Neuron-glia signaling in developing retina mediated by neurotransmitter spillover
There is rich research history demonstrating the important role glial cells play in developing and maintaining neural circuits ( Garrido , 2013 ) . In the mature brain , glial cells provide structural scaffolding and create physical barriers that compartmentalize synapses and limit diffusion of neurotransmitters . In addition , glial cells express a variety of neurotransmitter receptors . Activation of glial receptors causes increases in intracellular calcium that in turn may lead to the secretion of factors that influence circuit function ( Haydon and Nedergaard , 2015 ) . In the developing brain , glial cells secrete factors that are important for neuronal differentiation , migration , axonal pathfinding and synaptogenesis ( Clarke and Barres , 2013; Garrido , 2013; Chung et al . , 2015 ) . However , the mechanisms that are used for neuron-glial signaling in the developing CNS remain poorly defined . There are three types of glial cells in the retina: Müller cells ( MCs ) , astrocytes , and microglial cells . MCs , the principal glial cells of the retina , stretch radially across its thickness from the outer limiting membrane to the inner limiting membrane , sending processes throughout the inner and outer plexiform layers . MC processes envelop synapses of every neuron type , placing these cells in an ideal position to receive and respond to neuronal signals ( Newman and Reichenbach , 1996; Newman , 2004 , 2005; Reichenbach and Bringmann , 2013 ) . In adult retina , MCs maintain retinal homeostasis and integrity ( Byrne et al . , 2013 ) , and more recently they have been implicated in delivery of retinol to replenish cone opsin chromophore ( Xue et al . , 2015 ) . In zebrafish , MC ablation does not affect the laminar organization of the inner plexiform layer ( IPL , Randlett et al . , 2013 ) or the structure or organization of synapses in the outer plexiform layer ( Williams et al . , 2010 ) , in sharp contrast to ablation of MCs in developing ( Dubois-Dauphin et al . , 2000 ) and adult mice ( Byrne et al . , 2013; Shen et al . , 2012 ) , which leads to dramatic rearrangements retinal structure ( Byrne et al . , 2013 ) . However , unlike radial glial cells in the cortex and cerebellum , the role of MCs in retinal development has not been fully explored . MCs are the last retinal cell types to mature , integrating into neuronal circuits during the first two postnatal weeks ( Jadhav et al . , 2009; Williams et al . , 2010 ) when the inner retina is undergoing robust synaptogenesis . During this developmental period , the retina is spontaneously active , and ganglion cells ( the output cells of the retina ) exhibit depolarizations that spread laterally across the retina , a phenomenon termed retinal waves . These waves are mediated by circuits that change as the retina matures ( Blankenship and Feller , 2010 ) . From postnatal day 1 ( P1 ) to P9 , retinal waves are mediated by cholinergic circuits . As bipolar cells integrate into the circuit ( P10–P14 ) , the waves become mediated by glutamatergic signaling . At eye opening ( P14 ) , light activated signaling begins and retinal waves cease . Both cholinergic ( Ford et al . , 2012 ) and glutamatergic ( Blankenship and Feller , 2010; Firl et al . , 2013 ) retinal waves are associated with volume release of neurotransmitter , where the neurotransmitter is not restricted to the synaptic cleft , but spills out into the extracellular space , allowing activation of receptors on nearby ganglion cells . Hence , these waves provide a robust source of neurotransmitter that could mediate periodic activation of MCs during retinal development . Here we investigated if MCs participate in retinal waves as they integrate into neuronal circuits . We simultaneously performed whole-cell recordings of retinal ganglion cells ( RGCs ) and two-photon imaging of calcium indicators or glutamate sensors expressed specifically in MCs in order to investigate if neurotransmitters released during retinal waves reliably induce calcium transients in MCs . In addition , we determined which receptors mediate these neuron-glia interactions . Our results indicate that MCs express a complement of receptors that allow detection of neurotransmitters released during retinal waves , resulting in parallel activation of this glial network prior to eye opening .
MCs undergo a dramatic morphological transformation during the second postnatal week , a time when there are robust retinal waves . At P7 ( Figure 1B , bottom ) , a MC displays a simple morphology with its cell body residing in the inner nuclear layer ( INL ) and a thin , fiber-like process traversing the retina to form part of the inner and outer limiting membranes ( green signal in GLAST::tdTomato , middle panel , and bright punctas in GLASTCreER::GCaMP3 , right panel ) . By P11 ( Figure 1B , middle ) , MCs have extended many lateral processes ( yellow arrows ) into the IPL and these continue to grow into the adult form where they closely appose synapses and participate in removing neurotransmitters from the extracellular space ( Pow and Crook , 1996 ) . 10 . 7554/eLife . 09590 . 003Figure 1 . Morphology of Müller glial cells and their interactions with neurons change over development . ( A ) Left , Diagram of adult retinal cross-section illustrates layered circuitry ( OLM: outer limiting membrane; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer; ILM: inner limiting membrane ) and main cell types ( PR: photoreceptor; HC: horizontal cell; BC: bipolar cell; MC: Müller glial cell; AC: amacrine cell; GC: ganglion cell ) . Right , Orthogonal projection of two-photon Z-stacks shows GCaMP3 expression in MCs of a P22 GLASTCreER::GCaMP3 mouse retina . ( B ) Vibratome sections of GLASTCreER::tdTomato retinas show the structure of MCs ( green; tdTomato ) and the expression of vesicular glutamate transporter 1 ( VGLUT1 ) in bipolar cells ( magenta; anti-VGLUT1 ) at different ages . Blue stain is 4′ , 6-diamidino-2-phenylindoele ( DAPI ) for visualizing cell nuclei as landmarks . Rightmost images are XY planes of the IPLs in GLASTCreER::GCaMP3 retinas showing GCaMP3 signal at different ages . Note the expansion of MC lateral processes into the IPL with development . Yellow arrows indicate lateral processes of the Müller glial cells . ( C ) Left , Circuit diagram of the retina highlights cells recorded for figures C and D; labeling as in Figure 1A . Right , XY plane shows GCaMP3 signals of MCs in response to a retinal wave in a P9 GLASTCreER::GCaMP3 retina . Color scale indicates normalized changes in fluorescence during a retinal wave . ( D ) Simultaneous MC calcium imaging and retinal ganglion cell ( RGC ) whole-cell voltage-clamp recording ( Vm = −60 mV ) of a P9 GLASTCreER::GCaMP3 retina . Sample ∆F/F traces ( black traces ) from individual regions of interests ( ROIs ) ( that include stalks and processes of the MC population ) in response to neuronal waves recorded in a RGC ( grey trace ) . Histogram in middle denotes percentage over time of ROIs with responsive MCs . ( E ) Percentage of ROIs with responsive MCs during at least one retinal wave at different ages . P7: 1326 ROIs from 11 retinas; P9: 3027 ROIs from 14 retinas; P11: 872 ROIs from 6 retinas . Kruskal–Wallis one-way ANOVA , Dunn's post-hoc test . ***p < 0 . 001 and *p < 0 . 05 . See also Figure 1—figure supplement 1 and Video 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 09590 . 00310 . 7554/eLife . 09590 . 