Synaptic Reorganization Induced by Selective Photoablation of an Identified Neuron (original) (raw)

Dynamic Photo Stimulation Controlling Synaptic Input Patterns In Vitro by

Recent experimental and theoretical work indicates that both the intensity and the temporal structure of synaptic activity strongly modulate the integrative properties of single neurons in the intact brain. However, studying these effects experimentally is complicated by the fact that, in experimental systems, network activity is either absent, as in the acute slice preparation, or difficult to monitor and to control, as in in vivo recordings. Here, we present a new implementation of neurotransmitter uncaging in acute brain slices that uses functional projections to generate tightly controlled, spatio-temporally structured synaptic input patterns in individual neurons. For that, a set of presynaptic neurons is activated in a precisely timed sequence through focal photolytic release of caged glutamate with the help of a fast laser scanning system. Integration of synaptic inputs can be studied in postsynaptic neurons that are not directly stimulated with the laser, but receive input from the targeted neurons through intact axonal projections. Our new approach of dynamic photo stimulation employs functional synapses, accounts for their spatial distribution on the dendrites, and thus allows study of the integrative properties of single neurons with physiologically realistic input. Data obtained with our new technique suggest that, not only the neuronal spike generator, but also synaptic transmission and dendritic integration in neocortical pyramidal cells, can be highly reliable.

Controlling Synaptic Input Patterns In Vitro by Dynamic Photo Stimulation

Journal of Neurophysiology, 2005

Recent experimental and theoretical work indicates that both the intensity and the temporal structure of synaptic activity strongly modulate the integrative properties of single neurons in the intact brain. However, studying these effects experimentally is complicated by the fact that in experimental systems network activity is either absent, as in the acute slice preparation, or difficult to monitor and to control, as for in vivo recordings. Here, we present a new implementation of neurotransmitter uncaging in acute brain slices that uses functional projections to generate tightly controlled, spatio-temporally structured synaptic input patterns in individual neurons. For that, a set of presynaptic neurons is activated in a precisely timed sequence through focal photolytic release of caged glutamate with the help of a fast laser scanning system. Integration of synaptic inputs can be studied in postsynaptic neurons that are not directly stimulated with the laser, but receive input from the targeted neurons through intact axonal projections. Our new approach of dynamic photo stimulation employs functional synapses, accounts for their spatial distribution on the dendrites and allows, thus, to study the integrative properties of single neurons with physiologically realistic input. Data obtained with our new technique suggest that not only the neuronal spike generator, but also synaptic transmission and dendritic integration in neocortical pyramidal cells can be highly reliable.

Transfer of graded potentials at the photoreceptor-interneuron synapse

Journal of General Physiology, 1995

To characterize the transfer of graded potentials and the properties of the associated noise in the photoreceptor-interneuron synapse of the blowfly (CaUiphora vicina) compound eye, we recorded voltage responses of photoreceptors (R1-6) and large monopolar cells (LMC) evoked by: (a) steps of light presented in the dark; (b) contrast steps; and (c) pseudorandomly modulated contrast stimuli at backgrounds covering 6 log intensity units. Additionally, we made recordings from photoreceptor axon terminals. Increased light adaptation gradually changed the synaptic signal transfer from low-pass to band-pass filtering. This was accompanied by decreased synaptic delay and increased contrast gain, but the overall synaptic gain and the intrinsic noise (i.e., transmission noise) were reduced. Based on these results, we describe a descriptive synaptic model, in which the kinetics of the tonic transmitter (histamine) release from the photoreceptor axon terminals change with mean photoreceptor depolarization. During signal transmission, tonic transmitter release is augmented by voltage-dependent contrast-enhancing mechanisms in the photoreceptor axons that produce fast transients from the rising phases of the photoreceptor responses and add these enhanced voltages to the original photoreceptor responses. The model can predict the experimental findings and it agrees with the recently proposed theory of maximizing sensory information.

Photoinactivation of A Portion of A Neurone For Long-Term Studies of Its Role In Behaviour

Journal of Experimental Biology, 1989

An important method in the study of the neural basis of behaviour is the removal from a nerve circuit of a single cell in vivo and the subsequent search for changes in behaviour (Atkins et al. 1984; Comer, 1985). In such studies it can also be useful to remove only a part of a cell, such as a segment of its dendritic tree or axon. This can be done by filling the cell with the fluorescent dye Lucifer Yellow (LY) and irradiating the particular segment to be removed (Jacobs & Miller, 1985). However, for behavioural testing, it is often necessary to wait 1 day for the animal to recover from surgery. It is not yet clear in any system whether a small part of cell inactivated in this way repairs itself or remains inactive after 1 day. In this paper we examine this question of the permanence of LY photoinactivation (Miller & Selverston, 1979), and carry out behavioural tests showing the usefulness of this method.

