Retinal Ganglion Cells - Science topic
Retinal Ganglion Cells are neurons of the innermost layer of the retina, the internal plexiform layer. They are of variable sizes and shapes, and their axons project via the OPTIC NERVE to the brain. A small subset of these cells act as photoreceptors with projections to the SUPRACHIASMATIC NUCLEUS, the center for regulating CIRCADIAN RHYTHM.
Questions related to Retinal Ganglion Cells
I reprogramming glia in vitro into retinal ganglion cells. When I stain the induced neuron with retinal ganglion cells marker (Rbpms), I have a weak signal in nuclei, however, Rbpms should be in the cytosol. I check the control (only the 2nd antibody or Igg) but I did not see the same signal. The other marker of retinal ganglion cells (Brn3b) doesn't work and I am ordering from another producer.
Anyone experienced the same phenomenon and could give me an advice?
is there anyone has ever used glutamate to induce primary retinal ganglion cells death? what concentration do you use? or on neuronal PC12 cells? or other neuronal cells?
I've been doing CTB-Alexa injections for tracing from the eyes to the dLGN, and they have been working perfectly. I'd like to do intraocular injections of WGA-Alexa to trace from the dLGN to the visual cortex but haven't had much success to far. Does anyone have any experience with these injections and the most effective concentration of the WGA-Alexa dye to use? I think I may have been using too high a concentration so far.Thanks
I have been trying to record neuronal (ganglion cell) activity from the chick retina using a MEA 1060-BC-INV amplifier and a 60 channel MEA. The signal to noise ratio is low, and thus is hindering further analysis like spike sorting. Is there any tips for improving such recordings, and also is it necessary to have a perfusion setup for stable recordings, or can I manually change the medium periodically over the course of a recording? Will this affect the recording adversely with regard to there being too much disturbance during medium change?
I have sections of mouse retina that I'm staining with RBPMS and GFAP using a very standard Immunofluorescence protocol. GFAP positivity seems to consistently come up in some RGCs. This doesn't make sense. I've tried switching to TBS for the buffer, increasing the blocking incubation time (2 hrs), and using detergents (Triton (0.1%) and Tween (0.2%)).
Any thoughts? Why might this be happening?
Block: 10% Goat Serum, 1% BSA in PBS. 2 hours at RT
Primary: 1% GS, 1% BSA, primary Abs in PBS. O/N at 4*C.
Wash: 3x5min PBS
Secondary: 1% GS, 1% BSA, secondary Abs in PBS. 90 min at RT, protected from light.
Wash: 5min PBS, 10min PBS+DAPI, 5min PBS
Mounting: Antifade Gold
Primaries: GFAP (mouse, 1:100) and RBPMS (Guinea Pig, 1:100)
Secondaries: Goat anti-mouse (555 nm, 1:1000) and Goat anti-guinea pig (647 nm, 1:1000)
I used to patch RGCs in a transgenic mouse line and defined 8 cell types (PV-0 to PV-7). Several types were electrically coupled, as revealed by neurobiotin the patch pipette (see attached figure: "PV cells"). I no longer work directly on retina, but I'm still fascinated by this feature of RGCs.
PV-7 has an asymmetric dendritic field (but only weak direction selectivity when the stimulus is centred on receptive field centre). The Sanes group named them JAM-B cells. I observed small electrically-coupled neurons within the dendritic field of PV-7 neurons close toi the GCL (see attached figure: "DS and coupling"). I'm curious if it is now known what these coupled cells are and what they would contribute to visual responses?
I am following your project with great interest!
Also see Fig S1b in Farrow et al Neuron 2013
Cones and IpRGCs have a overlap in spectral sensitivity , what is the best method to assess IpRGCs response alone without cones response in a experimental setting whie performing EEG.
I've used Pubmed to look up my gene or protein of interest in RGCs and have come up with nothing. Additionally, my attempt to use the Allen Brain Atlas proved unfruitful.
