injection, retinas were fixed in 2% paraformaldehyde and cryoprotected in 30% sucrose solution. Whole retinas were processed for immunohistochemistry as previously described (61) using MAb210, a rat monoclonal antibody that recognizes 1,
3, and
5 nAChRs. MAb210 was generously provided by Dr. Jon Lindstrom. In the rabbit retina, most nAChRs recognized by MAb210 are coassembled with
2-nAChR subunits (35).
Imaging
Images were collected with a Leica TCS four-dimensional confocal microscope (Leica Microsystems, Mannheim, Germany) equipped with argon, krypton, helium-neon, and UV lasers as previously described (61). Images were typically collected with a x40 oil-immersion lens objective and a numerical aperture of 1.25. The magnification allowed for imaging of the entire cell and its dendritic field while allowing for a relatively detailed look at the dendrites. The number of optical sections and the step sizes were selected on the basis of the thickness of the tissue at the location of the cell in the retina, the size of the cell, and how completely the cell dendrites were injected. Step sizes ranged from 0.3 to 0.4 µM. Maximum projection images, image quantification, and cytofluorochrome (CF) masking were performed using Leica software.
The intensity of immunofluorescence within and between flat sections of retina depends on the level of the section, as well as on the relative expression and amount of dendritic labeling present in the background. Because of this variability, a standardized procedure for determining whether a cell is immunoreactive was used as previously described (61). Briefly, CF masking was used to define a standard for positive nAChR immunoreactivity (IR) for an injected cell soma and/or dendrites. Using Leica software, we generated a cytofluorogram for both channels (red and green) of the maximum projection images of the injected cell and the antibody labeling. The cytofluorogram represented the frequency distribution of each pixel color and intensity for all of the optical sections obtained for a given cell. Red pixel intensities ranged along the y-axis, and green pixel intensities ranged along the x-axis. Pixels that represented simultaneous fluorescence from both channels (yellow) fell along the range between the x- and y-axes of the frequency distribution. A standardized region of interest (ROI) was designated such that yellow pixel intensities were included in the ROI, while red and green pixels were excluded. The same ROI was used for each image stack obtained. Pixels that fell within the ROI were defined as areas of colocalization and were masked onto overlay images of single optical sections in white. The same image stacks that were used to create the Z stack images also were used for the masked single optical sections. Because masking was assessed for only single optical sections, the masked pixels that represented colocalization and the dendrites were within the same 400500 nM optical plane.
Image brightness and contrast were minimally adjusted to improve the signal-to-noise ratio using PhotoShop software (Adobe Systems, Mountain View, CA). In some cases, the background was subtracted from maximum projection images of the filled cell to improve the signal-to-noise ratio.
Morphological Identification
The morphological cell type was classified on the basis of previous studies (8, 9, 53, 58) using criteria that included dendritic arborization, the shape of individual dendrites, and the location of initial dendritic branch points.
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RESULTS |
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Figure 2 summarizes RT-PCR results for whole neural retinas. Products for the nAChR subunits 3,
4,
6,
7,
2,
3, and
4 were amplified. Sequence homology to each of the corresponding human cDNA sequences was 8293%. The second, lighter band at
600 bp in the
7 lane also has 90% sequence homology to human
7 nAChR cDNA. The final 357 bases of the sequence from this band align exactly with the last 357 bases of the sequence of the predicted 462-bp
7 product. Though these
7 sequences correspond to the region in which a functional
7 splice variant was identified in the rat
7 subunit (60), the region of discontinuity between the two rabbit products does not correspond to the insert reported for rat.
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Effects of Agonist Application on Cell Activity
There is evidence for 7-like, MLA-sensitive nAChR involvement in the responses of DS GCs (56), and transcripts for
7 nAChR subunits are present in extracts of whole retina. Thus we sought to determine whether other physiologically identified GC types expressed
7-like AChRs. More than 80 GCs were physiologically characterized and tested for responses to nicotinic agonists before synaptic blockade. Cells that responded to the application of nicotinic agonists included those with both sustained and transient responses to light onset and those with sustained and transient responses to light offset. Cells with morphology consistent with
-GCs (53, 58) predominated, although GCs with morphology consistent with brisk, transient, linear and nonlinear concentric (9) cells were also identified.
