Intact Retina Research Articles

Voltage-Gated Sodium Channels Improve Contrast Sensitivity of a Retinal Ganglion Cell

Voltage-gated channels in a retinal ganglion cell are necessary for spike generation. However, they also add noise to the graded potential and spike train of the ganglion cell, which may degrade its contrast sensitivity, and they may also amplify the graded potential signal. We studied the effect of blocking Na+ channels in a ganglion cell on its signal and noise amplitudes and its contrast sensitivity. A spot was flashed at 1-4 Hz over the receptive field center of a brisk transient ganglion cell in an intact mammalian retina maintained in vitro. We measured signal and noise amplitudes from its intracellularly recorded graded potential light response and measured its contrast detection thresholds with an "ideal observer." When Na+ channels in the ganglion cell were blocked with intracellular lidocaine N-ethyl bromide (QX-314), the signal-to-noise ratio (SNR) decreased (p < 0.05) at all tested contrasts (2-100%). Likewise, bath application of tetrodotoxin (TTX) reduced the SNR and contrast sensitivity but only at lower contrasts (< or = 50%), whereas at higher contrasts, it increased the SNR and sensitivity. The opposite effect of TTX at high contrasts suggested involvement of an inhibitory surround mechanism in the inner retina. To test this hypothesis, we blocked glycinergic and GABAergic inputs with strychnine and picrotoxin and found that TTX in this case had the same effect as QX-314: a reduction in the SNR at all contrasts. Noise analysis suggested that blocking Na+ channels with QX-314 or TTX attenuates the amplitude of quantal synaptic voltages. These results demonstrate that Na+ channels in a ganglion cell amplify the synaptic voltage, enhancing the SNR and contrast sensitivity.

Functional assessment of glutamate clearance mechanisms in a chronic rat glaucoma model using retinal ganglion cell calcium imaging

Using optical imaging of retinal ganglion cell (RGC) calcium dynamics in living intact retinal wholemount preparations, we tested whether RGCs in an experimental rat glaucoma model were more sensitive to exogenously applied glutamate as a result of deficient glutamate clearance mechanisms. In contrast to post-natal rat RGCs in purified cultures, in which the calcium influx induced by 200 microm NMDA and 10 microm glutamate was approximately equivalent, application of up to 500 microm glutamate did not affect calcium levels in RGCs in retinal wholemounts, even though the RGCs responded to 200 microm NMDA. Glutamate (500 microm) did elicit a RGC calcium response in retinal wholemounts when glutamate transporters were inhibited pharmacologically with DL-threo-beta-benzyloxyaspartate, confirming the presence of glutamate clearance mechanisms in this intact retina preparation. The effect of glutamate was then assessed on retinas from rats with chronically elevated intraocular pressure in one eye, produced by the injection of hypertonic saline into an episcleral vein. Application of up to 500 microm glutamate had no effect on RGC calcium levels, while millimolar concentrations of glutamate induced a calcium signal in RGCs that was indistinguishable from that in fellow control retinas. Therefore, there was no evidence for a global defect in glutamate uptake in this rat model of experimental glaucoma. Imaging glutamatergic calcium dynamics of RGCs in retinal wholemounts represents a novel methodology to probe glutamate transporter function and dysfunction in an intact CNS tissue system.

Characterization of Strychnine-sensitive Glycine Receptor in the Intact Frog Retina: Modulation by Protein Kinases

We studied 3H-glycine and 3H-strychnine specific binding to glycine receptor (GlyR) in intact isolated frog retinas. To avoid glycine binding to glycine uptake sites, experiments were performed at low ligand concentrations in a sodium-free medium. The binding of both radiolabeled ligands was saturated. Scatchard analysis of bound glycine and strychnine revealed a KD of 2.5 and 2.0 microM, respectively. Specific binding of glycine was displaced by beta-alanine, sarcosine, and strychnine. Strychnine binding was displaced 50% by glycine, and sarcosine. Properties of the strychnine-binding site in the GlyR were modified by sarcosine. Binding of both radioligands was considerably reduced by compounds that inhibit or activate adenylate cyclase and increased cAMP levels. A phorbol ester activator of PKC remarkably decreased glycine and strychnine binding. These results suggest modulation of GlyR in response to endogenous activation of protein kinases A and C, as well as protein phosphorylation modulating GlyR function in retina.