004Figure 1—figure supplement 1 . Comparison of two-photon calcium imaging signals in stalks and lateral processes of MCs . MC calcium transients were not compartmentalized within lateral processes , but were equally present in both lateral processes and stalks within the IPL . ( A ) Vibratome sections of GLASTCreER::TdTomato retina show the widespread expression across the slice ( left ) . Higher resolution image of boxed portion of retina ( right ) . Blue labeling: DAPI stained nuclei . ( B ) XY plane of an IPL shows MCs expressing GCaMP3 in a P9 retina ( top ) . ROI mask was generated as described in ‘Materials and methods’ for A ( middle ) . Stalks ( yellow ) and lateral processes ( red ) could be distinguished among all ROIs ( bottom ) . ROIs presenting a round-shaped area were defined as stalks; the rest were defined as lateral processes . Yellow and red rectangles distinguish stalks from lateral processes , respectively . ( C ) Simultaneous calcium imaging of MC stalks and lateral processes and whole-cell voltage-clamp recording of a RGC ( grey trace , Vm = −60 mV ) monitored in the same field of view in a P9 GLASTCreER::GCaMP3 retina . ( D ) Left , Comparison of averaged calcium signals ( ΔF/F ) from lateral processes , stalks and all ROIs ( stalks + lateral processes ) evoked by a neuronal wave indicated by the inward current ( black trace , bottom ) . The vertical dashed line denotes the beginning of the neuronal wave . Right , Graph summarizes these averaged calcium signals ( ΔF/F ) . Error bars are standard deviations . Stalks: 86 ROIs from 2 retinas; lateral processes: 94 ROIs from 2 retinas . ( E ) Graph summarizes percentage of ROIs with MC parts that respond to at least one retinal wave . Error bars are standard deviations . ( F ) Line scans across MCs show a GCaMP3 signal duration of ∼3–5 s . Our standard rate of acquisition is 0 . 74 or 1 . 7 Hz . Thus to determine if we were missing any shorter MC calcium transients and to compare the temporal resolution from our standard acquisition , we used line scans to image several MCs at 300 Hz and found that the duration of each calcium transient was still ∼3–5 s . Left; Fluorescent image at the level of the IPL with line scan location ( magenta ) ; X-time ( t ) plot below image shows 3 example ROIs drawn around MC stalks . Right; GCaMP3 fluorescent signals from these 3 ROIs . Line scan acquisition is ∼300 Hz . Calcium transients indicate a response to neuronal activity . Notably we did not observe any calcium transients with line scan that had durations shorter than our standard imaging rate . DOI: http://dx . doi . org/10 . 7554/eLife . 09590 . 004 Like other types of glial cells , MCs in the mature retina exhibit calcium transients that occur spontaneously or are evoked by various stimuli ( Metea and Newman , 2006; Reichenbach and Bringmann , 2013 ) . However , MC calcium signaling during retinal development remains unexplored . Thus , we first investigated if MC calcium transients during early development coincided with retinal waves . We visualized MC transients via two-photon calcium imaging of whole-mount retinas from mice expressing a genetically encoded calcium indicator specifically in MCs ( GLAST-CreER::ROSA26-lsl-GCaMP3 [GLAST-CreER::GCaMP3]; Figure 1A; see ‘Materials and methods’ , [Paukert et al . , 2014] ) . In the retina of GLAST-CreER mice , tamoxifen inducible Cre recombinase ( CreER ) is expressed by MCs ( Figure 1A ) . GLAST-CreER::GCaMP3 mice expressed sufficient GCaMP3 to detect MC calcium transients in the IPL as early as P7 . Retinal waves were identified by the occurrence of compound postsynaptic excitatory currents in RGCs . Simultaneous two-photon imaging of MCs and whole-cell recordings from RGCs ( Figure 1C , D ) showed periodic MC calcium transients in the stalks and lateral processes in the IPL ( Figure 1—figure supplement 1 , Video 1 ) that coincided with RGC compound postsynaptic excitatory currents ( Blankenship et al . , 2009 ) . Since the stalks and processes of MCs exhibited similar calcium responses , we pooled their results together throughout this study . We detected no wave-evoked calcium transients in other parts of MCs outside the IPL ( i . e . , in their somata , data not shown ) . The percentage of regions of interest ( ROIs , which correspond to compartments of individual MCs , see Figure 1—figure supplement 1 ) that responded to a wave ( termed responsive MCs ) was high at P7 ( 42 ± 10 . 2% , 1326 ROIs from 11 retinas ) and at P9 ( 48 ± 4 . 6% , 3027 ROIs from 14 retinas ) , but significantly lower at P11 ( 13 ± 2 . 2% , 872 ROIs from 6 retinas , Figure 1E ) . As MCs express a variety of neurotransmitter receptors , including glutamatergic and cholinergic receptors ( Wakakura and Yamamoto , 1994; Belmonte et al . , 2000 ) , MC calcium transients at different ages could be evoked by different neurotransmitters released during retinal waves . Thus , we next explored which transmitters modulated neuron-MC signaling at different developmental ages . 10 . 7554/eLife . 09590 . 005Video 1 . Wave-induced responses are shown as changes in fluorescence of the calcium indicator GCaMP3 expressed specifically in MCs in a P9 or P11 mouse retina in the presence of the glutamate uptake blocker DL-TBOA ( 25 μM ) . Electrophysiological recordings confirmed that calcium signals were correlated with RGC activity during retinal waves . Scale bars are 20 μm . Related to Figure 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 09590 . 005 Our primary hypothesis is that MC calcium transients are induced by neurotransmitters released from amacrine and bipolar cells ( the interneurons of the retina ) during retinal waves . To assess which neurotransmitters could elicit MC calcium transients during development , we first imaged MC calcium signals in the IPL in response to periodic , focal application of agonists that could be potentially involved in the neuron-glia interaction during P7 cholinergic waves ( Figure 2A–E ) . Control application of extracellular solution ( artificial cerebrospinal fluid [ACSF] ) did not evoke a MC response , indicating that the pressure injection itself did not evoke calcium transients through mechanical stimulation ( Figure 2C ) . When adenosine tri-phosphate ( ATP , 1 mM ) was applied , robust calcium transients were induced that were inhibited by the P2 receptor blocker suramin ( 100 μM; Figure 2D ) , as seen previously in the adult retina ( Uckermann et al . , 2002; Newman , 2004; Metea and Newman , 2006 ) . MCs also responded to acetylcholine ( ACh , 1 mM; Figure 2B , C; Video 2 ) , as described in cortical astrocytes ( Takata et al . , 2011 ) . These ACh-evoked MC calcium transients were reduced by the muscarinic ACh receptor antagonist atropine ( 50 µM; Figure 2E ) . Similar ACh- and ATP-evoked MC calcium transients were also observed at P9 ( during the transition from cholinergic to glutamatergic waves ) and at P11 ( during glutamatergic waves ) , indicating that MCs express multiple neurotransmitter receptors prior to eye opening ( Figure 2D , E ) . 10 . 7554/eLife . 09590 . 