Anton Nikolaev

Distinct Roles for Two Histamine Receptors (hclA and hclB) at the Drosophila Photoreceptor Synapse

Histamine (HA) is the photoreceptor neurotransmitter in arthropods, directly gating chloride channels on large monopolar cells (LMCs), postsynaptic to photoreceptors in the lamina. Two histamine-gated channel genes that could contribute to this channel in Drosophila are hclA (also known as ort) and hclB (also known as hisCl1), both encoding novel members of the Cys-loop receptor superfamily. Drosophila S2 cells transfected with these genes expressed both homomeric and heteromeric histamine-gated chloride channels. The electrophysiological properties of these channels were compared with those from isolated Drosophila LMCs. HCLA homomers had nearly identical HA sensitivity to the native receptors (EC50 = 25 μm). Single-channel analysis revealed further close similarity in terms of single-channel kinetics and subconductance states (∼25, 40, and 60 pS, the latter strongly voltage dependent). In contrast, HCLB homomers and heteromeric receptors were more sensitive to HA (EC50 = 14 and 1.2 μm, respectively), with much smaller single-channel conductances (∼4 pS). Null mutations of hclA (ortUS6096) abolished the synaptic transients in the electroretinograms (ERGs). Surprisingly, the ERG “on” transients in hclB mutants transients were approximately twofold enhanced, whereas intracellular recordings from their LMCs revealed altered responses with slower kinetics. However, HCLB expression within the lamina, assessed by both a GFP (green fluorescent protein) reporter gene strategy and mRNA tagging, was exclusively localized to the glia cells, whereas HCLA expression was confirmed in the LMCs. Our results suggest that the native receptor at the LMC synapse is an HCLA homomer, whereas HCLB signaling via the lamina glia plays a previously unrecognized role in shaping the LMC postsynaptic response.

Spikes in Retinal Bipolar Cells Phase-Lock to Visual Stimuli with Millisecond Precision

Summary Background The conversion of an analog stimulus into the digital form of spikes is a fundamental step in encoding sensory information. Here, we investigate this transformation in the visual system of fish by in vivo calcium imaging and electrophysiology of retinal bipolar cells, which have been assumed to be purely graded neurons. Results Synapses of all major classes of retinal bipolar cell encode visual information by using a combination of spikes and graded signals. Spikes are triggered within the synaptic terminal and, although sparse, phase-lock to a stimulus with a jitter as low as 2–3 ms. Spikes in bipolar cells encode a visual stimulus less reliably than spikes in ganglion cells but with similar temporal precision. The spike-generating mechanism does not alter the temporal filtering of a stimulus compared with the generator potential. The amplitude of the graded component of the presynaptic calcium signal can vary in time, and small fluctuations in resting membrane potential alter spike frequency and even switch spiking on and off. Conclusions In the retina of fish, the millisecond precision of spike coding begins in the synaptic terminal of bipolar cells. This neural compartment regulates the frequency of digital signals transmitted to the inner retina as well as the strength of graded signals.

Encoding of Luminance and Contrast by Linear and Nonlinear Synapses in the Retina

Summary Understanding how neural circuits transmit information is technically challenging because the neural code is contained in the activity of large numbers of neurons and synapses. Here, we use genetically encoded reporters to image synaptic transmission across a population of sensory neurons—bipolar cells in the retina of live zebrafish. We demonstrate that the luminance sensitivities of these synapses varies over 104 with a log-normal distribution. About half the synapses made by ON and OFF cells alter their polarity of transmission as a function of luminance to generate a triphasic tuning curve with distinct maxima and minima. These nonlinear synapses signal temporal contrast with greater sensitivity than linear ones. Triphasic tuning curves increase the dynamic range over which bipolar cells signal light and improve the efficiency with which luminance information is transmitted. The most efficient synapses signaled luminance using just 1 synaptic vesicle per second per distinguishable gray level.

Synaptic mechanisms of adaptation and sensitization in the retina

Sensory systems continually adjust the way stimuli are processed. What are the circuit mechanisms underlying this plasticity? We investigated how synapses in the retina of zebrafish adjust to changes in the temporal contrast of a visual stimulus by imaging activity in vivo. Following an increase in contrast, bipolar cell synapses with strong initial responses depressed, whereas synapses with weak initial responses facilitated. Depression and facilitation predominated in different strata of the inner retina, where bipolar cell output was anticorrelated with the activity of amacrine cell synapses providing inhibitory feedback. Pharmacological block of GABAergic feedback converted facilitating bipolar cell synapses into depressing ones. These results indicate that depression intrinsic to bipolar cell synapses causes adaptation of the ganglion cell response to contrast, whereas depression in amacrine cell synapses causes sensitization. Distinct microcircuits segregating to different layers of the retina can cause simultaneous increases or decreases in the gain of neural responses.

