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1From the Department of Physiology and the 2Neuroscience Program, Michigan State University, East Lansing, Michigan.
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Abstract |
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METHODS. A primate model of experimental glaucoma was combined with intracellular recording and staining techniques using an isolated retina preparation. Intrinsic electrical properties were examined by injection of depolarizing and hyperpolarizing currents. Visual responses were studied using drifting and counterphased gratings. Morphologic comparisons were made by injecting recorded cells with Neurobiotin and analyzing them quantitatively with a computer-based neuron reconstruction system.
RESULTS. Structurally, parasol cells from glaucomatous eyes had smaller somata and smaller, less complex dendritic arbors, resulting in a significant reduction in total dendrite length and surface area. Functionally, these neurons did not differ from normal in their mean resting membrane potentials, input resistances, or thresholds to electrical activation, but did differ in membrane time constants and spike duration. Parasol cells from both normal and glaucomatous eyes preferred low-spatial-frequency stimuli, but significantly fewer glaucoma-related cells were driven visually—in particular, by patterned stimuli. Glaucomatous cells also did not respond as well to visual stimuli presented at increased temporal frequencies.
CONCLUSIONS. Ganglion cells in the glaucomatous eye retain most of their normal intrinsic electrical properties, but are less responsive, both spatially and temporally, to visual stimuli. The reduction in visual responsiveness most likely results from significant changes in dendritic architecture, which affects their level of innervation by more distal retinal neurons.
Over the past several years, a number of studies have described the degenerative effects that chronic elevation of IOP and glaucoma have on fibers in the optic nerve, as well as the concomitant loss of ganglion cells that occurs within the retina itself.8 9 10 More recently, we combined the monkey model of experimental glaucoma with intracellular staining techniques to examine the pattern of degenerative changes that characterize glaucomatous neuropathy at the single cell level.11 The results of these studies indicate that in both midget and parasol cells, structural abnormalities at the level of the dendritic arbor represent the earliest signs of glaucoma-related retinal ganglion cell degeneration. These changes include a thinning of both proximal and distal dendritic processes, abrupt changes in dendrite thickness at branch points, and a general reduction in dendritic arbor complexity. Reductions in soma size and proximal axon diameter also occur, albeit slightly later in the degenerative process.
Because retinal ganglion cells receive all their input from more distal retinal elements through their dendrites,12 13 abnormalities in dendritic structure suggest a reduction in synaptic efficacy, and early functional deficits at the single-cell level. The primary goal of the present studies was, using parasol cells as the example, to determine the extent to which glaucoma-related changes in ganglion cell structure might also involve changes in the biophysical and visual response properties of single ganglion cells, thus indicating glaucoma-related visual dysfunction in advance of actual ganglion cell loss.
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Methods |
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In all the experimental animals with glaucoma (Table 1) , IOP was elevated by multiple injections of a sterile solution containing 3.6 x 105 latex microspheres (10 µm diameter, F-8836; Molecular Probes, Eugene, OR) into the anterior chamber of one eye.14 The fellow eyes, and those of eight untreated animals, served as normal control samples. Typically, 8 to 10 injections were needed to achieve a sustained elevation of IOP, and the frequency and size of subsequent injections were made based on biweekly measurements of IOP and the history of the eye’s responsiveness to the prior injections. All eyes were examined with the slit lamp before each injection. Fundus photographs were obtained every 3 to 4 weeks, depending on the clinical appearance of the optic disc compared with its appearance during the previous eye examination.