006Figure 2 . Volume release of acetylcholine ( ACh ) during P7 retinal waves induces calcium transients in MCs . ( A ) Diagram of retinal cross-section illustrates focal application of the agonist ( pink ) at the junction between the IPL and the GCL; labeling as in Figure 1 . ( B ) XY plane of the IPL from a P7 GLASTCreER::GCaMP3 retina showing fluorescent signals in MCs during a focal application of acetylcholine ( ACh , 1 mM , 100 ms ) pseudocolored as in Figure 1C . ( C ) Averaged MC calcium signals ( ΔF/F ) in a ROI evoked by a sequence of 3 focal applications of ACh ( solid line; 78 ROIs from 1 retina ) or artificial cerebrospinal fluid ( ACSF , dashed line; 429 ROIs from 3 retinas ) . Black arrows indicate agonist application . Shaded areas represent standard deviation . ( D ) Averaged MC calcium signals ( ΔF/F ) evoked by focal application of ATP in the absence and presence of the non-specific P2 receptor blocker suramin ( 100 μM ) at P7 , P9 and P11 . P7: 158 ROIs in control and 142 ROIs in suramin from 3 retinas; P9: 201 ROIs in control and 151 ROIs in suramin from 3 retinas; P11: 139 ROIs in control and 123 in suramin from 3 retinas . Black circle and error bars show mean ±SD . One-way ANOVA , Tukey post-hoc test ***p < 0 . 001; **p < 0 . 01 . ( E ) Averaged MC calcium signals ( ΔF/F ) evoked by focal applications of ACh in the absence and presence of the muscarinic cholinergic receptor blocker atropine ( 50 μM ) at P7 , P9 and P11 . P7: 256 ROIs in control and 396 ROIs in atropine from 3 retinas; P9: 234 ROIs in control and 264 ROIs in atropine from 3 retinas; P11: 183 ROIs in control and 121 ROIs in atropine from 3 retinas . One-way ANOVA , Tukey post-hoc test ***p < 0 . 001; **p < 0 . 01 . ( F–H ) Simultaneous MC calcium imaging ( black traces ) and RGC whole-cell voltage-clamp recording ( grey trace , Vm = −60 mV ) monitored in the same field of view from a P7 GLASTCreER::GCaMP3 retina in control solution ( F ) , in the presence of the muscarinic acetylcholine receptor blocker atropine ( 50 μM ) ( G ) , or in the presence of the non-selective P2 receptor antagonist suramin ( 100 μM ) ( H ) . Below the MC calcium transient traces are histograms showing the percentage of ROIs with responsive MCs over time . Left , Images show XY planes of the IPL with the MC fluorescent signals occurring at the time denoted by black arrows . Right , Plots summarize effects of suramin ( 350 ROIs in control and 390 ROIs in suramin from 6 retinas ) and atropine ( 267 ROIs in control and 64 ROIs in atropine from 4 retinas ) on the percentage of ROIs with responsive MCs per wave . Lines connect values from one experiment in control vs blocker . Black circle and error bars show mean ±SD . t-test **p < 0 . 01 . See also Video 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 09590 . 00610 . 7554/eLife . 09590 . 007Video 2 . Calcium transients ( ΔF/F ) in MCs expressing the calcium indicator GCaMP3 are shown in response to a series of focal applications of ATP or ACh ( 1 mM , 100 ms ) at P7 . Scale bars are 20 μm . White spots in video indicate when focal applications of agonist were applied . Related to Figure 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 09590 . 007 To explore the contribution of ACh and ATP to MC calcium transients that occur during P7 cholinergic waves , simultaneous two-photon laser scanning microscopy and whole-cell RGC recordings were performed . MC calcium transients ( Figure 2F , top black traces ) were correlated with RGC compound excitatory postsynaptic currents ( EPSCs ) that are known to be mediated by the volume release of ACh and the activation of neuronal nicotinic ACh receptors ( AChRs ) on RGCs ( Figure 2F , bottom grey traces ) ( Ford et al . , 2012 ) . Interestingly , this ACh is released by cholinergic amacrine cells that also release ATP ( Duarte et al . , 1999 ) . Thus , we tested the effect of AChR and ATP receptor blockers on wave-induced MC calcium transients . Although the wave-induced transients were blocked by the muscarinic ACh receptor ( mAChR ) antagonist atropine ( 50 µM; Figure 2G ) , they were not blocked by the ATP receptor antagonist suramin ( 100 μM; Figure 2H ) . Therefore , we conclude that at P7 , MC calcium transients are evoked by volume release of ACh , but not by ATP , and these transients are mediated by mAChRs on MCs . At P9 , circuits mediating retinal waves change from cholinergic to glutamatergic ( Blankenship and Feller , 2010 ) , and glutamatergic synapses between bipolar cells and their postsynaptic targets first appear ( Hoon et al . , 2014 ) . Thus , at P9 cholinergic and/or glutamatergic transmission could activate MC calcium transients . To determine the involvement of cholinergic transmission in MC signaling at this stage of development , we examined their response to focal application of ACh . Short applications of ACh induced calcium transients that were blocked by the mAChR blocker atropine at P9 ( Figure 2E ) , but bath application of atropine did not change the percentage of wave-induced MC calcium transients ( 57 ± 12 . 6% in control and 60 . 2 ± 7 . 8% in atropine; p = 0 . 54 , n = 4 ) . These results indicate that cholinergic signaling does not mediate neuron-MC signaling at this age . We then determined the role of glutamate in triggering these responses . Application of exogenous L-glutamate reliably evoked MC calcium transients ( Figure 3A–C; Video 3 ) . These responses were not blocked by atropine , ruling out the possibility that this agonist acted indirectly by stimulating release of ACh from amacrine cells . L-glutamate-evoked MC calcium transients were significantly reduced by a cocktail of ionotropic glutamate receptor antagonists ( iGluR: 20 μM AMPA/kainate receptor antagonist DNQX , 50 μM NMDA receptor antagonist D-AP5; Figure 3D ) , but were only slightly reduced by a mixture of metabotropic glutamate receptor antagonists ( mGluR: 8 μM LY341495 , 100 μM MCPG , 100 μM MTEP; Figure 3D ) . Blocking AMPA receptors produced substantial but incomplete inhibition of the L-glutamate-evoked response , whereas blocking NMDA receptors had no significant effect . These results suggest that glutamate elicits MC calcium transients primarily through activation of AMPA receptors , although a modest contribution of mGluRs is also observed . Notably , the reduction of L-glutamate-evoked calcium transients by glutamate receptor blockers occurred at all ages , not just during the period when glutamatergic waves predominate , indicating that MCs express glutamate receptors throughout this period of development . 10 . 7554/eLife . 09590 . 008Figure 3 . Wave-induced glial signaling is limited by extent of glutamate spillover . ( A ) Diagram of retinal cross-section illustrates focal application of the agonist ( green ) at the junction between the IPL and the GCL; labeling as in Figure 1 . ( B ) XY plane of the IPL from a P9 GLASTCreER::GCaMP3 retina shows fluorescent signals in MCs during short-application of L-glutamate ( 1 mM , 100 ms ) pseudocolored as in Figure 1C . ( C ) Averaged MC calcium signals ( ΔF/F ) in a ROI evoked by a sequence of focal applications of L-glutamate ( solid line; 145 ROIs from 3 retinas ) or ACSF ( dashed line; 78 ROIs from 1 retina ) . Black arrows represent agonist application . Shaded areas represent standard deviation . ( D ) Top , Averaged MC calcium signals ( ΔF/F ) evoked by focal applications of L-glutamate in the absence and presence of a cocktail of ionotropic glutamate receptor blockers ( iGluR blockers; 20 μM DNQX and 50 μM D-AP5 ) at P7 ( 462 ROIs from 3 retinas ) , P9 ( 708 ROIs from 4 retinas ) and P11 ( 1342 ROIs from 5 retinas ) . Bottom , Averaged MC calcium signals ( ΔF/F ) evoked by focal application of L-glutamate in the absence and presence of the ionotropic AMPA glutamate receptor blocker DNQX ( 20 μM; 1311 ROIs from 7 retinas ) , the ionotropic NMDA glutamate receptor blocker D-AP5 ( 50 μM; 907 ROIs from 4 retinas ) and a cocktail of metabotropic glutamate receptor blockers ( mGluR blockers; 8 μM LY341495 , 100 μM MCPG and 100 μM MTEP; 937 ROIs from 4 retinas ) at P9 . One-way ANOVA , Tukey post-hoc test ***p < 0 . 