Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

Retinal bipolar cells (BCs) transmit visual signals in parallel channels from the outer to the inner retina, where they provide glutamatergic inputs to specific networks of amacrine and ganglion cells. Intricate network computation at BC axon terminals has been proposed as a mechanism for complex network computation, such as direction selectivity, but direct knowledge of the receptive field property and the synaptic connectivity of the axon terminals of various BC types is required in order to understand the role of axonal computation by BCs. The present study tested the essential assumptions of the presynaptic model of direction selectivity at axon terminals of three functionally distinct BC types that ramify in the direction-selective strata of the mouse retina. Results from two-photon Ca2+ imaging, optogenetic stimulation, and dual patch-clamp recording demonstrated that 1) CB5 cells do not receive fast GABAergic synaptic feedback from starburst amacrine cells (SACs); 2) light-evoked and spontaneous Ca2+ responses are well coordinated among various local regions of CB5 axon terminals; 3) CB5 axon terminals are not directionally selective; 4) CB5 cells consist of two novel functional subtypes with distinct receptive field structures; 5) CB7 cells provide direct excitatory synaptic inputs to, but receive no direct GABAergic synaptic feedback from, SACs; and 6) CB7 axon terminals are not directionally selective, either. These findings help to simplify models of direction selectivity by ruling out complex computation at BC terminals. They also show that CB5 comprises two functional subclasses of BCs.

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Formation of retinal direction-selective circuitry initiated by starburst amacrine cell homotypic contact

eLife ◽

10.7554/elife.34241 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 13

Author(s):

Thomas A Ray ◽

Suva Roy ◽

Christopher Kozlowski ◽

Jingjing Wang ◽

Jon Cafaro ◽

...

Keyword(s):

Synapse Formation ◽

Ganglion Cells ◽

Plexiform Layer ◽

Surface Protein ◽

Amacrine Cells ◽

Direction Selectivity ◽

Mouse Retina ◽

Inner Plexiform Layer ◽

Starburst Amacrine Cells ◽

Developing Neurons

A common strategy by which developing neurons locate their synaptic partners is through projections to circuit-specific neuropil sublayers. Once established, sublayers serve as a substrate for selective synapse formation, but how sublayers arise during neurodevelopment remains unknown. Here, we identify the earliest events that initiate formation of the direction-selective circuit in the inner plexiform layer of mouse retina. We demonstrate that radially migrating newborn starburst amacrine cells establish homotypic contacts on arrival at the inner retina. These contacts, mediated by the cell-surface protein MEGF10, trigger neuropil innervation resulting in generation of two sublayers comprising starburst-cell dendrites. This dendritic scaffold then recruits projections from circuit partners. Abolishing MEGF10-mediated contacts profoundly delays and ultimately disrupts sublayer formation, leading to broader direction tuning and weaker direction-selectivity in retinal ganglion cells. Our findings reveal a mechanism by which differentiating neurons transition from migratory to mature morphology, and highlight this mechanism’s importance in forming circuit-specific sublayers.

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Spontaneous Oscillatory Activity of Starburst Amacrine Cells in the Mouse Retina

Journal of Neurophysiology ◽

10.1152/jn.00279.2005 ◽

2005 ◽

Vol 94 (3) ◽

pp. 1770-1780 ◽

Cited By ~ 20

Author(s):

Jerome Petit-Jacques ◽

Béla Völgyi ◽

Bernardo Rudy ◽

Stewart Bloomfield

Keyword(s):

Feedback Inhibition ◽

Glutamate Release ◽

Amacrine Cells ◽

Bipolar Cells ◽

Kainate Receptors ◽

Oscillatory Activity ◽

Mouse Retina ◽

Inner Plexiform Layer ◽

Experimental Conditions ◽

Starburst Amacrine Cells

Using patch-clamp techniques, we investigated the characteristics of the spontaneous oscillatory activity displayed by starburst amacrine cells in the mouse retina. At a holding potential of –70 mV, oscillations appeared as spontaneous, rhythmic inward currents with a frequency of ∼3.5 Hz and an average maximal amplitude of ∼120 pA. Application of TEA, a potassium channel blocker, increased the amplitude of oscillatory currents by >70% but reduced their frequency by ∼17%. The TEA effects did not appear to result from direct actions on starburst cells, but rather a modulation of their synaptic inputs. Oscillatory currents were inhibited by 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX), an antagonist of AMPA/kainate receptors, indicating that they were dependent on a periodic glutamatergic input likely from presynaptic bipolar cells. The oscillations were also inhibited by the calcium channel blockers cadmium and nifedipine, suggesting that the glutamate release was calcium dependent. Application of AP4, an agonist of mGluR6 receptors on on-center bipolar cells, blocked the oscillatory currents in starburst cells. However, application of TEA overcame the AP4 blockade, suggesting that the periodic glutamate release from bipolar cells is intrinsic to the inner plexiform layer in that, under experimental conditions, it can occur independent of photoreceptor input. The GABA receptor antagonists picrotoxin and bicuculline enhanced the amplitude of oscillations in starburst cells prestimulated with TEA. Our results suggest that this enhancement was due to a reduction of a GABAergic feedback inhibition from amacrine cells to bipolar cells and the resultant increased glutamate release. Finally, we found that some ganglion cells and other types of amacrine cell also displayed rhythmic activity, suggesting that oscillatory behavior is expressed by a number of inner retinal neurons.