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In Vitro Procedures
Immediately on enucleation, the anterior segment of each eye was removed and the posterior eyecup placed in a solution of aCSF saturated with a mixture of 95% O2 and 5% CO2 at 22°C. Before recording, each eyecup was flatmounted, ganglion cell layer up, in a chamber perfused with oxygenated aCSF (3–8 mL/min) at 36.5°C and allowed 30 minutes to acclimate in dim light. The chamber then was mounted on the stage of an upright microscope equipped with epifluorescence, and single ganglion cells, prestained with acridine orange (1 mM, A-6014; Sigma-Aldrich, St. Louis, MO), were targeted with a 40x water immersion objective with a working distance of 1.6 mm.11
Intracellular Recording and Labeling
Intracellular recordings were made using glass microelectrodes filled with 1 M potassium acetate, 2% Neurobiotin (SP-1120; Vector Laboratories, Burlingame, CA), and 0.2% pyranine (41218; Acros Organics, Fairlawn, NJ). The electrodes were beveled to a final impedance of 35 to 45 M
at 30 Hz. Recordings were made using a hydraulic microdrive, high-impedance intracellular amplifier (AxoClamp 2B) with bridge and current injection circuitry and software (pClamp6/8; Axon Instruments, Union City, CA). Neurobiotin was iontophoresed into cells with positive-current pulses (1–2 nA) of 100 to 200 ms duration, and labeled cells were revealed with an avidin biotin complex (ABC) kit (Vectastain, SK-4100; Vector Laboratories).
Biophysical Measurements
After optimal capacitance compensation, the extracellular response to a brief pulse of negative current (–1 nA/0.5 ms) was used to determine the response characteristic of each electrode. Typically, this yielded electrodes with time constants of 100 to 200 µs, considerably faster than those measured intracellularly (1–4 ms). Electrodes were used for only a single penetration and were discarded if the electrical characteristics changed during recording. Whole-cell input resistance (Rn) was determined from the slope of the I-V curve derived by delivering a series of hyperpolarizing current pulses (0 to –1 nA in 0.1-nA steps of 250-ms duration) through the recording electrode. Membrane time constants (
m) were derived by fitting a standard exponential to the voltage decay curve that resulted from application of a brief pulse of hyperpolarizing current (–1 nA, 0.5 ms). Thresholds and firing rates were determined by applying depolarizing current pulses (0 nA to +1 nA in 0.1-nA steps of 250-ms duration) through the recording electrode and measuring the onset of spike activity, as well as the maximum number of spikes per second.
Visual Stimulation
Visual stimuli included drifting and counterphased light and dark bars presented by a computer-driven image synthesizer (Picasso; Innisfree, Cambridge, MA) and a high-resolution CRT (model 608 w/P4 phosphor; Tektronics, Beaverton, OR) that was directed through the microscope camera port. The mean luminance of the monitor, as measured with a 1° luminance head (J1823; Tektronics) and a photometer (J17; Tektronics), was 40 cd/m2 at 60% contrast. Delivering the stimulus through the camera port and 40x water-immersion objective needed to target single cells reduced the luminance to approximately 1 cd/m2. Spatial frequencies of 0.08 to 3.6 cyc/deg, presented at temporal frequencies of 0.5 to 25 Hz (highest rate the image synthesizer could generate), were randomly interleaved, presented five times each, and the resultant binned visual responses averaged. Each trial included the presentation of a blank screen (0% contrast), which was used to correct for cell–cell differences in spontaneous activity. Ganglion cells were considered linear if they displayed a null point and nonlinear if they displayed a doubling response, when presented with the appropriate, sinusoidally modulated (2–4 Hz) grating.16
Cell Sampling, Mapping, Classification, and Analysis
Because the intracellular approach does not allow sampling of a large number of neurons over the entire retina, we focused on ganglion cells located in the superior and inferior regions of the midtemporal retina (4–8 mm from the fovea), the region considered clinically to be the most vulnerable to pressure-induced degeneration.17 18 19 We also focused on parasol cells, because of the greater ease of obtaining stable, long-term, recordings, their high-contrast gain,20 21 and previous work suggesting that these neurons may be most susceptible in the glaucomatous eye22 23 24 (but see Ref. 9 ). Individual cells, prestained with acridine orange, were selected randomly for analysis. Cells initially were identified anatomically by their large somata and phasic responses to a maintained visual stimulus; after injection, fixation, and processing, the labeled cells were confirmed as parasol cells by their characteristic dendritic morphologies.11 12 13 16 25 26 27 28 Before in vitro sampling, a map was made of the retinal blood vessel pattern and the approximate location of each recorded neuron. After the recording and processing, the retinal outline, optic disc, fovea, and location of each injected neuron were mapped with a computer-based stage digitizing system (AccuStage, Shoreview, MN). Cells labeled intracellularly with Neurobiotin were matched with their physiological data, reconstructed, and analyzed quantitatively by microscope (FX-A; Nikon, Tokyo, Japan), with a 100x oil-immersion objective and morphometric software (Neurolucida and NeuroExplorer; MicroBrightField, Inc., Colchester, VT). Distribution-based comparisons were made with the Mann-Whitney test, whereas mean data comparisons (presented as the mean ± SE) were made with a two-tailed Student’s t-test. All statistical comparisons were made by computer (SPSS software; SPSS, Chicago, IL), with P = 0.05 as the level of significance.
