optimized-protocol-for-retinal-wholemount-preparation-for-imaging

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

Weill Medical College of Cornell University

In the present study, we aimed to evaluate the pathways contributing to ATP release from mouse astrocytes during hypoosmotic stress. We first examined the expression of mRNAs for proteins constituting possible ATP-releasing pathways that have been suggested over the past several years. In RT-PCR analysis using both control and osmotically swollen astrocytes, amplification of cDNA fragments of expected size was seen for connexins (Cx32, Cx37, Cx43), pannexin 1 (Px1), the P2X7 receptor, MRP1 and MDR1, but not CFTR. Inhibitors of exocytotic vesicular release, gap junction hemi-channels, CFTR, MRP1, MDR1, the P2X7 receptor, and volume-sensitive outwardly rectifying chloride channels had no significant effects on the massive ATP release from astrocytes. In contrast, the hypotonicity-induced ATP release from astrocytes was most effectively inhibited by gadolinium (50 muM), an inhibitor of the maxi-anion channel, which has recently been shown to serve as a pathway for ATP release from several other cell types. Thus, we propose that the maxi-anion channel constitutes a major pathway for swelling-induced ATP release from cultured mouse astrocytes as well.

Maxi-anion channel as a candidate pathway for osmosensitive ATP release from mouse astrocytes in primary culture.

The maxi-anion channel with a large single-channel conductance of >300 pS, and unknown molecular identity, is functionally expressed in a large variety of cell types. The channel is activated by a number of experimental maneuvers such as exposing cells to hypotonic or ischemic stress. The most effective and consistent method of activating it is patch membrane excision. However, the activation mechanism of the maxi-anion channel remains poorly understood at present. In the present study, involvement of phosphorylation/dephosphorylation in excision-induced activation was examined. In mouse mammary fibroblastic C127 cells, activity of the channel was suppressed by intracellular application of Mg-ATP, but not Mg-5'-adenylylimidodiphosphate (AMP-PNP), in a concentration-dependent manner. When a cocktail of broad-spectrum tyrosine phosphatase inhibitors was applied, channel activation was completely abolished, whereas inhibitors of serine/threonine protein phosphatases had no effect. On the other hand, protein tyrosine kinase inhibitors brought the channel out of an inactivated state. In mouse adult skin fibroblasts (MAFs) in primary culture, similar maxi-anion channels were found to be activated on membrane excision, in a manner sensitive to tyrosine phosphatase inhibitors. In MAFs isolated from animals deficient in receptor protein tyrosine phosphatase (RPTP)zeta, activation of the maxi-anion channel was significantly slower and less prominent compared with that observed in wild-type MAFs; however, channel activation was restored by transfection of the RPTPzeta gene. Thus it is concluded that activation of the maxi-anion channel involves protein dephosphorylation mediated by protein tyrosine phosphatases that include RPTPzeta in mouse fibroblasts, but not in C127 cells.

Activation of maxi-anion channel by protein tyrosine dephosphorylation.

Neural oscillations play an important role in normal brain activity, but also manifest during Parkinson's disease, epilepsy, and other pathological conditions. The contribution of these aberrant oscillations to the function of the surviving brain remains unclear. In recording from retina in a mouse model of retinal degeneration (RD), we found that the incidence of oscillatory activity varied across different cell classes, evidence that some retinal networks are more affected by functional changes than others. This aberrant activity was driven by an independent inhibitory amacrine cell oscillator. By stimulating the surviving circuitry at different stages of the neurodegenerative process, we found that this dystrophic oscillator further compromises the function of the retina. These data reveal that retinal remodeling can exacerbate the visual deficit, and that aberrant synaptic activity could be targeted for RD treatment.

Network deficiency exacerbates impairment in a mouse model of retinal degeneration.

In this manuscript, we describe a protocol for capturing both physiological and structural properties of living neuronal tissue. An essential aspect of this method is its flexibility and fast turnaround time. It is a streamlined process that includes recording of electrophysiological neuronal activity, calcium imaging, and structural analysis. This is accomplished by placing intact tissue on a modified Millicell Biopore insert. The Biopore membrane suspends the tissue in the perfusion solution, allowing for complete access to nutrients, oxygen, and pharmacological agents. The ring that holds the membrane ensures its structural stability; forceps can be used to grip the ring without contacting the filter or the tissue, for easy transfer between multiple setups. We show that tissue readily adheres to the surface of the membrane, its entire surface is visible in transmitted light and accessible for recording and imaging, and remains responsive to physiological stimuli for extended periods of time.

A time and cost efficient approach to functional and structural assessment of living neuronal tissue.

Retinal degeneration leads to progressive photoreceptor cell death, resulting in vision loss. Subsequently, inner retinal neurons develop aberrant synaptic activity, compounding visual impairment. In retinal ganglion cells, light responses driven by surviving photoreceptors are obscured by elevated levels of aberrant spiking activity. Here, we demonstrate in rd10 mice that targeting disruptive neuronal circuitry with a gap junction antagonist can significantly reduce excessive spiking. This treatment increases the sensitivity of the degenerated retina to light stimuli driven by residual photoreceptors. Additionally, this enhances signal transmission from inner retinal neurons to ganglion cells, potentially allowing the retinal network to preserve the fidelity of signals either from prosthetic electronic devices, or from cells optogenetically modified to transduce light. Thus, targeting maladaptive changes to the retina allows for treatments to use existing neuronal tissue to restore light sensitivity, and to augment existing strategies to replace lost photoreceptors.

Block of gap junctions eliminates aberrant activity and restores light responses during retinal degeneration.

Spontaneous rhythmic activity is a hallmark feature of the developing retina, where propagating retinal waves instruct axonal targeting and synapse formation. Retinal waves cease around the time of eye-opening; however, the fate of the underlying synaptic circuitry is unknown. Whether retinal waves are unique to the developing retina or if they can be induced in adulthood is not known. Combining patch-clamp techniques with calcium imaging, we demonstrate that propagative events persist in adult mouse retina when it is deprived of inhibitory input. This activity originates in bipolar cells, resembling glutamatergic stage III retinal waves. We find that, as it develops, the network interactions progressively curtail this activity. Together, this provides evidence that the correlated propagative neuronal activity can be induced in adult retina following the blockade of inhibitory interactions.

Article	Year
Maxi-anion channel as a candidate pathway for osmosensitive ATP release from mouse astrocytes in primary culture. Cell research\| PubMed ID: 18414449	2008
Activation of maxi-anion channel by protein tyrosine dephosphorylation. American journal of physiology. Cell physiology\| PubMed ID: 19657061	2009
Network deficiency exacerbates impairment in a mouse model of retinal degeneration. Frontiers in systems neuroscience\| PubMed ID: 22383900	2012
A time and cost efficient approach to functional and structural assessment of living neuronal tissue. Journal of neuroscience methods\| PubMed ID: 23370309	2013
Block of gap junctions eliminates aberrant activity and restores light responses during retinal degeneration. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 23986234	2013
Correlated spontaneous activity persists in adult retina and is suppressed by inhibitory inputs. PloS one\| PubMed ID: 24204906	2013

Abduqodir H Toychiev

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