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Spontaneous Depolarization-Induced Action Potentials of ON-Starburst Amacrine Cells during Cholinergic and Glutamatergic Retinal Waves

Cells ◽

10.3390/cells9122574 ◽

2020 ◽

Vol 9 (12) ◽

pp. 2574

Author(s):

Rong-Shan Yan ◽

Xiong-Li Yang ◽

Yong-Mei Zhong ◽

Dao-Qi Zhang

Keyword(s):

Patch Clamp ◽

Spontaneous Activity ◽

Action Potentials ◽

Amacrine Cells ◽

Bipolar Cells ◽

Dependent Manner ◽

Cellular Mechanisms ◽

Retinal Waves ◽

Starburst Amacrine Cells ◽

Release Of Acetylcholine

Correlated spontaneous activity in the developing retina (termed “retinal waves”) plays an instructive role in refining neural circuits of the visual system. Depolarizing (ON) and hyperpolarizing (OFF) starburst amacrine cells (SACs) initiate and propagate cholinergic retinal waves. Where cholinergic retinal waves stop, SACs are thought to be driven by glutamatergic retinal waves initiated by ON-bipolar cells. However, the properties and function of cholinergic and glutamatergic waves in ON- and OFF-SACs still remain poorly understood. In the present work, we performed whole-cell patch-clamp recordings and Ca2+ imaging from genetically labeled ON- and OFF-SACs in mouse flat-mount retinas. We found that both SAC subtypes exhibited spontaneous rhythmic depolarization during cholinergic and glutamatergic waves. Interestingly, ON-SACs had wave-induced action potentials (APs) in an age-dependent manner, but OFF-SACs did not. Simultaneous Ca2+ imaging and patch-clamp recordings demonstrated that, during a cholinergic wave, APs of an ON-SAC appeared to promote the dendritic release of acetylcholine onto neighboring ON- and OFF-SACs, which enhances their Ca2+ transients. These results advance the understanding of the cellular mechanisms underlying correlated spontaneous activity in the developing retina.

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Evidence for novel transient clusters of cholinergic ganglion cells in the neonatal mouse retina

10.1101/2019.12.27.888792 ◽

2019 ◽

Author(s):

Jean de Montigny ◽

Vidhyasankar Krishnamoorthy ◽

Fernando Rozenblit ◽

Tim Gollisch ◽

Evelyne Sernagor

Keyword(s):

Ganglion Cells ◽

Amacrine Cells ◽

Bipolar Cells ◽

Neonatal Mouse ◽

Mouse Retina ◽

Retinal Ganglion ◽

Electrical Imaging ◽

Subplate Neurons ◽

Starburst Amacrine Cells ◽

Cholinergic Cell

AbstractWaves of spontaneous activity sweep across the neonatal mouse retinal ganglion cell (RGC) layer, driven by directly interconnected cholinergic starburst amacrine cells (the only known retinal cholinergic cells) from postnatal day (P) 0-10, followed by waves driven by glutamatergic bipolar cells. We found transient clusters of cholinergic RGC-like cells around the optic disc during the period of cholinergic waves. They migrate towards the periphery between P2-9 and then they disappear. Pan-retinal multielectrode array recordings reveal that cholinergic wave origins follow a similar developmental center-to-periphery pattern. Electrical imaging unmasks hotspots of dipole electrical activity occurring in the vicinity of wave origins. We propose that these activity hotspots are sites for wave initiation and are related to the cholinergic cell clusters, reminiscent of activity in transient subplate neurons in the developing cortex, suggesting a universal hyper-excitability mechanism in developing CNS networks during the critical period for brain wiring.