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Results |
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0.32 nA) after intracellular application of depolarizing current pulses (0–1 nA). Although they also showed similar whole cell input resistances (21.7 ± 1.5 M vs. 22.5 ± 1.9 M), the time constants of parasol cells from the glaucomatous eyes were, on average, significantly shorter than those recorded in normal parasol cells (2.2 ± 0.12 ms vs. 2.7 ± 0.14 ms, respectively). Similarly, although there was no difference in the mean spike amplitudes in these two populations of cells (46 mV), spike duration (measured as spike width at half height) was significantly longer in the glaucomatous versus normal parasol cells (0.5 ± 0.02 ms vs. 0.43 ± 0.02 ms).
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2.5–15 mV), the response itself was threefold more prevalent in the normal (48%) versus glaucomatous (16%) parasol cells. A similar decrease in the frequency of the sag response has been noted in cat motoneurons after axotomy.31
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Discussion |
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Although the biophysical properties of ganglion cells in the mammalian retina have been described previously in both slice and culture preparations, the work most relevant to the present study is that of O’Brien et al.,30 in which they used a similar isolated retina preparation to compare the intrinsic response properties of ganglion cells in the adult feline retina. Despite considerable variability within each response category, the large 
cells were readily distinguished by their mean input resistances and membrane time constants, which were at least four times lower than those of the other classes of ganglion cells studied (see Table 1 in O’Brien et al.30 ). In general, the biophysical characteristics of the cells reported by O’Brien et al. are highly comparable to those of the parasol cells examined in the current study. This is not surprising, considering that these two classes of ganglion cells often are considered anatomically and functionally similar. The extent to which our lower estimates of input resistance for parasol cells (21.7 M vs. 31.3 M) might reflect real, methodological (whole-cell patch versus sharp electrode), or sampling differences, requires further investigation. Preparation quality may also be a factor; however, this explanation seems unlikely based on the good membrane resting potentials, low activation thresholds, large amplitude spikes, and, in normal cells, strong visual responses. Although O’Brien et al. report membrane time constants for their cells that are approximately 67% longer than those obtained for our parasol cells (4.5 ms vs. 2.7 ms), it is important to note that they considered their time constant estimate to be an overestimate, because they excluded the fastest cells from their analysis. We did not, as the time course of the intracellular responses (1–4 ms) of these neurons were clearly different from the extracellular responses of the electrode alone (100–200 µs). O’Brien et al. also reported significantly higher spike frequencies for their cells in response to electrical stimulation (262 Hz vs. 108 Hz); however, they used a depolarizing current level that was approximately twice that used in the present study (1.8 nA vs. 1.0 nA). Our decision to limit the maximum level of stimulation to 1.0 nA was based on our initial experience that parasol cells from glaucomatous eyes often did not hold up well to electrical stimulation above 1.2 nA. Another factor affecting our spike-frequency measurements was the finding that not all parasol cells responded equally to depolarizing current pulses. Some produced only one to two spikes regardless of the level of depolarization; others started slowly, but quickly increased their rate of firing with increased stimulus intensity; and a third group of cells showed a high level of activity, even at the lowest levels of stimulation, and increased rapidly from there. Although we did not include the most phasic cells in our computations of spike frequency, we also did not restrict our estimates to only those with the highest firing rates, thus reducing the mean rate. One clear difference in the biophysical response properties of our parasol cells and the cells examined by O’Brien et al., was the presence of a sag response, or anomalous rectification, in about half the normal parasol cells studied. Although this response also was seen in many of the other classes of feline ganglion cells, it was not a prominent feature of cells, suggesting that ganglion cells differ with respect to the type and number of potassium channels they possess. At present, the role this hyperpolarizing-activated current plays in retinal function remains unknown. It appears, however, that the ion channels underlying this response are susceptible to the effects of glaucomatous neuropathy, as only one third of the parasol cells examined in the glaucomatous eyes displayed sag responses compared with normal.