Functional Properties of Endogenous Receptor- and Store-operated Calcium Influx Channels in HEK293 Cells

Activation of phospholipase C (PLC)-mediated signaling pathways in non-excitable cells causes the release of calcium (Ca2+) from inositol 1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores and activation of Ca2+ influx via plasma membrane Ca2+ channels. The properties and molecular identity of plasma membrane Ca2+ influx channels in non-excitable cells is a focus of intense investigation. In the previous studies we used patch clamp electrophysiology to describe the properties of Ca2+ influx channels in human carcinoma A431 cell lines. Now we extend our studies to human embryonic kidney HEK293 cells. By using a combination of Ca2+ imaging and whole cell and single channel patch clamp recordings we discovered that: 1) HEK293 cells contain four types of plasma membrane Ca2+ influx channels: ICRAC, Imin, Imax, and INS; 2) ICRAC channels are highly Ca2+-selective (PCa/Cs > 1000) and ICRAC single channel conductance is too small for single channel analysis; 3) Imin channels in HEK293 cells display functional properties identical to Imin channels in A431 cells, with single channel conductance of 1.2 pS for divalent cations, 10 pS for monovalent cations, and divalent cation selectivity PBa/K = 20; 4) Imin channels in HEK293 cells are activated by InsP3 and inhibited by phosphatidylinositol 4,5-bisphosphate, but store-independent; 5) when compared with Imin, Imax channels have higher conductance for divalent (17 pS) and monovalent (33 pS) cations, but less selective for divalent cations (PBa/K = 4), 6) Imax channels in HEK293 cells can be activated by InsP3 or by Ca2+ store depletion; 7) INS channels are non-selective (PBa/K = 0.4) and display a single channel conductance of 5 pS; and 8) INS channels are not gated by InsP3 but activated by depletion of intracellular Ca2+ stores. Our findings provide novel information about endogenous Ca2+ channels supporting receptor-operated and store-operated Ca2+ influx pathways in HEK293 cells.

Regulation of the Miniature Plasma Membrane Ca2+ Channel I min by Inositol 1,4,5-Trisphosphate Receptors

I min is a plasma membrane-located, Ca2+-selective channel that is activated by store depletion and regulated by inositol 1,4,5-trisphosphate (IP3). In the present work we examined the coupling betweenI min and IP3 receptors in excised plasma membrane patches from A431 cells. I minwas recorded in cell-attached mode and the patches were excised into medium containing IP3. In about 50% of experiments excision caused the loss of activation of I minby IP3. In the remaining patches activation ofI min by IP3 was lost upon extensive washes of the patch surface. The ability of IP3 to activateI min was restored by treating the patches with rat cerebellar microsomes reach in IP3 receptors but not by control forebrain microsomes. The re-activatedI min had the same kinetic properties asI min when it is activated by Ca2+-mobilizing agonists in intact cells and by IP3 in excised plasma membrane patches and it was inhibited by the I crac inhibitor SKF95365. We propose that I min is a form ofI crac and is gated by IP3receptors.

Network Adaptation Improves Temporal Representation of Naturalistic Stimuli in Drosophila Eye: I Dynamics

Because of the limited processing capacity of eyes, retinal networks must adapt constantly to best present the ever changing visual world to the brain. However, we still know little about how adaptation in retinal networks shapes neural encoding of changing information. To study this question, we recorded voltage responses from photoreceptors (R1–R6) and their output neurons (LMCs) in the Drosophila eye to repeated patterns of contrast values, collected from natural scenes. By analyzing the continuous photoreceptor-to-LMC transformations of these graded-potential neurons, we show that the efficiency of coding is dynamically improved by adaptation. In particular, adaptation enhances both the frequency and amplitude distribution of LMC output by improving sensitivity to under-represented signals within seconds. Moreover, the signal-to-noise ratio of LMC output increases in the same time scale. We suggest that these coding properties can be used to study network adaptation using the genetic tools in Drosophila, as shown in a companion paper (Part II).

A Synaptic Mechanism for Temporal Filtering of Visual Signals

Synaptic volume matters! The size of the presynaptic compartment of retinal bipolar cells controls the amplitude, speed, and adaptation of synaptic transmission.

Imaging Neuronal Activity in the Optic Tectum of Late Stage Larval Zebrafish

The zebrafish is an established model to study the development and function of visual neuronal circuits in vivo, largely due to their optical accessibility at embryonic and larval stages. In the past decade multiple experimental paradigms have been developed to study visually-driven behaviours, particularly those regulated by the optic tectum, the main visual centre in lower vertebrates. With few exceptions these techniques are limited to young larvae (7–9 days post-fertilisation, dpf). However, many forms of visually-driven behaviour, such as shoaling, emerge at later developmental stages. Consequently, there is a need for an experimental paradigm to image the visual system in zebrafish larvae beyond 9 dpf. Here, we show that using NBT:GCaMP3 line allows for imaging neuronal activity in the optic tectum in late stage larvae until at least 21 dpf. Utilising this line, we have characterised the receptive field properties of tectal neurons of the 2–3 weeks old fish in the cell bodies and the neuropil. The NBT:GCaMP3 line provides a complementary approach and additional opportunities to study neuronal activity in late stage zebrafish larvae.