001; **p < 0 . 01 and *p < 0 . 05 . ( E–H ) Simultaneous MC calcium imaging ( black traces ) and RGC whole-cell voltage-clamp recording ( grey trace , Vm = −60 mV ) monitored in the same field of view at P9 ( E ) and P11 ( G ) in control solution and in the presence of 25 μM DL-TBOA . Below calcium traces are histograms showing the percentage of ROIs with responsive MCs over time . Plots summarize DL-TBOA effects on the percentage of ROIs with responsive MCs per retinal wave at P9 ( F; 3027 ROIs in control and 3549 ROIs in DL-TBOA from 8 retinas ) and P11 ( H; 872 in control and 1342 in DL-TBOA from 6 retinas ) . Lines connect values from one experiment in control vs DL-TBOA . Black circle and error bars show mean ±SD . t-test , ***p < 0 . 001 . See also Video 3 and Video 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 09590 . 00810 . 7554/eLife . 09590 . 009Video 3 . Calcium transients ( ΔF/F ) in MCs expressing the calcium indicator GCaMP3 are shown in response to a series of focal applications ( 100 ms ) of ACSF or L-glutamate ( 1 mM ) at P9 . Scale bars are 20 μm . White spots in video indicate when focal applications of agonist were applied . Related to Figure 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 09590 . 009 To study MC calcium transients evoked by glutamatergic waves , we combined two-photon laser scanning microscopy with whole-cell recordings from RGCs at P9 and P11 . At P9 , wave-induced MC calcium transients were correlated with compound EPSCs in RGCs ( Figure 3E , F ) that are mediated by release of glutamate from bipolar cells and by activation of ionotropic glutamate receptors on RGCs ( Bansal et al . , 2000; Wong et al . , 2000; Zhou and Zhao , 2000 ) . However , at P11 , wave-induced MC calcium transients were dramatically reduced ( Figures 1E , 3G , H ) although robust glutamate spillover occurs at this age ( Blankenship et al . , 2009; Firl et al . , 2013 ) and exogenous glutamate induced MC calcium transients ( Figure 3B–D ) . We hypothesize that this reduction of MC activity is a consequence of the upregulation of glutamate transporters at P11 ( Pow and Barnett , 2000; Pannicke et al . , 2002 ) , which may limit the amount of glutamate that reaches MC membranes to induce calcium transients . To test this hypothesis , we blocked glutamate transporters by bath applying DL-TBOA ( 25 µM ) , an agent previously shown to increase extracellular glutamate levels ( Blankenship et al . , 2009; Firl et al . , 2013 ) . At P11 , DL-TBOA drastically increased the percentage of responsive MCs from 13 ± 2 . 2% to 61 ± 4 . 3% ( Figure 3G , H; 872 ROIs in control and 1342 ROIs in DL-TBOA from 6 retinas ) . Interestingly , at P9 , DL-TBOA did not significantly change the percentage of responsive MCs , increasing only slightly from 48 ± 4 . 5% to 58 ± 10 . 0% ( Figure 3E , F; 3027 ROIs in control and 3549 ROIs in DL-TBOA from 8 retinas ) . We conclude that at ages earlier than P11 , transporters do not strongly regulate glutamate spillover , but by P11 , they mediate significant glutamate uptake , decreasing the amount of glutamate that reaches MC receptors . We expressed the membrane-bound intensity-based glutamate sensing fluorescent reporter iGluSnFR ( Marvin et al . , 2013 ) either in MCs or neurons ( Borghuis et al . , 2013 ) to test directly if extracellular glutamate was reaching the membrane of MCs during retinal waves . The iGluSnFR signal is proportional to the amount of glutamate reaching the membrane ( Marvin et al . , 2013 ) ; thus , the lack of fluorescence in the INL where MC cell bodies are located indicates that during early development there is little glutamate release outside of the IPL . To detect glutamate signals during retinal waves , we simultaneously performed two-photon imaging of iGluSnFR signals in the IPL and whole-cell voltage clamp recordings from RGCs ( Figure 4; Figure 4—figure supplement 1 ) . At both P9 and P11 , the processes of neurons that expressed iGluSnFR exhibited large glutamate transients that propagated throughout the field of view with each retinal wave ( Figure 4A , C; 160 ROIs from 5 retinas at P9 and 160 ROIs from 5 retinas at P11; Video 4 ) , consistent with the key role played by glutamate in inducing retinal waves at these ages . In contrast , although many MCs expressing iGluSnFR displayed glutamate transients during P9 waves ( 38 ± 4% , 1023 ROIs from 5 retinas , Figure 4B , C ) , the percentage of responsive MCs decreased dramatically by P11 ( 6 ± 2% , 269 ROIs from 5 retinas at P11; Figure 4B , C ) . Inhibiting glutamate transporters with DL-TBOA ( 25 µM ) increased the percentage of responsive MCs at P11 ( 31 ± 7% , 822 ROIs from 5 retinas; Video 5 ) and reduced the inter-event intervals for MC calcium and glutamate transients ( Figure 4D; Figure 4—source data 1 ) , indicating that glutamate uptake limits spillover-induced activation of MCs at this age . DL-TBOA exerted a minimal effect on MC calcium and glutamate transients at P9 ( Figure 4B–D ) . These findings corroborate the hypothesis that the lower expression of glutamate transporters at P9 enables robust entrainment of MCs during retinal waves . 10 . 7554/eLife . 09590 . 010Figure 4 . Glutamate released during neuronal waves reaches MC membrane at P9 but not at P11 . ( A ) Left , Diagram of a retinal cross-section illustrates neuronal expression of iGluSnFR ( green ) in the IPL and simultaneous voltage-clamp recording of a RGC ( grey ) . XY plane of the IPL shows iGluSnFR expression in neuronal membranes at P11 . Right , Simultaneous imaging of AAV9-2YF-hSynapsin-iGluSnFR signals in neuronal membranes ( green traces ) and whole-cell voltage-clamp recording of a RGC ( grey trace , Vm = −60 mV ) monitored in the same field of view at P11 in control and in the presence of 25 μM DL-TBOA . Above the whole-cell voltage-clamp trace are histograms showing the percentage of ROIs within a neuronal iGluSnFR signal . ( B ) Left , Diagram of a retinal cross-section illustrates glial expression of iGluSnFR ( green ) and simultaneous voltage-clamp recording of a RGC ( grey ) . XY plane of the IPL shows iGluSnFR expression in MCs . Right , Simultaneous imaging of ShH10-CMV-iGluSnFR signals in MCs ( green traces ) and whole-cell voltage-clamp recording of a RGC ( grey trace , Vm = −60 mV ) monitored in the same field of view at P9 and P11 in control and in the presence of 25 μM DL-TBOA . Above each whole-cell voltage-clamp trace are histograms showing the percentage of ROIs with glial iGluSnFR signals in response to retinal waves . ( C ) Plot summarizes DL-TBOA effects on the participation of neuronal ( 160 ROIs from 5 retinas at P9 and 160 ROIs from 5 retinas at P11 ) and MC ( 1023 ROIs from 5 retinas at P9 and 1201 ROIs from 5 retinas at P11 ) ROIs per retinal wave . Lines connect values from one experiment in control vs DL-TBOA . Black circle and error bars are mean ±SD . t-test ***p < 0 . 001 . ( D ) Cumulative probability distribution of inter-transient intervals of iGluSnFR ( green traces ) and GCaMP3 ( black traces ) signals in MC ROIs at P9 and P11 . Control in solid lines and DL-TBOA in dashed lines . See also Figure 4—figure supplement 1 and Videos 4 , 5 . DOI: http://dx . doi . org/10 . 7554/eLife . 09590 . 01010 . 7554/eLife . 09590 . 011Figure 4—source data 1 . Cumulative probability distributions of inter-transient intervals of iGluSnFR and GCaMP3 signals in MC ROIs for each experiment at P9 and P11 , and in absence or in presence of DL-TBOA . DOI: http://dx . doi . org/10 . 7554/eLife . 09590 . 01110 . 7554/eLife . 09590 . 