M opsin phosphorylation in intact mammalian retinas

The deactivation of visual pigments involved in phototransduction is critical for recovering sensitivity after exposure to light in rods and cones of the vertebrate retina. In rods, phosphorylation of rhodopsin by rhodopsin kinase (GRK1) and the subsequent binding of visual arrestin completely terminates phototransduction. Although signal termination in cones is predicted to occur via a similar mechanism as in rods, there may be differences due to the expression of related but distinct gene products. While rods only express GRK1, cones in some species express only GRK1 or GRK7 and others express both GRKs. In the mouse, cone opsin is phosphorylated by GRK1, but this has not been demonstrated in mammals that express GRK7 in cones. We compared cone opsin phosphorylation in intact retinas from the 13-lined ground squirrel (GS) and pig, cone- and rod-dominant mammals, respectively, which both express GRK7. M opsin phosphorylation increased during continuous exposure to light, then declined between 3 and 6 min. In contrast, rhodopsin phosphorylation continued to increase during this time period. In GS retina homogenates, anti-GS GRK7 antibody blocked M opsin phosphorylation by 73%. In pig retina homogenates, only 20% inhibition was observed, possibly due to phosphorylation by GRK1 released from rods during homogenization. Our results suggest that GRK7 phosphorylates M opsin in both of these mammals. Using an in vitro GTPgammaS binding assay, we also found that the ability of recombinant M opsin to activate G(t) was greatly reduced by phosphorylation. Therefore, phosphorylation may participate directly in the termination of phototransduction in cones by decreasing the activity of M opsin.

Responses of Müller glia to retinal injury in adult zebrafish

In an effort to identify the cellular events that enable neuronal regeneration in the vertebrate retina, the identity and characteristics of mitotic and apoptotic cells were examined in lesioned retinas of adult zebrafish. Following lesion a complex spatiotemporal pattern of mitosis was observed, including a delayed entry of Müller glia into the cell cycle. Characteristics of these proliferative Müller glia indicated they might serve as a stem/precursor cell of regenerated retina. The results suggested a model of retinal regeneration in which lesions are filled, in part, by a localized en place cytogenesis within intact retina surrounding the lesion site.

Site-specific Phosphorylation of Phosducin in Intact Retina: DYNAMICS OF PHOSPHORYLATION AND EFFECTS ON G PROTEIN βγ DIMER BINDING

Phosducin (Pdc) is a G protein beta gamma dimer (G beta gamma) binding protein, highly expressed in retinal photoreceptor and pineal cells, yet whose physiological role remains elusive. Light controls the phosphorylation of Pdc in a cAMP and Ca(2+)-dependent manner, and phosphorylation in turn regulates the binding of Pdc to G(t)beta gamma or 14-3-3 proteins in vitro. To directly examine the phosphorylation of Pdc in intact retina, we prepared antibodies specific to the three principal phosphorylation sites (Ser-54, Ser-73, and Ser-106) and measured the kinetics of phosphorylation/dephosphorylation during light/dark adaptation and the subsequent effects on G(t)beta gamma binding. Ser-54 phosphorylation increased slowly (t((1/2)) approximately 90 min) during dark adaptation to approximately 70% phosphorylated and decreased rapidly (t((1/2)) approximately 2 min) during light adaptation to less than 20% phosphorylated. Ser-73 phosphorylation increased much faster during dark adaptation (t((1/2)) approximately 3 min) to approximately 50% phosphorylated and decreased more slowly during light adaptation (t((1/2)) approximately 9 min) to less than 20% phosphorylated. The Ca(2+) chelator BAPTA-AM blocked Ser-54 phosphorylation during dark adaptation but had no effect on Ser-73 phosphorylation. In contrast, Ser-106 was not phosphorylated in either the light or dark. Importantly, G beta gamma binding to Pdc was enhanced by Ca(2+) chelation and the binding kinetics closely paralleled those of Ser-54 dephosphorylation, indicating that Ser-54 phosphorylation controls G(t)beta gamma binding in vivo. These results suggest a pivotal role of Ser-54 and Ser-73 phosphorylation in determining the interactions of Pdc with its binding partners, G(t)beta gamma and 14-3-3 protein, which may regulate the light-dependent translocation of the photoreceptor G protein.