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Synaptic Regulation of the Light-Dependent Oscillatory Currents in Starburst Amacrine Cells of the Mouse Retina

Journal of Neurophysiology ◽

10.1152/jn.01399.2007 ◽

2008 ◽

Vol 100 (2) ◽

pp. 993-1006 ◽

Cited By ~ 8

Author(s):

Jerome Petit-Jacques ◽

Stewart A. Bloomfield

Keyword(s):

Metabotropic Glutamate Receptors ◽

Light Stimulus ◽

Amacrine Cells ◽

Kainate Receptors ◽

Mouse Retina ◽

Light Responses ◽

Axon Terminals ◽

Oscillatory Component ◽

Starburst Amacrine Cells ◽

Initial Peak

Responses of on-center starburst amacrine cells to steady light stimuli were recorded in the dark-adapted mouse retina. The response to spots of dim white light appear to show two components, an initial peak that correspond to the onset of the light stimulus and a series of oscillations that ride on top of the initial peak relaxation. The frequency of oscillations during light stimulation was three time higher than the frequency of spontaneous oscillations recorded in the dark. The light-evoked responses in starburst cells were exclusively dependent on the release of glutamate likely from presynaptic bipolar axon terminals and the binding of glutamate to AMPA/kainate receptors because they were blocked by 6-cyano-7-nitroquinoxalene-2,3-dione. The synaptic pathway responsible for the light responses was blocked by AP4, an agonist of metabotropic glutamate receptors that hyperpolarize on-center bipolar cells on activation. Light responses were inhibited by the calcium channel blockers cadmium ions and nifedipine, suggesting that the release of glutamate was calcium dependent. The oscillatory component of the response was specifically inhibited by blocking the glutamate transporter with d-threo-β-benzyloxyaspartic acid, suggesting that glutamate reuptake is necessary for the oscillatory release. GABAergic antagonists bicuculline, SR 95531, and picrotoxin increased the amplitude of the initial peak while they inhibit the frequency of oscillations. TTX had a similar effect. Strychnine, the blocker of glycine receptors did not affect the initial peak but strongly decreased the oscillations frequency. These inhibitory inputs onto the bipolar axon terminals shape and synchronize the oscillatory component.

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Bistratified starburst amacrine cells in Sox2 conditional knockout mouse retina display ON and OFF responses

Journal of Neurophysiology ◽

10.1152/jn.00322.2018 ◽

2018 ◽

Vol 120 (4) ◽

pp. 2121-2129 ◽

Cited By ~ 1

Author(s):

Todd L. Stincic ◽

Patrick W. Keeley ◽

Benjamin E. Reese ◽

W. Rowland Taylor

Keyword(s):

Transcription Factor ◽

Plexiform Layer ◽

Amacrine Cells ◽

Bipolar Cells ◽

Conditional Knockout ◽

Physiological Data ◽

Mouse Retina ◽

Inner Plexiform Layer ◽

Environmental Signals ◽

Starburst Amacrine Cells

Cell-intrinsic factors, in conjunction with environmental signals, guide migration, differentiation, and connectivity during early development of neuronal circuits. Within the retina, inhibitory starburst amacrine cells (SBACs) comprise ON types with somas in the ganglion cell layer (GCL) and dendrites stratifying narrowly in the inner half of the inner plexiform layer (IPL) and OFF types with somas in the inner nuclear layer (INL) and dendrites stratifying narrowly in the outer half of the IPL. The transcription factor Sox2 is crucial to this subtype specification. Without Sox2, many ON-type SBACs destined for the GCL settle in the INL while many that reach the GCL develop bistratified dendritic arbors. This study asked whether ON-type SBACs in Sox2-conditional knockout retinas exhibit selective connectivity only with ON-type bipolar cells or their bistratified morphology allows them to connect to both ON and OFF bipolar cells. Physiological data demonstrate that these cells receive ON and OFF excitatory inputs, indicating that the ectopically stratified dendrites make functional synapses with bipolar cells. The excitatory inputs were smaller and more transient in Sox2-conditional knockout compared with wild type; however, inhibitory inputs appeared largely unchanged. Thus dendritic stratification, rather than cellular identification, may be the major factor that determines ON vs. OFF connectivity. NEW & NOTEWORTHY Conditional knockout of the transcription factor Sox2 during early embryogenesis converts a monostratifying starburst amacrine cell into a bistratifying starburst cell. Here we show that these bistratifying starburst amacrine cells form functional synaptic connections with both ON and OFF bipolar cells. This suggests that normal ON vs. OFF starburst connectivity may not require distinct molecular specification. Proximity alone may be sufficient to allow formation of functional synapses.

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Formation of retinal direction-selective circuitry initiated by starburst amacrine cell homotypic contact

10.1101/235978 ◽

2017 ◽

Author(s):

Thomas A. Ray ◽

Suva Roy ◽

Christopher Kozlowski ◽

Jingjing Wang ◽

Jon Cafaro ◽

...

