J. Simon Wiegert

Long-term depression triggers the selective elimination of weakly integrated synapses

Significance Long-term plasticity, the ability of synapses to change their strength, is considered a possible basis of learning and memory. Extending the study of individual synapses to timescales relevant for behavior has been challenging: although rats can retain specific memories for several weeks, intracellular recordings that monitor the strength of individual synapses are typically limited to 1 h. Here we introduce an all-optical approach to stimulate and monitor synaptic function for 7 d. We show that long-term depression of hippocampal synapses significantly shortens their lifetime. While synapses with a high probability of transmitter release often recovered from depression, unreliable synapses were selectively removed from the circuit. Thus, specific activity patterns not only modulate synaptic weights but alter hippocampal connectivity. Long-term depression (LTD) weakens synaptic transmission in an activity-dependent manner. It is not clear, however, whether individual synapses are able to maintain a depressed state indefinitely, as intracellular recordings rarely exceed 1 h. Here, we combine optogenetic stimulation of identified Schaffer collateral axons with two-photon imaging of postsynaptic calcium signals and follow the fate of individual synapses for 7 d after LTD induction. Optogenetic stimulation of CA3 pyramidal cells at 1 Hz led to strong and reliable depression of postsynaptic calcium transients in CA1. NMDA receptor activation was necessary for successful induction of LTD. We found that, in the days following LTD, many depressed synapses and their “neighbors” were eliminated from the hippocampal circuit. The average lifetime of synapses on nonstimulated dendritic branches of the same neurons remained unaffected. Persistence of individual depressed synapses was highly correlated with reliability of synaptic transmission, but not with spine size or the amplitude of spine calcium transients. Our data suggest that LTD initially leads to homogeneous depression of synaptic function, followed by selective removal of unreliable synapses and recovery of function in the persistent fraction.

An improved chloride-conducting channelrhodopsin for light-induced inhibition of neuronal activity in vivo.

Channelrhodopsins are light-gated cation channels that have been widely used for optogenetic stimulation of electrically excitable cells. Replacement of a glutamic acid in the central gate with a positively charged amino acid residue reverses the ion selectivity and produces chloride-conducting ChRs (ChloCs). Expressed in neurons, published ChloCs produced a strong shunting effect but also a small, yet significant depolarization from the resting potential. Depending on the state of the neuron, the net result of illumination might therefore be inhibitory or excitatory with respect to action potential generation. Here we report two additional amino acid substitutions that significantly shift the reversal potential of improved ChloC (iChloC) to the reversal potential of endogenous GABAA receptors. As a result, light-evoked membrane depolarization was strongly reduced and spike initiation after current injection or synaptic stimulation was reliably inhibited in iChloC-transfected neurons in vitro. In the primary visual cortex of anesthetized mice, activation of iChloC suppressed spiking activity evoked by visual stimulation. Due to its high operational light sensitivity, iChloC makes it possible to inhibit neurons in a large volume of brain tissue from a small, point-like light source.

AMPA receptors gate spine Ca2+ transients and spike-timing-dependent potentiation

Spike timing-dependent long-term potentiation (t-LTP) is the embodiment of Donald Hebb’s postulated rule for associative memory formation. Pre- and postsynaptic action potentials need to be precisely correlated in time to induce this form of synaptic plasticity. NMDA receptors have been proposed to detect correlated activity and to trigger synaptic plasticity. However, the slow kinetic of NMDA receptor currents is at odds with the millisecond precision of coincidence detection. Here we show that AMPA receptors are responsible for the extremely narrow time window for t-LTP induction. Furthermore, we visualized synergistic interactions between AMPA and NMDA receptors and back-propagating action potentials on the level of individual spines. Supralinear calcium signals were observed for spike timings that induced t-LTP and were most pronounced in spines well isolated from the dendrite. We conclude that AMPA receptors gate the induction of associative synaptic plasticity by regulating the temporal precision of coincidence detection.

Layer-specific optogenetic activation of pyramidal neurons causes beta-gamma entrainment of neonatal networks.

Coordinated activity patterns in the developing brain may contribute to the wiring of neuronal circuits underlying future behavioural requirements. However, causal evidence for this hypothesis has been difficult to obtain owing to the absence of tools for selective manipulation of oscillations during early development. We established a protocol that combines optogenetics with electrophysiological recordings from neonatal mice in vivo to elucidate the substrate of early network oscillations in the prefrontal cortex. We show that light-induced activation of layer II/III pyramidal neurons that are transfected by in utero electroporation with a high-efficiency channelrhodopsin drives frequency-specific spiking and boosts network oscillations within beta–gamma frequency range. By contrast, activation of layer V/VI pyramidal neurons causes nonspecific network activation. Thus, entrainment of neonatal prefrontal networks in fast rhythms relies on the activation of layer II/III pyramidal neurons. This approach used here may be useful for further interrogation of developing circuits, and their behavioural readout.