Nitric oxide modulates the transfer function between cones and horizontal cells during changing conditions of ambient illumination

It has been suggested that nitric oxide (NO) serves as a retinal neuromodulator, adjusting retinal function to changing conditions of adaptation. We tested this hypothesis in the intact turtle retina by recording the photoresponses of L-cones and L1-horizontal cells, while changing retinal NO level and background illumination. Raising the retinal level of NO, by adding an NO donor (sodium nitroprusside) or the precursor for NO synthesis (L-arginine), induced response augmentation in L-cones and L1-horizontal cells. Lowering retinal level of NO by adding L-NAME, an inhibitor of NO synthesis, reduced the amplitudes of the photoresponses in these retinal neurons. The transfer function between L-cones and L1-horizontal cells, constructed from the photoresponses of these cells, was modified by NO and by background lights. The nonlinear transfer function, characteristic of the dark-adapted retina, became linear and of low gain when the retinal NO level was increased or by increasing the level of ambient illumination. In contrast, inhibiting NO synthesis in the light-adapted retina induced nonlinearity in the cone-to-horizontal cell transfer function similar to that seen in the dark-adapted state. NADPH diaphorase histochemistry, conducted on isolated retinal cells, demonstrated activity in cone inner segments and distal process of Müller cells. These findings support the hypothesis that NO synthesis in the distal turtle retina is triggered by background illumination, and that NO acts to adjust the modes of visual information processing in the outer plexiform layer to the conditions required during continuous background illumination.

Retinal bipolar cells: Contrast encoding for sinusoidal modulation and steps of luminance contrast

Contrast encoding for sinusoidal modulations of luminance contrast was investigated by intracellular recording in the intact salamander retina. In what appears to be the first study of this kind for vertebrate bipolar cells, responses of the central receptive-field mechanism of cone-driven cells to modulation of 3 Hz were analyzed quantitatively via both signal averaging and a Fast Fourier Transform (FFT) while the retina was light adapted to 20 cd/m2. Depolarizing and hyperpolarizing bipolar cells showed very similar encoding. Both responded with sinusoidal waveforms whose amplitude varied linearly with modulation depths ranging up to 7-8%. The slope of the modulation/response curve was very steep in this range. Thus, the contrast gain was high, reaching values of 6-7, and the half-maximal response was achieved at modulations of 9% or less. At modulations above approximately 15%, the responses typically showed strong compressive nonlinearity and the waveform was increasingly distorted. At maximum modulation, the higher harmonics of the FFT constituted about 30% of the amplitude of the fundamental. Measurements were also made for cones and horizontal cells. Both cell types showed predominantly linear responses and low contrast gain, in marked contrast to bipolar cells. These results suggest that the high contrast gain and strong nonlinearity of bipolar cells largely arise postsynaptic to cone transmitter release. Further experiments were performed to compare responses to contrast steps versus those to sinusoidal modulation. In the linear range, we show that the contrast gains of cones and horizontal cells are low and virtually identical for both steps and sinusoidal modulations. In bipolar cells, on the other hand, the contrast gain is about two times greater for steps than that for the 3-Hz sine waves. These results suggest that mechanisms intrinsic to bipolar cells act like a high-pass filter with a short time constant to selectively emphasize contrast transients over slower changes in contrast.