Keyword(s):

Synapse Formation ◽

Ganglion Cells ◽

Plexiform Layer ◽

Surface Protein ◽

Amacrine Cells ◽

Direction Selectivity ◽

Mouse Retina ◽

Inner Plexiform Layer ◽

Starburst Amacrine Cells ◽

Developing Neurons

Impact statementSelective synapse formation in a retinal motion-sensitive circuit is orchestrated by starburst amacrine cells, which use homotypic interactions to initiate formation of a dendritic scaffold that recruits projections from circuit partners.SUMMARYA common strategy by which developing neurons locate their synaptic partners is through projections to circuit-specific neuropil sublayers. Once established, sublayers serve as a substrate for selective synapse formation, but how sublayers arise during neurodevelopment remains unknown. Here we identify the earliest events that initiate formation of the direction-selective circuit in the inner plexiform layer of mouse retina. We demonstrate that radially-migrating newborn starburst amacrine cells establish homotypic contacts on arrival at the inner retina. These contacts, mediated by the cell-surface protein MEGF10, trigger neuropil innervation resulting in generation of two sublayers comprising starburst-cell dendrites. This dendritic scaffold then recruits projections from circuit partners. Abolishing MEGF10-mediated contacts profoundly delays and ultimately disrupts sublayer formation, leading to broader direction tuning and weaker direction-selectivity in retinal ganglion cells. Our findings reveal a mechanism by which differentiating neurons transition from migratory to mature morphology, and highlight this mechanism’s importance in forming circuit-specific sublayers.

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An extracellular biochemical screen reveals that FLRTs and Unc5s mediate neuronal subtype recognition in the retina

eLife ◽

10.7554/elife.08149 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 27

Author(s):

Jasper J Visser ◽

Yolanda Cheng ◽

Steven C Perry ◽

Andrew Benjamin Chastain ◽

Bayan Parsa ◽

...

Keyword(s):

Visual Processing ◽

Ganglion Cells ◽

Expression Patterns ◽

Plexiform Layer ◽

Amacrine Cells ◽

Extracellular Proteins ◽

Mouse Retina ◽

Inner Plexiform Layer ◽

Starburst Amacrine Cells

In the inner plexiform layer (IPL) of the mouse retina, ~70 neuronal subtypes organize their neurites into an intricate laminar structure that underlies visual processing. To find recognition proteins involved in lamination, we utilized microarray data from 13 subtypes to identify differentially-expressed extracellular proteins and performed a high-throughput biochemical screen. We identified ~50 previously-unknown receptor-ligand pairs, including new interactions among members of the FLRT and Unc5 families. These proteins show laminar-restricted IPL localization and induce attraction and/or repulsion of retinal neurites in culture, placing them in an ideal position to mediate laminar targeting. Consistent with a repulsive role in arbor lamination, we observed complementary expression patterns for one interaction pair, FLRT2-Unc5C, in vivo. Starburst amacrine cells and their synaptic partners, ON-OFF direction-selective ganglion cells, express FLRT2 and are repelled by Unc5C. These data suggest a single molecular mechanism may have been co-opted by synaptic partners to ensure joint laminar restriction.

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Measurement of spontaneous neuronal membrane activity by time lapse confocal microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100141238 ◽

1995 ◽

Vol 53 ◽

pp. 972-973

Author(s):

R H. Selinfreund ◽

A. H. Cornell-Bell

Keyword(s):

Membrane Potential ◽

Confocal Microscopy ◽

Patch Clamp ◽

Electrical Activity ◽

Time Lapse ◽

Neuronal Firing ◽

Neuronal Membrane ◽

Pituitary Cells ◽

Patch Clamp Recording ◽

Spontaneous Electrical Activity

Cellular electrophysiological properties are normally monitored by standard patch clamp techniques . The combination of membrane potential dyes with time-lapse laser confocal microscopy provides a more direct, least destructive rapid method for monitoring changes in neuronal electrical activity. Using membrane potential dyes we found that spontaneous action potential firing can be detected using time-lapse confocal microscopy. Initially, patch clamp recording techniques were used to verify spontaneous electrical activity in GH4\C1 pituitary cells. It was found that serum depleted cells had reduced spontaneous electrical activity. Brief exposure to the serum derived growth factor, IGF-1, reconstituted electrical activity. We have examined the possibility of developing a rapid fluorescent assay to measure neuronal activity using membrane potential dyes. This neuronal regeneration assay has been adapted to run on a confocal microscope. Quantitative fluorescence is then used to measure a compounds ability to regenerate neuronal firing.The membrane potential dye di-8-ANEPPS was selected for these experiments. Di-8- ANEPPS is internalized slowly, has a high signal to noise ratio (40:1), has a linear fluorescent response to change in voltage.

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