012Figure 4—figure supplement 1 . Methodological tools to study neuron-glia interaction mediated by glutamate spillover in whole mount retinas . ( A ) Orthogonal projection of two-photon Z-stacks shows expression of the intensity-based glutamate sensor iGluSnFR in ganglion and amacrine cell membranes of a P9 wild-type mouse retina . AAV9-2YF-hSynapsin1-iGluSnFR was tail vein injected at P2 . Yellow solid and dashed lines indicate the XY planes chosen to illustrate iGluSnFR expression in neuronal membranes in the IPL and GCL , respectively ( middle panel ) . Magenta overlay indicates an iGluSnFR-expressing ON-OFF ganglion cell targeted for whole-cell recording and filled with a red fluorescent dye ( Alexa 594 ) . Right panel: Z-projection and X-Z projection of this ON-OFF RGC ( magenta ) and the neuronal dendritic network expressing iGluSnFR ( green ) . ( B ) Orthogonal projection of two-photon Z-stacks shows iGluSnFR expression in MCs of a P9 wild-type mouse retina . ShH10-CMV-iGluSnFR was intravitreally injected at P4 . Yellow solid and dashed lines indicate the XY planes chosen to illustrate iGluSnFR expression in MC membranes in the IPL and GCL , respectively ( right panel ) . DOI: http://dx . doi . org/10 . 7554/eLife . 09590 . 01210 . 7554/eLife . 09590 . 013Video 4 . Wave-induced fluorescence changes of the glutamate sensor iGluSnFR expressed in RGCs and amacrine cells in a P11 mouse retina are shown in the presence of the glutamate uptake blocker DL-TBOA ( 25 μM ) . Electrophysiological recordings confirmed that glutamate signals were correlated with RGC activity during retinal waves . Scale bars are 10 μm . Related to Figure 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 09590 . 01310 . 7554/eLife . 09590 . 014Video 5 . Wave-induced fluorescence changes of the intensity-based glutamate sensing fluorescent reporter iGluSnFR expressed specifically in MCs in a P11 mouse retina are shown in the presence of the glutamate uptake blocker DL-TBOA ( 25 μM ) . Electrophysiological recordings confirmed that glutamate signals were correlated with RGC activity during retinal waves . Scale bars are 20 μm . Related to Figure 4 . DOI: http://dx . doi . org/10 . 7554/eLife . 09590 . 014 Several lines of evidence support the hypothesis that MC calcium transients depend upon the direct activation of MC glutamate receptors during the period of glutamatergic retinal waves . First , MCs are known to express AMPARs ( Wakakura and Yamamoto , 1994; Sullivan and Miller , 2010 ) . Second , glutamate-induced MC calcium transients depend upon the activation of ionotropic AMPARs ( Figure 3D ) . Third , the frequencies of glutamate and calcium transients in MCs are indistinguishable , indicating that MC calcium transients are driven by glutamate reaching the MC membranes ( Figure 4D; Figure 4—source data 1 ) . Establishing that wave-induced MC calcium transients are mediated by ionotropic glutamate receptors on MCs requires selective inhibition of these receptors; however , this approach is complicated by the fact that depolarization of RGCs during retinal waves also depends on AMPAR activation . Since the AMPAR antagonist DNQX may have an effect on glutamate release from bipolar cells , we tried to elucidate its effects in this circuit . In the presence of DNQX , we simultaneously monitored EPSCs from RGCs with whole-cell recordings and glutamate release from bipolar cells with neuronal iGluSnFR imaging ( Figure 5A , B ) . As described previously ( Blankenship et al . , 2009 ) , DNQX ( 20 µM ) significantly decreased the amplitude of the postsynaptic current in RGCs . However , it did not change the amount of wave-induced glutamate release from bipolar cells monitored using iGluSnFR ( Figure 5A ) . With this knowledge , we used DNQX to determine the role of AMPARs in wave-induced MC calcium transients . These experiments were conducted in the presence of DL-TBOA to maximize the amount of glutamate spillover . We found that DNQX significantly decreased the percentage of responsive MCs during waves ( Figure 5C , Figure 5—figure supplement 1A ) , whereas the NMDAR antagonist D-AP5 ( 50 µM ) had no significant effect on MC activity ( Figure 5—figure supplement 1B , C; 703 ROIs in control and 656 ROIs in D-AP5 from 2 retinas ) . Our results strongly suggest that glutamate acts directly on MC AMPARs to increase intracellular calcium . However , it will be necessary to selectively delete AMPARs from MCs to establish their contribution to MC calcium signaling during this period of development . 10 . 7554/eLife . 09590 . 015Figure 5 . Neuron-glia interaction is mediated by ionotropic AMPA glutamate receptors . ( A ) Left , Diagram of a retinal cross-section illustrates neuronal expression of iGluSnFR ( green ) in the IPL and simultaneous voltage-clamp recording of a RGC ( grey ) . Right , Simultaneous imaging of AAV9-2YF-hSynapsin1-iGluSnFR signals in neuronal membranes ( green traces ) and whole-cell voltage-clamp recording of a RGC ( grey trace , Vm = −60 mV ) monitored in the same field of view at P9 in control and in the presence of 20 μM DNQX . Above each whole-cell voltage-clamp trace are histograms showing the percentage of ROIs with responsive neuronal iGluSnFR signals . ( B ) Graph summarizes the effect of DNQX on the change in volume release of glutamate ( ΔF/F ) and on the change in amplitude of the RGC excitatory postsynaptic currents . Each open circle plots the value from one experiment . Note , DNQX does not modify the amount of glutamate released from bipolar cells . Black circle and error bars are mean ±SD . t-test , **p < 0 . 01 . ( C ) Left , Diagram of a retinal cross-section illustrates glial expression of GCaMP3 ( black ) and simultaneous voltage-clamp recording of a RGC ( grey ) . Right , Graph summarizes the effect of DNQX on the percentage of ROIs with responsive glia at P9 in the presence of DL-TBOA ( 25 µM ) . Lines connect values from one experiment in control ( 974 ROIs ) , DL-TBOA ( 1154 ROIs ) , DL-TBOA+DNQX ( 397 ROIs ) and wash ( 987 ROIs ) . Data collected from 5 retinas . Black circle and error bars are mean ±SD . One-way ANOVA , Tukey post-hoc test ***p < 0 . 001 . See also Figure 5—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 09590 . 01510 . 7554/eLife . 09590 . 016Figure 5—figure supplement 1 . The AMPA receptor antagonist DNQX , but not the NMDA receptor antagonist D-AP5 , decreases neuron-glia interaction at P9 . ( A ) Simultaneous calcium imaging of MCs ( black traces ) and whole-cell voltage-clamp recording of a RGC ( grey trace , Vm = −60 mV ) monitored in the same field of view in control , in the presence of 25 µM DL-TBOA , in the presence of 25 µM DL-TBOA + 20 µM DNQX and after wash . Below the whole-cell voltage-clamp trace are histograms showing the percentage of ROIs with responsive MCs in each condition . See also Figure 5 for quantification . ( B ) Simultaneous calcium imaging of MCs ( black traces ) and whole-cell voltage-clamp recording of a RGC ( grey trace , Vm = −60 mV ) monitored in the same field of view during the first minutes of DL-TBOA application ( black arrow ) , after 8–10 min of the drug application , and after adding D-AP5 and waiting for 10 min before recording . Below each whole-cell voltage-clamp trace are histograms showing the percentage of ROIs with responsive MCs in each condition . ( C ) Plot summarizes effect of D-AP5 ( 50 μM ) on the proportion of responsive ROIs at P9 ( 318 ROIs in control , 390 ROIs in DL-TBOA and 341 ROIs in DL-TBOA + D-AP5 from 2 retinas ) . Lines connect values from one experiment in control , DL-TBOA , DL-TBOA + D-AP5 , and wash . Black circle and error bars are mean ±SD . DOI: http://dx . doi . org/10 . 