Adenosine A1-receptor modulation of glutamate-induced calcium influx in rat retinal ganglion cells.

Although adenosine receptors (A(1)-Rs and A(2)-Rs) have been identified in the mammalian retina, the role of adenosine in this tissue is not fully understood. The purpose of this work was to investigate the action of adenosine on glutamate-induced calcium influx in rat retinal ganglion cells (RGCs) and to determine whether adenosine modulates RGC voltage-gated calcium channels. Purified RGC cultures were generated from neonatal rats with a two-step panning procedure. Isolated RGCs were loaded with the ratiometric calcium-indicator dye fura-2, and the effect of adenosine (and related agonists and antagonists) on intracellular calcium levels ([Ca(2+)](i)) during exposure to glutamate (10 microM with 10 microM glycine) was assessed. The effect of adenosine on calcium channel currents was also studied in isolated RGCs with whole-cell patch-clamp techniques. In addition, the effect of adenosine on [Ca(2+)](i) was investigated in fura dextran-loaded RGCs in an intact adult rat retina preparation. In isolated RGCs, adenosine (10 and 100 microM) significantly reduced the glutamate-induced increase in [Ca(2+)](i) ( approximately 30%). The effect of adenosine was blocked by the A(1)-R antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), but not by the A(2)-R antagonist 3,7-dimethyl-1-propargylxanthine (DMPX). Adenosine (10 microM) inhibited calcium channel currents by 43%, and again this effect was blocked by DPCPX, but not DMPX. Adenosine (100 microM) also significantly reduced the elevation of [Ca(2+)](i) in RGCs in the intact retina during exposure to N-methyl-d-aspartate (NMDA; 100 microM). Adenosine can inhibit glutamate-induced calcium influx and voltage-gated calcium currents in rat RGCs through A(1)-R activation. This work supports a role for adenosine as a neuromodulator of mammalian RGCs.

Invulnerability of retinal ganglion cells to NMDA excitotoxicity

NMDA excitotoxicity has been proposed to mediate the death of retinal ganglion cells (RGCs) in glaucoma and ischemia. Here, we reexamine the effects of glutamate and NMDA on rat RGCs in vitro and in situ. We show that highly purified RGCs express NR1 and NR2 receptor subunits by Western blotting and immunostaining, and functional NMDA receptor channels by whole-cell patch-clamp recording. Nevertheless, high concentrations of glutamate or NMDA failed to induce the death of purified RGCs, even after prolonged exposure for 24 h. RGCs co-cultured together with ephrins, astrocytes, or mixed retinal cells were similarly invulnerable to glutamate and NMDA, though their NMDA currents were 4-fold larger. In contrast, even a short exposure to glutamate or NMDA induced the rapid and profound excitotoxic death of most hippocampal neurons in culture. To determine whether RGCs in an intact retina are vulnerable to excitotoxicity, we retrogradely labeled RGCs in vivo using fluorogold and exposed acutely isolated intact retinas to high concentrations of glutamate or NMDA. This produced a substantial and rapid loss of amacrine cells; however, RGCs were not affected. Nonetheless, RGCs expressed NMDA currents in situ that were larger than those reported for amacrine cells. Interestingly, the NMDA receptors expressed by RGCs were extrasynaptically localized both in vitro and in situ. These results indicate that RGCs in vitro and in situ are relatively invulnerable to glutamate and NMDA excitotoxicity compared to amacrine cells, and indicate that important, as yet unidentified, determinants downstream of NMDA receptors control vulnerability to excitotoxicity.