With upstream inputs to GCs intact, agonist application evoked variable responses. Therefore, the effects on each component were tallied separately. Application of choline or nicotine enhanced the light response of 27 GCs (P < 0.02), while the light responses of 22 other GCs were not affected by either choline or nicotine. Agonist application enhanced the maintained firing of 41 GCs (P < 0.025) and had no effect on the maintained firing rates of 15 GCs. Interestingly, nicotine and choline suppressed the light responses and/or the maintained firing rates of 25 GCs (P < 0.001). For nine cells, this suppression was either preceded or followed by excitation. Typically, the cells that were suppressed by agonist application were those with high firing rates, although in some cases, the light responses of cells without maintained firing were blocked by agonist application (Fig. 3). Puff application of DI H2O had no effect on either light responses or maintained firing rates.
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To test the contribution of muscarinic AChRs to choline-induced suppression, 3 µM atropine was bath applied (n = 6) before choline puff. Atropine blocked the choline-induced suppression in two GCs and decreased the choline-induced suppression without completely eliminating it in four GCs (P < 0.01), suggesting that activation of muscarinic AChRs contributes to, but is only partly responsible for, choline-induced suppression. In four of the six cases, atropine also increased GC-maintained firing (P < 0.001).
Ten GCs with responses that were suppressed by agonist application were injected with fluorescent dye and processed immunohistochemically. Eight of these cells were immunoreactive for non-7 nAChRs. Figure 3 shows an example of MLA-insensitive blockade of light responses by choline. The light response of this sustained off cell was blocked by puff application of choline. Bath application of 15 nM MLA did not rescue the light response. However, under cobalt blockade, the cell responded to puff application of 2 µM nicotine, indicating that the cell did express nAChRs. Consistent with the response to nicotine application, immunohistochemical processing revealed that the cell expressed non-
7 nAChRs.
The effects of nicotine and choline on light responses and maintained firing suggest that ganglion cell firing can be modulated by the activation of both muscarinic AChRs and nAChRs. Both HMB and MLA consistently decreased the maintained firing rates of cells, suggesting that functional nAChRs contributed to tonic ganglion cell activation. However, because these effects could be mediated either by receptors upstream of the GCs or by receptors on the GCs themselves, we next studied the responses to choline under synaptic blockade.
We used the partially subtype-specific 7 agonist choline to test the hypothesis that GCs with transient or sustained responses to light stimuli directly express
7 nAChRs. Synaptic isolation of GCs by adding 2 mM cobalt to the superfusate blocks Ca2+-mediated synaptic transmission and ensures that GC responses are mediated by receptors expressed directly by the GC. The responses of 31 physiologically identified GCs tested for responses to choline while under synaptic blockade are summarized in Table 3. Cells with similar physiological and pharmacological responses were collapsed into subgroups. Fifteen GCs responded to puff application of choline in concentrations ranging from 200 µM to 400 mM. The reported P value is the largest obtained from within each group. Figure 4 shows a typical choline-responsive cell, a transient on GC that responded to 400 mM choline before (Fig. 4C) and after synaptic blockade (Fig. 4E). Choline-induced spiking after synaptic blockade of light response and upstream inputs to the cell demonstrated the expression of putative
7 nAChRs directly by the ganglion cell. As shown using CF masking, MAb210 IR indicated the expression of non-
7 nAChRs (Fig. 4, FH). Two additional choline-responsive cells were also MAb210-IR, suggesting the expression of a second nAChR subtype. Cells that responded to choline application while under synaptic blockade included GCs from each of the four main physiological groups (i.e., transient on and off and sustained on and off). One GC that did not respond to choline was MAb210-IR, suggesting that this cell expressed only non-
7-containing nAChRs. Pharmacological responses of a subgroup of cells were consistent with this interpretation because six GCs responded to nicotine but not to choline.