7554/eLife . 09590 . 016 Previous studies have shown that mGluRs contribute to neuron-astrocyte signaling in the brain , particularly during early development ( Grosche et al . , 1999; Kirischuk et al . , 1999 ) . To determine the extent that mGluRs are involved in wave-induced MC calcium transients in the retina we performed two experiments . First , we conducted simultaneous whole-cell recordings from RGCs and two-photon calcium imaging of MC activity in the presence of mGluR antagonists ( 8 μM LY341495 , 100 μM MCPG , 100 μM MTEP ) . In these conditions , there was a small , but not significant , decrease in the percentage of responsive MCs at P9 ( Figure 6D ) , consistent with the contribution of mGluRs to MC calcium transients evoked by exogenous L-glutamate ( Figure 3B–D ) . Second , we imaged the spontaneous activity of MCs in retinas isolated from mice that lack IP3R2 ( IP3R2-KO mice ) , which has been shown to be required for metabotropic receptor-induced calcium signaling in adult MCs ( Lipp et al . , 2009 ) , using a fluorescent calcium dye ( Figure 6A , B ) . The density of MC labeling was not dramatically different between GCaMP3 expressing mice vs MCs loaded with organic dyes , although this was not compared quantitatively . Wave-like calcium transients persisted in IP3R2-KO MCs and a similar percentage of MCs participated in these events ( Figure 6C , D ) indicating that IP3–dependent calcium signaling contributes minimally to MC calcium transients during retinal waves . Together , these findings suggest that volume release of glutamate during retinal waves evokes MC calcium transients primarily by activating AMPA receptors on MCs . 10 . 7554/eLife . 09590 . 017Figure 6 . mGluR and mAChRs contribute minimally to wave-induced MC calcium transients at P9 . ( A ) Schematic of mGluR-mediated pathway for increases in intracellular calcium in Müller glial cell . ( B ) XY plane of the IPL shows Cal-520 loading from an IP3R2-KO P9 retina . The whole field of view was divided in 16 ROI represented by the white rectangles . ( C ) Example traces of spontaneous MC calcium signals ( ΔF/F ) in IP3R2-KO retinas . ( D ) Summary of the effects of genetic and pharmacological manipulations of GPCR signaling in MCs using IP3R2-KO mice , mAChR antagonist ( 50 µM atropine ) , and mGluR antagonists ( 8 μM LY341495 , 100 μM MCPG , 100 μM MTEP ) . DOI: http://dx . doi . org/10 . 7554/eLife . 09590 . 017
In this study we elucidated the spatio-temporal characteristics and molecular pathways by which neurons and MCs communicate in the developing retina . We found that retinal waves were correlated with calcium transients in a large proportion of MCs , demonstrating that spontaneous activity encompasses both neuronal and glial networks at this age . Our physiological results indicate that MCs express multiple neurotransmitter receptor types throughout this period , allowing neuronal control of MC activity to be sustained as neuronal synchronization shifts from being dependent on cholinergic to glutamatergic signaling . The proportion of MCs that responded to waves with calcium transients decreased significantly from P9 to P11 , despite the presence of retinal waves . This age-dependent decline in MC activation during retinal waves was caused by an increase in glutamate transporter expression , which restricted glutamate spillover from nearby synapses limiting activation of MC AMPARs . These data indicate that MCs in the developing retina use multiple signaling pathways to participate in spontaneous , correlated activity with neurons during a crucial period of retinal development . Many different signaling mechanisms have been implicated in inducing calcium transients in astroglial cells throughout the peripheral and central nervous system ( Volterra et al . , 2014; Haydon and Nedergaard , 2015; Nimmerjahn and Bergles , 2015 ) . The robustness and mechanisms of signaling vary tremendously , even at the same synapses , with age , preparation , and state of arousal . Our data indicate that MCs in the retina prior to eye opening are capable of responding to different neurotransmitters with increases in intracellular calcium ( Figures 2A , 3A ) . Neurotransmitter-evoked calcium transients were observed in the stalks and lateral processes of MCs that transverse the IPL during a time when there is robust synaptogenesis . These multiple receptors allow MCs to detect neurotransmitter released during retinal waves whether it is ACh or glutamate , thereby extending the developmental period over which neuron-glia signaling occurs . The neuron-glia signaling we observed between neurons and MCs in the retina during glutamatergic waves is similar to signaling that occurs in Bergmann glial cells ( BGs ) , a type of radial astroglial cell found in the cerebellum . BGs express calcium permeable AMPARs that are activated by glutamate released at parallel and climbing fiber synapses ( Bergles et al . , 1997; Clark and Barbour , 1997; Piet and Jahr , 2007 ) , raising the possibility that AMPAR expression may be a conserved properties of radial-type astroglial cells . Although the role of AMPARs in BG activity is still controversial ( Müller et al . , 1996; Iino et al . , 2001; Nimmerjahn et al . , 2009 ) , in vivo studies indicate that calcium signaling in BGs during locomotion depends on the activation of AMPARs ( Nimmerjahn et al . , 2009 ) and disruption of this signaling ( Iino et al . , 2001; Saab et al . , 2012 ) , results in prolonged synaptic events that are associated with altered motor behavior and motor learning . Moreover , the mechanism of interaction between neurons and MCs we observed during cholinergic retinal waves is similar to previous findings in visual cortex and hippocampus , where cholinergic neuron stimulation leads to astrocyte calcium transients via activation of muscarinic acetylcholine receptors and require IP3R2 ( Chen et al . , 2012; Navarrete et al . , 2012 ) . Whether neuronal activity during retinal waves guides the development of MCs is still unclear . MCs exhibit dramatic morphological changes during the period from P7–P11 as their fine processes grow laterally into the IPL and ensheath synapses . Ensheathment of synapses by astrocytes has been shown to promote functional isolation of synaptic signaling by creating a barrier to diffusion of neurotransmitters and by placing neurotransmitter transporters near sites of release . It is possible that ablation of AMPARs in MCs would result in retraction of the lateral processes away from synapses , as observed in BGs after inhibition of calcium permeable AMPARs ( Iino et al . , 2001 ) or genetic ablation of GluA1 and GluA4 ( Saab et al . , 2012 ) . Our findings in the developing retina differ from studies performed in the adult retina , in which glutamate does not evoke calcium transients in MCs ( Newman and Zahs , 1997; Newman , 2005 ) , suggesting that glutamaterigic signaling through AMPARs in these cells may be limited to the early stages of synapse formation and refinement . Astrocytes have also been shown to play critical roles in promoting the refinement and maturation of neural circuits ( Stevens , 2008; Clarke and Barres , 2013; Chung et al . , 2015 ) by secreting synaptogenic factors , such as thrombospondin , hevin , protocadherins , BDNF , and TGF-beta , which are critical for synapse formation between RGCs and their central targets in the brain ( reviewed in Chung and Barres , 2012; Clarke and Barres , 2013 ) . In addition , astrocytes play an