Central vision

The clinical assessment of vision by means of optotypes does by no means test just two-point resolution, since a correct naming of the letters or digits requires a preceding visual object recognition. Cortical lesions can massively deteriorate vision up to a "Seelenblindheit" in spite of intact optics and retina. There are different processing levels involved in the analysis which can be individually defective, leading to disorders from visual indiscrimination to agnosia or anomia.

Spike generator limits efficiency of information transfer in a retinal ganglion cell.

The quality of the signal a retinal ganglion cell transmits to the brain is important for preception because it sets the minimum detectable stimulus. The ganglion cell converts graded potentials into a spike train with a selective filter but in the process adds noise. To explore how efficiently information is transferred to spikes, we measured contrast detection threshold and increment threshold from graded potential and spike responses of brisk-transient ganglion cells. Intracellular responses to a spot flashed over the receptive field center of the cell were recorded in an intact mammalian retina maintained in vitro at 37 degrees C. Thresholds were measured in a single-interval forced-choice procedure with an ideal observer. The graded potential gave a detection threshold of 1.5% contrast, whereas spikes gave 3.8%. The graded potential also gave increment thresholds approximately twofold lower and carried approximately 60% more gray levels. Increment threshold "dipped" below the detection threshold at a low contrast (<5%) but increased rapidly at higher contrasts. The magnitude of the "dipper" for both graded potential and spikes could be predicted from a threshold nonlinearity in the responses. Depolarization of the cell by current injection reduced the detection threshold for spikes but also reduced the range of contrasts they can transmit. This suggests that contrast sensitivity and dynamic range are related in an essential trade-off.

Fate of multipotent neural precursor cells transplanted into mouse retina selectively depleted of retinal ganglion cells

In some parts of the CNS, depletion of a particular class of neuron might induce changes in the microenvironment that influence the differentiation of newly grafted neural precursor cells. This hypothesis was tested in the retina by inducing apoptotic retinal ganglion cell (RGC) death in neonatal and adult female mice and examining whether intravitreally grafted male neural precursor cells (C17.2), a neural stem cell (NSC)-like clonal line, become incorporated into these selectively depleted retinae. In neonates, rapid RGC death was induced by removal of the contralateral superior colliculus (SC), in adults, delayed RGC death was induced by unilateral optic nerve (ON) transection. Cells were injected intravitreally 6–48 h after SC ablation (neonates) or 0–7 days after ON injury (adults). Cells were also injected into non-RGC depleted neonatal and adult retinae. At 4 or 8 weeks, transplanted cells were identified using a Y-chromosome marker and in situ hybridisation or by their expression of the lacZ reporter gene product Escherichia coli β-galactosidase (β-gal). No C17.2 cells were identified in axotomised adult-injected eyes undergoing delayed RGC apoptosis ( n = 16). Donor cells were however stably integrated within the retina in 29% (15/55) of mice that received C17.2 cell injections 24 h after neonatal SC ablation; 6–31% of surviving cells were found in the RGC layer (GCL). These NSC-like cells were also present in intact retinae, but on average, there were fewer cells in GCL. In SC-ablated mice, most grafted cells did not express retinal-specific markers, although occasional donor cells in the GCL were immunopositive for β-III tubulin, a protein highly expressed by, but not specific to, developing RGCs. Targeted rapid RGC depletion thus increased cell incorporation into the GCL, but grafted C17.2 cells did not appear to differentiate into an RGC phenotype.

Spontaneous Activity of Morphologically Identified Ganglion Cells in the Developing Ferret Retina

Whole-cell patch-clamp recordings were made from morphologically identified ganglion cells in the intact retina of developing ferrets. As early as 3 d after birth, all ganglion cells exhibited bursts of spontaneous activity, with the interval between bursts gradually decreasing with maturity. By 2 weeks after birth, ganglion cells could be morphologically differentiated into three major classes (alpha, beta, and gamma), and at this time each cell class was characterized by a distinct pattern of spontaneous activity. Dual patch-clamp recordings from pairs of neighboring cells revealed that cells of all morphological classes burst in a coordinated manner, regardless of cell type. These observations suggest that a common mechanism underlies the bursting patterns exhibited by all ganglion cell classes, and that class-specific firing patterns emerge coincident with retinal ganglion cell morphological differentiation.