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Because immunohistochemical data suggested the expression of both 7 and non-
7 nAChRs by GCs, and because positive responses to choline do not rule out coexpression of other nAChR subtypes, we processed a subset of these cells for single-cell multiplex RT-PCR to determine whether mRNA transcripts for
7 and/or
3 nAChRs were present. Six of the choline-responsive cells were harvested after recording and processed for multiplex, single-cell RT-PCR. Consistent with the pharmacological responses, two of these cells expressed
7 mRNA. Figure 5, AC, shows an example of one such cell. This cell responded transiently at light extinction and responded to puff application of choline after synaptic blockade of upstream inputs. Four other GCs that responded to choline concentrations ranging from 400 µM to 10 mM expressed
3 and
7 mRNA products (Fig. 5, D and E), providing additional indirect evidence that subsets of GCs express more than one nAChR subtype. To rule out false-positive results, four choline-negative cells were processed for single-cell RT-PCR. Neither
3 nor
7 mRNA products were amplified from these cells.
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Although micromolar choline is a specific agonist for 7 nAChRs (52), the range of choline concentrations that we tested under synaptic blockade ranged from nonspecific to
7 specific. Because higher concentrations of choline may also affect muscarinic AChRs (19),
3
4 nAChRs (4), or
4
4 nAChRs (4, 19, 71), that choline was acting on
7 nAChRs was confirmed by blockade of choline-induced responses by 1530 nM MLA. Each of the GCs included in this set of experiments first responded to the application of choline while under synaptic blockade.
After recording the positive response to choline application while under synaptic blockade, 1530 nM MLA was bath applied. MLA completely blocked the responses to choline application for 9 of 13 GCs tested, confirming that the majority of excitatory responses elicited by choline concentrations ranging from 200 µM to 400 mM were mediated solely by 7 nAChRs. The incomplete blockade of the choline responses of four cells by nanomolar concentrations of MLA suggests that choline may activate additional choline-sensitive receptors in a small subset of GCs. These results are summarized in Table 4. The MLA-insensitive choline-activated receptors are not muscarinic, because bath application of 3 µM atropine had no effect on the choline-induced responses elicited under synaptic blockade (n = 2). Blockade of the choline-induced and MLA- and atropine-insensitive responses by HMB (n = 1) indicated that the MLA-insensitive responses were mediated by nicotinic receptors. Four of the above-mentioned GCs were injected with fluorescent dye and processed for immunohistochemistry. Each of these four GCs demonstrated IR consistent with the expression of non-
7 nAChRs.
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Some of our data provided indirect evidence of the expression of multiple nAChR subtypes. These included detection of transcripts for both 7 and
3 nAChR subunits, immunohistochemical evidence of non-
7 nAChRs by choline-responsive cells, and incomplete blockade by MLA of agonist responses. Because neither the presence of mRNA nor the presence of immunolabeling confirmed functional receptor expression, we next chose to test directly for the presence of more than one functional subtype. Seven GCs were tested for responses to nicotine after complete blockade of choline responses by 30 nM MLA. These results are summarized in Table 5. Figure 6 is a representative example of the direct evidence that a single GC expressed multiple functional nAChR subtypes. Although the peak light responses of this cell were slightly decreased by puff application of choline before synaptic blockade, choline-induced spiking was additive with light-induced spiking, such that choline application before synaptic blockade resulted in a significant increase in total spikes compared with light responses alone (Fig. 6, B and C). After synaptic isolation by the substitution of Ames medium containing 2 mM cobalt, this cell demonstrated an MLA-sensitive response to choline, indicating the expression of
7 nAChRs (Fig. 6, DF). After complete blockade of the MLA-sensitive nAChRs, nicotine application elicited firing (Fig. 6G), indicating the presence of additional MLA-insensitive nAChRs.