important role in refining nascent circuits in the visual system by engulfing unused or inappropriate synapses ( Chung et al . , 2015 ) . Our findings indicate that robust signaling occurs between retinal neurons and MCs between the ages P7 and P11 , a time period when there is robust synaptogenesis in the retina ( Hoon et al . , 2014 ) . The close lineage relationship and functional similarities between astrocytes and MCs increase the possibility that MCs perform similar roles in the retina , particularly in regions such as the IPL where they are the sole astroglial cell . Previous studies have shown that changes in intracellular calcium can trigger the release of so-called gliotransmitters such as ATP , glutamate and D-serine from astrocytes that can influence the activity of surrounding neurons ( Araque et al . , 2014; Newman , 2015 ) . In the retina , activation of AMPARs promotes the release of D-serine , a co-agonist of NMDA receptors in ganglion cells , that has been shown to play an important role in synaptic plasticity ( Stevens , 2008; Sullivan and Miller , 2010 ) , raising the possibility that MC activity induced during retinal waves participates in synaptic refinement through release of gliotransmitters and/or synaptogenic factors . Selective manipulation of MC calcium signaling during this period of remarkable refinement will help to further define their roles in the development of distinct retinal circuits .
GLASTCreER::GCAMP3 mice were generated by cross breeding GLAST-CreER BAC transgenic mice [Tg ( Slc1a3-cre/ERT ) 1Nat/J] ( de Melo et al . , 2012 ) to ROSA26-lsl-GCaMP3 reporter mice ( Paukert et al . , 2014 ) . GLASTCreER::tdTomato mice were generated by crossing GLASTCreERBAC mice to mice that ubiquitously express tdTomato preceded by a loxP-flanked stop cassette ( B6;129S6-Gt ( ROSA ) 26Sortm9 ( CAG-tdTomato ) Hze/J , from The Jackson Laboratory , Bar Harbor , ME ) . The gene targeting and generation of IP3R2 knock-out mice have been previously described ( Li et al . , 2005 ) . All animal procedures were approved by the University of California ( UC ) Berkeley and Johns Hopkins University's Animal Care and use Committees and conformed to the NIH Guide for the Care and Use of Laboratory Animals , the Public Health Service Policy , and the SFN Policy on the Use of Animals in Neuroscience Research . Intraperitoneal injection of 0 . 5 mg of 4-hydroxytamoxifen ( 50:50 E and Z isomers , Sigma-Aldrich , St Louis , MO ) at 4 and 2 days before each experiment reliably induced the expression of GCaMP3 in MCs by P5 , P7 or P11 . For the glutamate experiments , the intensity-based glutamate sensing fluorescent reporter iGluSnFR ( Borghuis et al . , 2013; Marvin et al . , 2013 ) was specifically expressed either in MCs or in retinal neurons ( ganglion and amacrine cells ) . Expression of iGluSnFR in MCs was accomplished by intravitreally injecting C57BL/6J mice ( The Jackson Laboratory , Bar Harbor , ME ) of either sex at P4 with a capsid-modified adeno-associated virus incorporating a CMV promoter driving expression of the iGluSnFR cDNA ( ShH10-CMV-iGluSnFR; 1 μl ) ( Klimczak et al . , 2009; Dalkara et al . , 2011 ) . Expression of iGluSnFR in neurons was performed via tail vein injection at P2 of adeno-associated virus serotype 9 carrying 2 tyrosine mutations ( Dalkara et al . , 2011 ) under control of the human synapsin-1 promoter ( AAV9-2YF-hSynapsin1-iGluSnFR , 10 μl ) from P2 C57BL/6J mice . All AAV vectors were produced according to the methods described in Flannery and Visel ( 2013 ) . Calcium indicator Cal-520-AM ( AAT Bioquest , Sunnyvale , CA; Figure 6 ) was bath loaded into MCs using methods analogous to other organic dyes ( Newman and Zahs , 1997; Bansal et al . , 2000 ) . Isolated retinas were incubated with Cal-520 ( 10 µM ) prepared in ACSF containing 1% DMSO and 0 . 02% pluronic acid for 30 min in an oxygenated chamber at 32°C . The specificity of calcium signals in MCs was achieved by using the AM version of the organic dye that restricts loading to glial cells in developing retina and by two-photon imaging in the IPL . Specificity of loading into MCs was confirmed by comparison with images obtained from GLASTCreER::R26-lsl-tdTomato mice ( GLASTCreER::tdTomato mice ) . The evidence that expression was restricted to MCs was based on morphology . MCs have a distinct morphology , in which they traverse the entire retina with lateral processes expanding from their stalks at the level of IPL and OPL . Our two-photon and confocal images of tdTomato immunofluorescence from GLASTCreER::tdTomato mice confirmed these morphological features ( Figure 1B ) . The animals were anesthetized with isoflurane inhalation and decapitated . After enucleation of the eyes , the retinas were dissected in oxygenated ( 95% O2–5% CO2 ) ACSF ( containing [in mM] 119 . 0 NaCl , 26 . 2 NaHCO3 , 11 glucose , 2 . 5 KCl , 1 . 0 K2HPO4 , 2 . 5 CaCl2 , and 1 . 3 MgCl2 ) . The isolated retinas were then mounted ganglion cell side up on filter paper ( Millipore , Billerica , MA ) and transferred into the recording chamber of an upright microscope for simultaneous imaging and electrophysiological recording . Retinas were fixed in 4% paraformaldehyde and cut into 120 μm sections with a vibratome . Sections were washed in PBS ( 10 min ) and then a blocking solution ( 2% donkey serum , 1% bovine serum albumin , 0 . 2% Triton X-100; 45 min at room temperature ) . Sections were then incubated in primary antibodies at 4°C overnight ( 1:200 guinea-pig anti-VGLUT1 , Chemical International , Temecula , CA ) . Retinas were washed in blocking solution ( 3 times , 10 min ) . The sections were subsequently incubated in a secondary antibody ( 1:200 Alexa fluor 647 anti-guinea-pig , Invitrogen , Grand Island , NY; 45 min ) and then washed in blocking solution ( 3 times , 10 min ) . Sections were mounted on slides in anti-fade medium containing 4′ , 6-diamidino-2-phenylindole and imaged with a Zeiss LSM 780 confocal microscope . Note , the labeling of MC somas from the GLASTCreER::tdTomato mice is more apparent than in the GLASTCreER::GCaMP3 mice because it was visualized via immunofluorescence , which significantly improved signal-to-noise . In addition , tdTomato confocal images were acquired from retinal slices , while GCaMP3 two-photon images were acquired from whole mount flat retinas , where signal strength was reduced as a function of depth . Two-photon imaging of neurons and MCs in the IPL was performed using either a custom-modified two-photon microscope ( Fluoview 300 , Olympus America , Melville , NY ) or a custom-built two-photon microscope . Time series images were acquired using Olympus 60× , 1 NA , LUMPlanFLN objectives , and two-photon excitation of GCAMP3 was evoked with an ultrafast pulsed laser ( Chameleon Ultra , Coherent , Santa Clara , CA ) tuned to 920 nm on both microscopes . For imaging Cal520 ( Figure 6 ) the laser was tuned to 820 nm . The microscope was controlled by Fluoview Viewer software or ScanImage software ( version 3 . 8 , www . scanimage . org ) . Images ( 256 × 256 pixels ) were acquired at 0 . 74 or 1 . 7 Hz at 2 or 4 ms/line . Scan parameters were [pixels/line × lines/frame ( frame rate in Hz ) ]: [256 × 256 ( 0 . 74 − 1 . 7 ) ] , at 2–4 ms/line . Line scans were obtained at 300 Hz and down-sampled to 30 Hz for presentation in Figure 1—figure supplement 1 . Whole-cell voltage clamp recordings were made from whole-mount retinas continuously superfused in oxygenated ACSF ( 32–34°C ) at a rate of 2–4 ml/min . Retinas were visualized under infrared illumination ( 870 nm ) . Voltage-clamp recordings from somas of ganglion cells ( holding potential of −60/−65 mV ) were obtained using glass microelectrodes of 4–5 MΩ ( PC-10 pipette puller; Narishige , East Meadow , NY ) filled with an internal solution containing ( in mM ) : 110 CsMeSO4 , 2 . 