Selective Photostimulation of Genetically ChARGed Neurons

Neuron, 2002

Program is encoded in DNA, and the responsive subset of neurons can therefore be restricted genetically (Crick, 1999; Memorial Sloan-Kettering Cancer Center 1275 York Avenue Zemelman and Miesenbö ck, 2001) to certain cell types (through cell-type specific promoters) or circuit ele-New York, New York 10021 ments (through viral vectors that spread through synaptic contacts). Localizing the susceptibility to stimulation is an inversion of the logic of existing stimulation meth-Summary ods, which, whether electrical (Pine, 1980; Regehr et al., 1989; Kovacs, 1994; Fromherz and Stett, 1995; Colicos To permit direct functional analyses of neural circuits, we have developed a method for stimulating groups et al., 2001) or photochemical (Farber and Grinvald, 1983; Callaway and Katz, 1993; Dalva and Katz, 1994; of genetically designated neurons optically. Coexpression of the Drosophila photoreceptor genes encoding Denk, 1994; Pettit et al., 1997; Matsuzaki et al., 2001), must narrowly localize the stimulus to avoid indiscrimi-arrestin-2, rhodopsin (formed by liganding opsin with retinal), and the ␣ subunit of the cognate heterotri-nate responses. Since sensitivity to light is built into each target neuron, advance knowledge of its spatial meric G protein-an explosive combination we term "chARGe"-sensitizes generalist vertebrate neurons coordinates is unnecessary. Large numbers of neurons can be addressed simultaneously and precisely, without to light. Illumination of a mixed population of neurons elicits action potentials selectively and cell-autono-undesirable cross-talk to neighboring cells that are functionally distinct. mously in its genetically chARGed members. In contrast to bath-applied photostimulants or caged neurotransmitters, which act indiscriminately throughout Results and Discussion the illuminated volume, chARGe localizes the responsiveness to light. Distributed activity may thus be fed Because the photoreceptors of vertebrate and invertedirectly into a circumscribed population of neurons in brate eyes are naturally equipped with genetically enintact tissue, irrespective of the spatial arrangement coded "receivers" that allow them to respond to light, of its elements. our search for sensitizing components that could be transplanted to nonphotoreceptor cells concentrated on Introduction them. Phototransduction in invertebrates (Montell, 1999; Hardie and Raghu, 2001) differs in two fundamental re-Reconstitution of biological function with pure agentsspects from that in vertebrates (Stryer, 1991; Burns and catalysis by purified enzymes, rescue of mutant pheno-Baylor, 2001): the origin and polarity of the photoreceptypes by isolated genes-offers compelling insights into tor current, and the mechanism that regenerates the causality and mechanism. The instances in neuroscilight-sensitive chromophore, 11-cis retinal, from the ence where artificial stimulation of neurons has elicited bleached all-trans isomer. The first difference is due to sensations or movements (Penfield and Rasmussen, the activation of distinct classes of heterotrimeric G 1950) or influenced decisions (Salzman et al., 1990) are proteins by photoexcited metarhodopsins. Vertebrate no exception: they demonstrate most vividly and directly metarhodopsins signal through transducin to cGMP the fundamental link between mental activity and its phosphodiesterase. As cGMP is consumed, cGMPneuronal substrate. Due to the technical difficulty of gated cation channels carrying a depolarizing dark curstimulating functionally circumscribed but anatomically rent close, and the photoreceptor hyperpolarizes dispersed groups of neurons, however, most artificial (Stryer, 1991; Burns and Baylor, 2001). Invertebrate stimuli are directed at single, isolated neurons or illmetarhodopsins, in contrast, couple to a member of the defined clusters rather than the precisely delineated, G q/11 class of heterotrimeric G proteins (Lee et al., 1990), coherent ensembles thought to be important for nervous which activates phospholipase C (PLC) (Inoue et al., system function. The ability to feed synthetic activity 1985; Bloomquist et al., 1988; Neer, 1995). In a poorly directly into such ensembles would provide a powerful understood mechanism, a product of PLC-inositoltool for mapping functional connections and determin-1,4,5-triphosphate (IP 3), diacylglycerol, or an indirect ing the response characteristics of circuits and systems, metabolite-opens cation channels in the plasma memas well as for unveiling behaviorally relevant information brane (Montell and Rubin, 1989; Hardie and Minke, 1992; carried in distributed neural representations. Phillips et al., 1992; Hardie and Raghu, 2001), and the Here we describe a general method for stimulating photoreceptor depolarizes (Hardie, 1991; Ranganathan functionally circumscribed ensembles of neurons in inet al., 1991). tact tissue, in virtually any three-dimensional arrange-Binding of arrestin inactivates metarhodopsin (Stryer, ment or anatomical location. The method uses a broadly 1991; Byk et al., 1993; Montell, 1999; Burns and Baylor, 2001) and initiates the biochemical cycle that regenerates 11-cis retinal. The vertebrate cycle consists of a