Proteomic profiling of primary retinal Müller glia cells reveals a shift in expression patterns upon adaptation to in vitro conditions.

Cultured primary retinal Müller glia cells (RMG), a glia cell spanning the entire neuroretina, have recently gained increased attention, especially with respect to their presumed in vivo role in supporting photoreceptor function and survival. Cultured RMG cells, however, are at risk to lose much of their in vivo features. To determine the conditions of isolated primary RMG cells best corresponding with their physiological role in the intact retina, we profiled the respective proteomes of RMG freshly isolated from intact pig eye, as well as from cultured material at different timepoints. Protein samples were separated by high-resolution two-dimensional electrophoresis (2-DE), and isolated proteins were identified by matrix-assisted laser desorption ionization time-of- flight (MALDI-TOF) peptide mass fingerprint. Compared with freshly isolated RMG, the in vitro protein expression patterns remain relatively stable for the first 3 days in culture but change dramatically thereafter. Proteins involved in specific RMG physiological functions, such as glycolysis, transmitter recycling, CO2 siphoning, visual pigment cycle, and detoxification, are either downregulated or absent. In contrast, cytoskeletal proteins, as well as proteins involved in motility and in proliferation, are upregulated during culture. In the present report, we show for the first time, on a systematic level, that profound changes in the RMG proteome reflect transdifferentiation from a multifunctional, highly differentiated glial cell to a dedifferentiated fibroblast-like phenotype in culture.

Topographic Plasticity in Primary Visual Cortex Is Mediated by Local Corticocortical Connections

The placement of monocular laser lesions in the adult cat retina produces a lesion projection zone (LPZ) in primary visual cortex (V1) in which the majority of neurons have a normally located receptive field (RF) for stimulation of the intact eye and an ectopically located RF (displaced to intact retina at the edge of the lesion) for stimulation of the lesioned eye. Animals that had such lesions for 14-85 d were studied under halothane and nitrous oxide anesthesia with conventional neurophysiological recording techniques and stimulation of moving light bars. Previous work suggested that a candidate source of input, which could account for the development of the ectopic RFs, was long-range horizontal connections within V1. The critical contribution of such input was examined by placing a pipette containing the neurotoxin kainic acid at a site in the normal V1 visual representation that overlapped with the ectopic RF recorded at a site within the LPZ. Continuation of well defined responses to stimulation of the intact eye served as a control against direct effects of the kainic acid at the LPZ recording site. In six of seven cases examined, kainic acid deactivation of neurons at the injection site blocked responsiveness to lesioned-eye stimulation at the ectopic RF for the LPZ recording site. We therefore conclude that long-range horizontal projections contribute to the dominant input underlying the capacity for retinal lesion-induced plasticity in V1.

Melanopsin and rod-cone photoreceptive systems account for all major accessory visual functions in mice.

In the mammalian retina, besides the conventional rod-cone system, a melanopsin-associated photoreceptive system exists that conveys photic information for accessory visual functions such as pupillary light reflex and circadian photo-entrainment. On ablation of the melanopsin gene, retinal ganglion cells that normally express melanopsin are no longer intrinsically photosensitive. Furthermore, pupil reflex, light-induced phase delays of the circadian clock and period lengthening of the circadian rhythm in constant light are all partially impaired. Here, we investigated whether additional photoreceptive systems participate in these responses. Using mice lacking rods and cones, we measured the action spectrum for phase-shifting the circadian rhythm of locomotor behaviour. This spectrum matches that for the pupillary light reflex in mice of the same genotype, and that for the intrinsic photosensitivity of the melanopsin-expressing retinal ganglion cells. We have also generated mice lacking melanopsin coupled with disabled rod and cone phototransduction mechanisms. These animals have an intact retina but fail to show any significant pupil reflex, to entrain to light/dark cycles, and to show any masking response to light. Thus, the rod-cone and melanopsin systems together seem to provide all of the photic input for these accessory visual functions.