Does anyone know of a user-friendly site to look up expression profiles in mouse RGCs?
I'm interested in locating the myelin transition zone in serial cross sections of the mouse optic nerve. What antibody would you recommend for use in lightly fixed cryostat sections?
thanks in advance!
The ability to detect photons near the sensory threshold will probably be compromised by the fact that the organism/retina also emits electromagnetic waves (e.g. as heat). Hecht et al (1942) used a dark adaptation of only 30 min, which still permitted light sensitivity - so the retina had probably not reached equilibrium with the environment. Dodt and Echte (1961) used a dark adaptation of 4 hours, still without the dark adapted retina ever reaching equilibrium with the environment. I had used Dodt's original setup in his lab at Bad Nauheim and retinal ganglion cells seemed sensitive even after 12 hours of dark adaptation. Are there any more enlightening estimates on the visual threshold (irradiance, photon flux, etc) below which the dark-adapted mammalian retina would not be sensitive to visible light because of environmental and self-induced photon noise? This should be near the thermal noise of the retina in the visible range. So a different way to phrase the question would be "what is the thermal noise level of the retina in the visible wavelengths?" ideally with the minimal realistic photon flux of the environment taken also into consideration.
I'm looking for H&E stained slides of retina to find how does the retina look at different stages of retinal ganglion cell death in diseases like glaucoma. Haven't found much on google.
I am trying to use a Nissl/Cresyl Violet stain to visualize ganglion cells in flat-mounted bird retinas. However, I don't think I'm seeing any individual cells after staining - all I've got is fairly uniform purple/pink retinal tissue. I believe that at the magnification I'm looking (200x) the cells should be visible. I'm new to these techniques and would really appreciate any input.
The retinas have been fixed in 4% PFA and stored in PB buffer until dissection. They're bleached in hydrogen peroxide and mounted on gelatin-subbed slides. My staining procedure is:
Xylene 10 min
100% ethanol 3 min
90% ethanol 2 min
70% ethanol 2 min
20% ethanol 2 min
ddH2O 2 min
Cresyl Violet 20 min
ddH2O brief rinse
20% ethanol 30 sec
70% ethanol 30 sec
90% ethanol 30 sec
Differentiation Solution (900:1 95% ethanol & glacial acetic acid) 1 min
100% ethanol 3 min
Xylene 4 min
I'm attaching a photo taken at 200x magnification. The scale bar is not accurate. Any help would be much appreciated, thanks!
I'm looking into options for how to purify retinal ganglion cells, and recently came across neural tracers. It seems as though the preferred method is to inject into the brain, although some studies use postmortem tissue. Does anyone have experience with using the optic nerve from enucleated eyes to stain the retinal ganglion cells? I am hoping to FACS purify and then do RNA/DNA analysis.
Is the time required for staining of the ganglion cells from the optic nerve going to be prohibatively long, or long enough to justify delivering an injection or dye sponge to the areas of interest in the brain? it seems most studies leave the crystal or dye for at least a week, is there a way to enhance the diffusion and marking of the cells?
I am looking for a current clamp recording from starburst amacrine cells of the vertebrates' retina. I have already found a similar study (Figure1, A Unique Role for Kv3 Voltage-Gated Potassium Channels in Starburst Amacrine Cell Signaling in Mouse Retina - Ozaita et al) and I am looking for another one to cross-validate the findings.
Ganglion cells in the retina have relatively small receptive fields (RF) (~1deg or less). If we assume that these neurons work as spatial filters for local contrast, we should expect that spatial frequencies (SF) stimuli lower than ~0.5cyc/deg would be hardly detected by these neurons because the local contrast within their RF is very small. However, signals with SF lower than 0.5cyc/deg are encoded by the retinal output and made accessible to higher level visual areas with wider RF (the neurons in these areas could therefore detect these kind of stimuli).
How are low spatial frequency signals transmitted then?