Preparation of Slice Cultures from Rodent Hippocampus

This protocol describes the preparation of hippocampal slice cultures from rat or mouse pups using sterile conditions that do not require the use of antibiotics or antimycotics. Combining very good optical and electrophysiological accessibility with a lifetime approaching that of the intact animal, many fundamental questions about synaptic plasticity and long-term dynamics of network connectivity can be addressed with this preparation.

Single-Cell Electroporation of Neurons

Single-cell electroporation allows the transfection of a small number of neurons in an organotypic culture with a single plasmid or a defined mixture of plasmids. Desired protein expression levels can vary depending on the experimental goals (e.g., high expression levels are needed for optogenetic experiments); however, when too much protein is expressed, cellular toxicity and cell death may arise. To a large degree, protein expression can be controlled by adjusting the concentration of plasmid DNA in the electroporation pipette. Here, we present a protocol for transfecting individual neurons in hippocampal slice cultures by electroporation. Essentially, a patch-clamp setup is required that includes an upright microscope with infrared differential interference contrast or Dodt contrast with a camera and a specialized amplifier that is able to deliver large-voltage pulses to the electroporation pipette.

Stimulating Neurons with Heterologously Expressed Light-Gated Ion Channels

Heterologous expression of ion channels that can be directly gated by light has made it possible to stimulate almost any excitable cell with light. Optogenetic stimulation has been particularly powerful in the neurosciences, as it allows the activation of specific, genetically defined neurons with precise timing. Organotypic hippocampal slice cultures are a favored preparation for optogenetic experiments. They can be cultured for many weeks and, after transfection with optogenetic actuators and sensors, allow the study of individual synapses or small networks. The absence of any electrodes allows multiple imaging sessions over the course of several days and even chronic stimulation inside the incubator. These timescales are not accessible in electrophysiological experiments. Here, we introduce the production of organotypic hippocampal slice cultures and their transduction or transfection with optogenetic tools. We then discuss the options for light stimulation.

Automated analysis of spine dynamics on live CA1 pyramidal cells

Dendritic spines may be tiny in volume, but are of major importance for neuroscience. They are the main receivers for excitatory synaptic connections, and their constant changes in number and in shape reflect the dynamic connectivity of the brain. Two-photon microscopy allows following the fate of individual spines in brain slice preparations and in live animals. The diffraction-limited and non-isotropic resolution of this technique, however, makes detection of such tiny structures rather challenging, especially along the optical axis (z-direction). Here we present a novel spine detection algorithm based on a statistical dendrite intensity model and a corresponding spine probability model. To quantify the fidelity of spine detection, we generated correlative datasets: Following two-photon imaging of live pyramidal cell dendrites, we used serial block-face scanning electron microscopy (SBEM) to reconstruct dendritic ultrastructure in 3D. Statistical models were trained on synthetic fluorescence images generated from SBEM datasets via point spread function (PSF) convolution. After the training period, we tested automatic spine detection on real two-photon datasets and compared the result to ground truth (correlative SBEM data). The performance of our algorithm allowed tracking changes in spine volume automatically over several hours. Using a second fluorescent protein targeted to the endoplasmic reticulum, we could analyze the motion of this organelle inside individual spines. Furthermore, we show that it is possible to distinguish activated spines from non-stimulated neighbors by detection of fluorescently labeled presynaptic vesicle clusters. These examples illustrate how automatic segmentation in 5D (x, y, z, t, λ) allows us to investigate brain dynamics at the level of individual synaptic connections.

How (not) to silence long-range projections with light

Inhibitory optogenetic tools prevent action potential generation during illumination. A study explores the possibility of squelching already propagating action potentials locally at axon terminals before they trigger neurotransmitter release.

Viral Vector-Based Transduction of Slice Cultures

Transgenes can be introduced into the cells of organotypic slice cultures using different delivery methods, such as biolistic transfection, electroporation, and viral vector-based transduction. These methods produce different patterns of transgene expression. Local injection of recombinant adeno-associated virus (rAAV) produces a small cluster of transgene-expressing neurons around the injection site. Expression in individual cells varies with the distance from the injection site, indicating that many neurons take up several rAAV particles. The serotype and promoter also play a role in transgene expression. Here, we present a protocol for the transduction of previously prepared hippocampal slice cultures with rAVV.