A high-throughput on-line microdialysis-capillary assay for D-serine.

A high-throughput method is described for the analysis of D-serine and other neurotransmitters in tissue homogenates. Analysis is performed by microdialysis-capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection in a sheath flow detection cell. Sample pretreatment is not required as microdialysis sampling excludes proteins and cell fragments. Primary amines are derivatized on-line with o-phthaldialdehyde (OPA) in the presence of beta-mercaptoethanol followed by on-line CE-LIF analysis. Under the separation conditions described here, D-serine is resolved from L-serine and other primary amines commonly found in biological samples. Each separation requires less than 22 s. Eliminating the need for sample pretreatment and performing the high-speed CE analysis on-line significantly reduces the time required for D-serine analysis when compared with traditional methods. This method has been used to quantify D-serine levels in larval tiger salamander retinal homogenates, as well as dopamine, gamma-amino-n-butyric acid (GABA), glutamate and L-aspartate. D-serine release from an intact retina was also detected.

Estrogens reduce the expression of YKL-40 in the retina: implications for eye and joint diseases.

To identify modifications in the gene expression profile of the ocular posterior segment in ovariectomized (OVX) mice with and without substitutive estradiol therapy and to select differentially expressed genes that could be relevant to the natural history of human age-related macular degeneration (AMD). Chorioretinal tissues from two groups of 25 treated and untreated OVX mice were analyzed by using cDNA array technology. The expression level of selected genes was confirmed in triplicate by RT-PCR and related to the estrogenic status of the animals. Expression of the YKL-40 gene was further investigated in intact or diseased human retinas and in a murine model of experimental choroidal neovascularization (CNV), using laser pressure catapulting. Of the approximately 10,000 genes screened, only YKL-40 expression was significantly downregulated by 17-beta-estradiol. YKL-40 was expressed in intact human neural retina and in the RPE. The expression of YKL-40 was upregulated in experimental CNV and in neovascular membranes extracted from patients affected by the exudative form of AMD. These observations indicate that YKL-40 expression in the retina is modulated by serum levels of estradiol. This protein could be relevant to the development of AMD and is also a new mediator to take into account when evaluating the broad consequences of hormonal replacement therapy.

Demonstration of Cholinergic Ganglion Cells in Rat Retina: Expression of an Alternative Splice Variant of Choline Acetyltransferase

Acetylcholine acts as a neurotransmitter in the retina. Although previous physiological studies have indicated that some retinal ganglion cells may be cholinergic, several immunohistochemical studies using antibodies to choline acetyltransferase (ChAT) have stained only amacrine cells but not ganglion cells. Recently, we identified a splice variant of ChAT mRNA, lacking exons 6-9, in rat peripheral nervous system. The encoded protein was designated as ChAT of a peripheral type (pChAT), against which an antiserum was raised. In the present study, we examined expression of pChAT in rat retina, both at the protein level by immunohistochemistry using the antiserum and at the mRNA level by RT-PCR. Immunohistochemistry revealed that although no positive neurons were found in untreated intact retinas, many neurons became immunoreactive for pChAT after intravitreal injection of colchicine. Damage of the optic nerve was also effective in disclosing positive cells. Such positive neurons were shown to be ganglion cells by double labeling with a retrograde tracer that had been injected into the contralateral superior colliculus. Western blot analysis and RT-PCR revealed a corresponding band to the pChAT protein and to the amplified pChAT gene fragment, respectively, in retinal samples. In addition, ChAT activity was definitely detected in retinofugal fibers of the optic nerve. These results indicate the presence of cholinergic ganglion cells in rat retina.