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DISCUSSION |
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Although the main source of choline is from dietary intake, choline is constitutively present in many parts of the nervous system and is actively transported to the brain (46). When choline is taken up by the high-affinity choline transporter on cholinergic neurons, it is used as a precursor to ACh (11, 25). Choline is also present at synaptic sites as a product of the hydrolysis of ACh. Choline plasma levels are reportedly stable at 10 µM (46), although extracellular choline concentrations in the brain can be sufficient to activate 7 nAChRs (5). Thus, at physiological concentrations, choline can act as a specific endogenous ligand for
7 nAChRs (4, 52). This may allow the responses of GCs to differ, depending on the availability of ACh, choline, and the specific nAChR subtypes expressed. For example, low concentrations of choline could contribute to tonic depolarization or maintained firing of GCs, while light-evoked ACh release might contribute to either transient components mediated by
7 nAChRs, or more sustained components, mediated by non-
7 nAChRs, of light-evoked firing by given GCs. If pChAT immunohistochemistry (70) reflects additional ACh release in the retina, subsets of GCs may both release and respond to ACh, adding to the complexity of information processing by GCs.
Effects of Agonists and Antagonists on Light Responses
Previous studies have provided evidence for functional -containing (52) and
7-like nAChRs in rabbit retinal GCs (56). Immunohistochemical studies also show nAChR expression by amacrine cells (21, 22, 35) and limited sets of bipolar cells (69). Our studies with the use of nicotinic agonists in concert with muscarinic antagonists explored the contribution of AChRs to the light responses and maintained firing of retinal ganglion cells. Choline-induced suppression appeared to be mediated in part by muscarinic AChRs, with a portion mediated by nAChRs. In contrast, nAChRs, including those that are MLA sensitive, contribute to GC-maintained activity. Thus nAChR-mediated tonic excitation may serve to balance tonic suppression mediated by muscarinic AChRs. Taken together, the data indicate that the mechanism underlying the maintained firing is distinct from that of the suppression.
However, a small proportion of the choline-induced suppression may be mediated by non-7 nAChRs as evidenced by nicotine-induced suppression and choline-induced suppression that was reduced by HMB. Interestingly, choline doses >10 mM can inhibit the responses of
4
4 nAChRs to ACh (71), raising the possibility that the second type of agonist-induced suppression is mediated by
4
4 nAChRs. This possibility requires further study but is supported by the presence of both
4 and
4 mRNA transcripts in whole retina extracts.
Effects of Agonists and Antagonists under Synaptic Blockade
The responses of GCs to choline application while under synaptic blockade, in conjunction with the presence of 7 mRNA in harvested GCs, support the hypothesis that functional
7 nAChRs are expressed by multiple GC types. However,
3 mRNA transcripts were also amplified in a number of individual GCs. Because choline can act as a partial agonist for
3
4 nAChRs, and because we used choline in concentrations that ranged from
7-specific to nonspecific (52, 4, 19), it was necessary to confirm
7 nAChR activation. Blockade of choline responses by 1530 nM MLA in all but four GCs tested confirmed
7 nAChR activation. Although MLA at nanomolar concentrations is a competitive antagonist for both
6 and
7 nAChRs (39, 50), there are no reports of choline-induced activation of
6-containing nAChRs in the literature. Although the incomplete blockade of choline responses by MLA may have been due to incomplete antagonist penetration, it instead may suggest a third functional nAChR subtype, e.g., one that is choline sensitive but MLA insensitive.
A choline-sensitive nAChR that is insensitive to low concentrations of MLA has been described in chick brain (25). Although the subunit composition of the chick nAChR has not yet been identified, it may be analogous to the receptor that mediated the MLA-insensitive choline responses in rabbit retina. An alternative analog for our choline-sensitive, MLA-insensitive nAChR subtype may be the slowly desensitizing nAChR subtype described in rat hippocampal neurons (64). A subset of neurons in the rat stratum oriens have slow rise times and longer decay kinetics, together with statistically correlated 7 and
2 mRNA expression. In addition,
7 and
2 subunits can form functional heteromeric channels in oocyte expression systems (36). Because the pharmacology of these receptors is not yet known, extended protein studies are needed to explore the potential for the expression of an
7 heteromer in retina.