Parasol cells in the normal eyes responded best to gratings of low spatial frequency, drifting at 8 to 10 Hz. When the temporal frequency response functions, which were limited to a maximum of 25 Hz by the pattern generator, were extended to the baseline, the projected temporal cutoff frequency for these neurons was approximately 45 Hz. Although the temporal response functions were similar in shape to those derived in vivo (i.e., band-pass), the peak temporal frequencies of the parasol cells examined by us were only approximately one half that reported previously.32 This most likely reflects the significant reduction in image brightness imposed by presenting the visual stimuli through the microscope camera port and 40x objective (40 cd/m2 at video monitor vs. 1 cd/m2 at retina) and not our use of an isolated retina preparation. Previous studies have shown that decreasing stimulus luminance and contrast results in a shift of the temporal frequency response function toward lower optimal frequencies and response magnitudes.20 21 32 Although we may have reduced this effect by using a lower-power objective (e.g., 4x), it was our experience that switching back and forth between the 40x water immersion and a dry objective often resulted in loss of the targeted cell. In addition, visual responsiveness is highly sensitive to focal quality at the tissue level, and this is monitored most reliably with the higher-power objective. The reduction in retinal luminance also guided our basis for focusing on the visual responses of parasol cells, whose sensitivities to luminance contrast are 8 to 10 times greater than those of midget ganglion cells.20 21 32
In a previous study, Smith et al.33 used the primate model of glaucoma to examine, in vivo, the visual response properties of retinal target neurons in the dorsal lateral geniculate nucleus (LGN), after long-term (20–52 months) elevation of IOP. They concluded that visual deficits in long-term glaucoma result primarily from ganglion cell loss and not a reduction in the functional capacity of surviving neurons. Although the results of the present study appear to contradict this conclusion, there are several important differences between these two studies. First, many of the magnocellular LGN neurons sampled by Smith et al.33 had receptive fields, and thus received their retinal input, from parasol cells located near central retina (0–15°). By contrast, because of the limited number of cells that could be studied per eye intracellularly, we restricted our retinal sampling to mid-temporal retina (15–30°), the region considered clinically to be most vulnerable to glaucoma-related injury17 18 19 ; ganglion cells located in these regions give rise to the arcuate fibers that form the superior and inferior poles of the optic nerve head, regions shown to be affected most often in glaucomatous eyes.17 18 19 In this respect, it is important to note that Smith et al.,33 reported their greatest reductions in retinal innervation were associated with LGN receptive field locations outside the macular region, but in regions associated with the arcuate fibers. Thus, it is likely that some of the differences between these two studies result from differences in the locations of the retinal cells targeted. Second, except for animals with higher mean levels of IOP (37–53 mm Hg vs. 23–53 mm Hg; Smith et al.), the changes in cup-disc ratio for most of the animals examined by Smith et al.33 were more modest (0.2–0.4) than those determined for the animals used in our study (0.4–0.9), suggesting that the eyes of our animals had experienced, on average, a more significant level of degeneration. Of note, these investigators state that in three animals with severe nerve damage, they were able to drive only 20/194 units encountered through the glaucomatous eye and that, in many passes, they were unable to drive any cells by the affected eye. Although they ascribe this to a loss of retinal ganglion cell innervation to this region of the LGN, it also is possible that, to some extent, these silent regions represent areas of the nucleus that continue to receive retinal input, but that the ganglion cells providing the input no longer respond normally to the visual stimuli used. This could form the basis for the LGN neurons they encountered that showed spontaneous activity, but could not be influenced by visual stimulation. An interesting comparison would have been the relation between the location and encounter rate of LGN neurons determined physiologically and that derived from the histologic reconstructions