The simplest way would be that ganglion cells would respond not only to local contrast but also to local luminance. In this way low SF signals would be encoded in the ganglion cells output as a "place code". In other words every ganglion cell output could represent a "pixel" of the un-filtered luminance pattern projected on the retina preserving the low SF information.
Could you please suggest any sort of material addressing this question?
Currently, I use CD11b (abcam) and CD90 (BD) antibodies to purification of retinal ganglion cells by immunopanning. But I think the purification rate is low. Could you give me suggestion or information about the primary antibodies used in immunopanning.
Since the beginning of the 21st century, we know it exists other photo-receptors in the retina like the ipRGCs. These cells are not only implicated in the regulation of circadian rhythm but also in the contrast/motions detection. This diversity of functions can be explain by the different types of ipRGCs. It exist 5 types of ipRGCs (from M1 to M5) which have been characterized by their electrophysiological profile. Does anyone know if it exist specific molecular markers to differentiate these five ipRGCs types ?
I'm aware that inhibitory synapses are GABA and glycine and excitatory are glutamate. Gephyrin looks like a reasonable option for inhibitory - i.e. stains glycine and some GABA. Wondering if PSD-95 is genuinely excitatory specific?
Definitely could do with a protocol for these kinds of antibodies in 10-12 micron retinal sections if anyone could point me towards good articles or other resources.
I usually patch bipolar and amacrine cells in retinal slices, but I am trying to do some wholemount stuff at the moment. I have varying degrees of success with either clearing the membrane with an empty, broken pipette first, or just applying a lot of positive pressure on the recording pipette, punching through, and then recording. The latter technique seem to work reasonably well but if I have neurobiotin in the pipette it labels all the surrounding Muller cells. I know everyone does this differently, so I'd really like to hear different suggestions.
To support my in vitro experimental data, I start to try retina explant culture to check the axonal outgrowth of retina ganglia cell. I have tried many times with WT E18 mouse. The isolation of retina is good, and were cut into pieces round 200~500um. retina was put on the glass surface coated with laminin- (40 µg/ml) and poly-D-lysine- (100 µg/ml). The maintenance medium is containing Neural Basal medium and 1% N2 neuronal supplement (Gibco BRL), 1% penicillin-streptomycin (Gibco BRL), and 5 µg/ml insulin, 100 µg/ml transferrin, 20 nM progesterone, 0.1 mM putrescine, 30 nM selenium, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.4 mM glutamine. The problem is that the outgrowth rate of axon is very very low ( round 200~300 um round DIV10) compared to the previous report. some report said after 24h there is quite obvious axon out. some literatures used the collagen as matrix. some literatures just used glass coverslip as growth surface. I tried DRG explant culture with the same coating condition and medium condition, and the result was pretty good.
I'm interested in immuno-labelling ganglion cells in mouse retina. I am mostly interested in antibodies that would also stain the dendrites (inner plexiform layer), but it's fine if it also labels the soma and axons. I see from the literature that Thy1 is often used, but I have struggled to get any of these antibodies to work in mouse (some work in rat).
Does anyone have the product info for Thy1 or other antibodies that work for this purpose in mouse?
In whole mount preparations of the retina around postnatal day 6 appeared a layer of hyaloids vessel/inner limiting membrane (I guess this layer is it?) preventing the access to retinal ganglion cells (RGCs) for patch clamp experiments, only some RGCs are patchable. In later developmental stages this problem rises. The same problem appeared in Ca2+-imaging experiments, when retinae/RGCs are loaded with Fura2, this hyaloid vessel layer/inner limiting membrane prevents the loading of RGCs with the dye. How is it possible to avoid this problem? How to remove this layer without destroying the retina to get an good access to RGCs in more mature retinal whole mount preparations?
I have searched for quite a bit, but have not found much. Does anybody know of an antibody for specifically identifying retinal ganglion cells, which works in birds (or one which works in all amniotes/vertebrates)?
I have only found a few papers in which they used an anti-Brn3A antibody. Does anybody have experience with this?