While each physiologically defined GC group (sustained off, sustained on, transient off, and transient on cells) contained cells with MLA-sensitive choline responses, the expression of more than one nAChR subtype by single GCs appeared to be correlated with specific physiological groups. Sustained cells had a slightly higher incidence of incomplete blockade by MLA. Other sustained GCs responded to nicotine application after MLA blockade of choline responses. However, this apparent association may simply reflect that there were fewer transient than sustained cells tested in this study.
The larger groups of physiologically defined GC likely consisted of multiple GC types, because there were several dendritic morphologies within some of our physiological groups. The most common morphology identified in injected cells was consistent with -GCs (see Ref. 53), although cells with morphologies consistent with G4 brisk sustained GCs (58), brisk transient GCs, and nonstreak concentric GCs (9) also were identified. Subsets of cells with each of the identified morphologies demonstrated responses to choline application while under synaptic blockade.
Responses to choline under synaptic blockade were more sustained than might have been predicted for 7 nAChRs.
7 nAChRs expressed in oocytes and native
7 nAChRs expressed in brain areas such as the hippocampus rapidly desensitize (1, 2). In contrast, the responses observed in our retinal preparations had time courses that regularly lasted longer than the period of drug application. These results are consistent with the literature in noting that low-dose choline activation can prolong the
7 responses (65). Although we tested a wide range of choline concentrations, our effective choline concentrations likely were much lower than the concentrations loaded into the pipette (i.e., 200 µM400 mM) because of the additional dilution in the superfusate covering the retina. For example, nicotine applied via a rapid solution exchange system at a distance of 20 µm from the cell reportedly can be diluted by
46% by the time the nicotine reaches the cell (20). Assuming a similar diffusion rate for choline, our effective concentrations ranged from
100 µM to
200 mM. In addition, although Di Angelantonio and Nistri (20) calculated the diffusion of nicotine from a point source to dissociated cells in culture, in our experiments, the rate of diffusion was also likely increased by the 3 ml/min superfusate flow. Diffusion into the retina is further hampered by the inner limiting membrane, which may result in even lower effective concentrations. This responsiveness of GCs in these studies argues for receptors with extraordinary sensitivity to even the lowest agonist concentrations loaded into the pipette.
GC responses were not due to mechanical displacement of the cells by pipette ejection pressure, because vehicle controls (see MATERIALS AND METHODS) did not elicit cell firing. In addition, if the agonist-induced firing were a function of mechanical displacement or an effect of the Azure B, then we would not have been able to demonstrate negative responses or antagonist-induced blockade of positive responses.
Expression of Multiple nAChR Subtypes by Individual GCs
The RT-PCR data from whole rabbit retina demonstrated the presence of mRNA for multiple nAChR subunits. Subsets of GCs in our study displayed direct and indirect evidence of expression of more than one nAChR subtype.
Lines of evidence for the expression of multiple nAChR subtypes are demonstrated by the subset of GCs that responded to choline application under synaptic blockade and contained mRNA for both the 3 and the
7 nAChR subunits. Moreover, some GCs that responded to choline application under synaptic blockade have been shown to demonstrate immunoreactivity consistent with the expression of
3
2 nAChRs. Definitive physiological evidence of functional expression of more than one nAChR subtype was provided by the response to nicotine by synaptically isolated GCs after complete blockade of choline-sensitive nAChRs.
The current study has demonstrated conclusively that 7 nAChRs activated by choline are capable of modulating the response properties of multiple GC types in adult rabbit retina. Additional GCs demonstrated physiological responses, mRNA expression, and immunohistochemistry consistent with the expression of
3-containing nAChRs, while other GCs expressed both
3-containing and
7 nAChRs. In addition to demonstrating functional expression of
7 nAChRs in the retina, we have shown that subsets of GCs express multiple functional nAChR subtypes and that muscarinic AChRs are active in the modulation of GC response properties.
Thus GC responses are shaped by 7 and non-
7 nAChRs expressed by GCs themselves, as well as by choline-sensitive nAChRs expressed by upstream cells. The expression of functional
7 nAChRs has implications beyond that of modulating the response properties of GCs. Activation of
7 nAChRs has been shown to play a role in cytoprotection in a porcine model of retinal glutamate excitotoxicity (68) and in other experimental assays (31, 32).
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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