8 NaCl , 4 EGTA , 5 TEA-Cl , 4 adenosine 5′-triphosphate ( magnesium salt ) , 0 . 3 guanosine 5′-triphosphate ( trisodium salt ) , 20 HEPES and 10 phosphocreatine ( disodium salt ) , pH 7 . 2 and 290 mOsm . The liquid junction potential correction for this solution was −13 mV . Signals were acquired using pCLAMP 9 recording software and a Multiclamp 700 A amplifier ( Molecular Devices , Sunnyvale , CA ) , sampled at 20 kHz and low-pass filtered at 2 kHz . RGC dendritic stratification was visualized by including 20 μM Alexa Fluor 594 ( Invitrogen , Grand Island , NY ) in the intracellular solution . The dendritic morphology of dye-injected RGCs was reconstructed by two-photon imaging with the laser tuned to 780 nm . Images ( RGCs and whole retina ) were acquired at z intervals of 0 . 5 μm using a 60× objective ( Olympus 60× , 1 NA , LUMPlanFLN ) . Images were later reconstructed from image stacks with ImageJ . For pharmacology experiments , recordings were performed 10 min after perfusion of ACSF with pharmacological agents at the following concentrations into the recording chamber: 25 μM DL-TBOA , 50 μM D-AP5 , 20 μM DNQX , 100 μM MCPG , 100 μM MTEP , 8 μM LY341495 , 50 μM atropine , and 100 μM suramin hexasodium salt . All compounds were purchased from Tocris Bioscience , Minneapolis , MN and prepared in dH2O and diluted to the final concentration in ACSF . DL-TBOA was prepared in 0 . 1% DMSO . Direct activation of receptors on MCs was accomplished by repeated short applications ( 100 ms ) of L-glutamate ( 1 mM , Sigma-Aldrich , St Louis , MO ) , acetylcholine ( ACh , 1 mM , Sigma-Aldrich , St Louis , MO ) , ATP ( 1 mM , Tocris Bioscience , Minneapolis , MN ) , or ACSF to the IPL via a pipette that was similar to the recording pipette and a Picospritzer ( PV830 Pneumatic PicoPump , World Precisions Instruments , Sarasota , FL ) set at 20–30 psi . Videos were analyzed offline using SARFIA ( freely available on http://www . igorexchange . com/project/SARFIA ) , a suite of macros running in Igor Pro ( Wavemetrics , Portland , OR ) ( Dorostkar et al . , 2010 ) Prior to analysis , images were registered to correct movements in the X and Y directions . Videos were rejected if the plane of focus altered significantly during imaging acquisition . For GLASTCreER::GCaMP3 and ShH10-CMV-iGluSnFR recordings , ROIs containing both stalks and lateral processes of MCs were chosen using a filtering algorithm based on a Laplace operator and segmented by applying a threshold , as described in detail in ( Dorostkar et al . , 2010 ) . This algorithm defined most or all of the ROIs that an experienced observer would recognize by eye . For AAV9-2YF-hSynapsin1-iGluSnFR recordings , the entire field of view ( 256 × 256 pixels ) was divided into 16 identical squares and each square was considered one ROI . In both cases , individual ROI responses were then normalized as the relative change in fluorescence ( ∆F/F ) , smoothed by binomial Gaussian filtering , and analyzed to detect activity using custom-made scripts based on a first derivate detection algorithm . A threshold set at ∼2 times ( when imaging at 0 . 74 Hz ) or at ∼4 times ( when imaging at 1 . 7 Hz ) the standard deviation of the time derivative trace was used to detect changes in fluorescence within the ROIs . The reliability of this algorithm to detect calcium and glutamate activity was first tested by comparing the results with manual activity detection . For the experiments using focal application of agonists , a threshold set at ∼2–3 times the standard deviation of the derivate trace was used to detect activity . In this paper , the fluorescent intensity of ROIs is reported as the average intensity across all pixels within its area . Fluorescent responses are reported as normalized increases as follows:ΔF/F= ( F−Fo ) /Fo , where F is the instantaneous fluorescence induced by a spontaneous activity or by a focal application of the agonist and Fo is the baseline fluorescence . Group measurements are expressed as mean ±SD . We used t tests to compare two groups and one-way ANOVA to compare more than two groups . The level of significance was set at p < 0 . 05 .
Neuron-glia interactions play a critical role in the maturation of neural circuits; however , little is known about the pathways that mediate their communication in the developing CNS . We investigated neuron-glia signaling in the developing retina , where we demonstrate that retinal waves reliably induce calcium transients in Müller glial cells ( MCs ) . During cholinergic waves , MC calcium transients were blocked by muscarinic acetylcholine receptor antagonists , whereas during glutamatergic waves , MC calcium transients were inhibited by ionotropic glutamate receptor antagonists , indicating that the responsiveness of MCs changes to match the neurotransmitter used to support retinal waves . Using an optical glutamate sensor we show that the decline in MC calcium transients is caused by a reduction in the amount of glutamate reaching MCs . Together , these studies indicate that neurons and MCs exhibit correlated activity during a critical period of retinal maturation that is enabled by neurotransmitter spillover from retinal synapses .
A structure at the back of the eye known as the retina is essential for vision . When light hits the retina , cells called neurons produce electrical signals that lead to the release of chemicals known as neurotransmitters . When these chemicals reach a neighboring neuron , a corresponding electrical signal is produced in this cell . In this way , information about what we can see is ultimately transmitted to the brain . The retina also contains cells that are not neurons , such as Müller glial cells . These cells span the thickness of the retina , and appear well placed to interact with all of the different types of neuron in the retina . Other types of glial cells help the neural circuits in the brain to develop , but it is not clear whether Müller cells perform the same role in the developing retina . Newborn mice do not open their eyes until around two weeks after they are born , during which time their retina continues to develop . The retinal neurons are already active throughout this period , and spontaneously generate periodic bursts of electrical activity that spreads across the developing retina in waves . Now , Rosa , Bos et al . show that in newborn mice , these waves also trigger brief , or ‘transient’ , increases in the concentration of calcium ions inside Müller cells . Exactly how the Müller cells respond depends on which neurotransmitters are released during waves . A few days before the mice open their eyes , the number of calcium transients in Müller cells decreases sharply . At the same time , neurons continue to spontaneously release waves of one particular neurotransmitter called glutamate . Rosa , Bos et al . used a sensor that showed where glutamate is found in the developing retina . This revealed that the decrease in calcium transients as the retina matures is due to less glutamate reaching the Müller cells . These findings reveal that Müller cells are involved in the spontaneous electrical activity seen in the retina during a critical period of retinal development . The next challenge is to determine how this neuronal-glial cell signaling helps the retina to mature .