of Smith et al.33 A third factor that may have contributed to the different conclusions of these two studies is the method of cell sampling. In the approach used by Smith et al. the LGN neurons, and thus the retinal afferents, analyzed were identified by their ability to respond to presentation of a visual stimulus. This may have caused a bias toward those cells that retained relatively normal levels of retinal innervation. By contrast, the retinal neurons in the present study were selected randomly through direct visualization, thus removing any functional bias from the selection process. Finally, it is important to note the different time frames of the two studies. The animals examined by Smith et al. had their IOP elevated by laser treatment,34 35 36 then received little additional intervention over a 20- to 58-month survival period, during which IOP was allowed to decline slowly to normal. The animals in this study had their IOP elevated by repeated (sometimes biweekly) intraocular injections of latex microspheres.14 While these injections were moderated carefully so as not to induce acute spikes in IOP, we did see a higher frequency, although not necessarily higher magnitude, of fluctuations in IOP in our animals compared with those of Smith et al. (see their Fig. 1 in Ref. 33 ). Furthermore, the IOPs of the animals used in our study were not reduced to normal (16 mm Hg) until 24 to 48 hours before examination of the retina. As noted by Smith et al., it is possible that their data reflect a system that has stabilized with time, whereas the results of the present study more closely reflect short-term changes in retinal function. Thus, while the data presented herein do not refute the conclusion of Smith et al., that visual deficits in glaucoma result primarily from ganglion cell loss, they do indicate that abnormal visual function by surviving ganglion cells also must be considered a contributing factor.
Regardless the mechanism underlying visual dysfunction, it is important to note that many of the glaucoma-related ganglion cells showed a high degree of normalcy in their intrinsic membrane properties, suggesting that, despite relatively significant changes in morphology, their cell membranes are basically intact. This is not surprising, considering the wide range of effects seen with respect to the intrinsic membrane properties of neurons from other systems after axonal injury.37 The extent to which this may represent a resistance to injury by these neurons is difficult to assess, since glaucomatous changes typically are not uniform across the retina, spatially or in degree.38 39 Although it was our intent that by combining the intracellular recording and labeling techniques we would be able to identify a specific structure-function deficit in the glaucomatous eyes, no such relation was obvious. The decreased spatial–temporal responsiveness of ganglion cells from these animals appears to result from global changes in dendritic structure, which then have a negative impact on synaptic integrity. Variations among degenerating neurons most likely reflect complex differences in the number, type, and distribution of synaptic inputs along the dendrites of different neurons,40 41 their differential influences on the response properties of those cells, and the spatial–temporal pattern of degeneration induced by the disease.
Thus, the data presented here suggest that decreases in the spatial–temporal response properties of single ganglion cells contribute to the spatial and temporal deficits identified psychophysically in glaucoma.42 43 44 45 46 47 48 49 50 51 These functional deficits most likely derive from pressure-induced changes in the structural integrity and synaptic organization of the affected neurons, but not necessarily changes in their intrinsic neuronal membrane properties.
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Acknowledgements |
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Footnotes |
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Submitted for publication July 15, 2004; revised April 8, 2005; accepted April 27, 2005.
Disclosure: A.J. Weber, None; C.D. Harman, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Arthur J. Weber, Department of Physiology, 2193 Biomedical and Physical Sciences Building, Michigan State University, East Lansing, MI 48824; weberar{at}msu.edu.
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-motorneurones. Brain Res. 1985;326:392–395.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
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