Spatially selective photoconductive stimulation of live neurons

Frontiers in Cellular Neuroscience, 2014

Synaptic activity is intimately linked to neuronal structure and function. Stimulation of live cultured primary neurons, coupled with fluorescent indicator imaging, is a powerful technique to assess the impact of synaptic activity on neuronal protein trafficking and function. Current technology for neuronal stimulation in culture include chemical techniques or microelectrode or optogenetic based techniques. While technically powerful, chemical stimulation has limited spatial resolution and microelectrode and optogenetic techniques require specialized equipment and expertise. We report an optimized and improved technique for laser based photoconductive stimulation of live neurons using an inverted confocal microscope that overcomes these limitations. The advantages of this approach include its non-invasive nature and adaptability to temporal and spatial manipulation. We demonstrate that the technique can be manipulated to achieve spatially selective stimulation of live neurons. Coupled with live imaging of fluorescent indicators, this simple and efficient technique should allow for significant advances in neuronal cell biology.

Quantitative Analysis of Synaptic Release at the Photoreceptor Synapse

Biophysical Journal, 2010

Exocytosis from the rod photoreceptor is stimulated by submicromolar Ca 2þ and exhibits an unusually shallow dependence on presynaptic Ca 2þ. To provide a quantitative description of the photoreceptor Ca 2þ sensor for exocytosis, we tested a family of conventional and allosteric computational models describing the final Ca 2þ-binding steps leading to exocytosis. Simulations were fit to two measures of release, evoked by flash-photolysis of caged Ca 2þ : exocytotic capacitance changes from individual rods and postsynaptic currents of second-order neurons. The best simulations supported the occupancy of only two Ca 2þ binding sites on the rod Ca 2þ sensor rather than the typical four or five. For most models, the on-rates for Ca 2þ binding and maximal fusion rate were comparable to those of other neurons. However, the off-rates for Ca 2þ unbinding were unexpectedly slow. In addition to contributing to the high-affinity of the photoreceptor Ca 2þ sensor, slow Ca 2þ unbinding may support the fusion of vesicles located at a distance from Ca 2þ channels. In addition, partial sensor occupancy due to slow unbinding may contribute to the linearization of the first synapse in vision.

Optogenetic analysis of synaptic function

Nature Methods, 2008

We introduce optogenetic investigation of neurotransmission (OptIoN) for time-resolved and quantitative assessment of synaptic function via behavioral and electrophysiological analyses. We photo-triggered release of acetylcholine or c-aminobutyric acid at Caenorhabditis elegans neuromuscular junctions using targeted expression of Chlamydomonas reinhardtii Channelrhodopsin-2. In intact Channelrhodopsin-2 transgenic worms, photostimulation instantly induced body elongation (for c-aminobutyric acid) or contraction (for acetylcholine), which we analyzed acutely, or during sustained activation with automated image analysis, to assess synaptic efficacy. In dissected worms, photostimulation evoked neurotransmitter-specific postsynaptic currents that could be triggered repeatedly and at various frequencies. Lightevoked behaviors and postsynaptic currents were significantly (P r 0.05) altered in mutants with pre-or postsynaptic defects, although the behavioral phenotypes did not unambiguously report on synaptic function in all cases tested. OptIoN facilitates the analysis of neurotransmission with high temporal precision, in a neurotransmitter-selective manner, possibly allowing future investigation of synaptic plasticity in C. elegans.

Optogenetic Control of Synaptic Composition and Function

Neuron, 2017

The molecular composition of the postsynaptic membrane is sculpted by synaptic activity. During synaptic plasticity at excitatory synapses, numerous structural, signaling, and receptor molecules concentrate at the postsynaptic density (PSD) to regulate synaptic strength. We developed an approach that uses light to tune the abundance of specific molecules in the PSD. We used this approach to investigate the relationship between the number of AMPA-type glutamate receptors in the PSD and synaptic strength. Surprisingly, adding more AMPA receptors to excitatory contacts had little effect on synaptic strength. Instead, we observed increased excitatory input through the apparent addition of new functional sites. Our data support a model where adding AMPA receptors is sufficient to activate synapses that had few receptors to begin with, but that additional remodeling events are required to strengthen established synapses. More broadly, this approach introduces the precise spatiotemporal co...

Light Stimulation Parameters Determine Neuron Dynamic Characteristics

Applied Sciences

Optogenetics is a recently developed technique that is widely used to study neuronal function. In optogenetic experiments, neurons encode opsins (channelrhodopsins, halorhodopsins or their derivatives) by means of viruses, plasmids or genetic modification (transgenic lines). Channelrhodopsin are light activated ion channels. Their expression in neurons allows light-dependent control of neuronal activity. The duration and frequency of light stimulation in optogenetic experiments is critical for stable, robust and reproducible experiments. In this study, we performed systematic analyses of these parameters using primary cultures of hippocampal neurons transfected with channelrhodopsin-2 (ChR2). The main goal of this work was to identify the optimal parameters of light stimulation that would result in stable neuronal activity during a repeated light pulse train. We demonstrated that the dependency of the photocurrent on the light pulse duration is described by a right-skewed bell-shape...