Juan Burrone

Multiple forms of synaptic plasticity triggered by selective suppression of activity in individual neurons

The rules by which neuronal activity causes long-term modification of synapses in the central nervous system are not fully understood. Whereas competitive or correlation-based rules result in local modification of synapses, homeostatic modifications allow neuron-wide changes in synaptic strength, promoting stability. Experimental investigations of these rules at central nervous system synapses have relied generally on manipulating activity in populations of neurons. Here, we investigated the effect of suppressing excitability in single neurons within a network of active hippocampal neurons by overexpressing an inward-rectifier potassium channel. Reducing activity in a neuron before synapse formation leads to a reduction in functional synaptic inputs to that neuron; no such reduction was observed when activity of all neurons was uniformly suppressed. In contrast, suppressing activity in a single neuron after synapses are established results in a homeostatic increase in synaptic input, which restores the activity of the neuron to control levels. Our results highlight the differences between global and selective suppression of activity, as well as those between early and late manipulation of activity.

Activity-dependent relocation of the axon initial segment fine-tunes neuronal excitability

In neurons, the axon initial segment (AIS) is a specialized region near the start of the axon that is the site of action potential initiation. The precise location of the AIS varies across and within different neuronal types, and has been linked to cells’ information-processing capabilities; however, the factors determining AIS position in individual neurons remain unknown. Here we show that changes in electrical activity can alter the location of the AIS. In dissociated hippocampal cultures, chronic depolarization with high extracellular potassium moves multiple components of the AIS, including voltage-gated sodium channels, up to 17 μm away from the soma of excitatory neurons. This movement reverses when neurons are returned to non-depolarized conditions, and depends on the activation of T- and/or L-type voltage-gated calcium channels. The AIS also moved distally when we combined long-term LED (light-emitting diode) photostimulation with sparse neuronal expression of the light-activated cation channel channelrhodopsin-2; here, burst patterning of activity was successful where regular stimulation at the same frequency failed. Furthermore, changes in AIS position correlate with alterations in current thresholds for action potential spiking. Our results show that neurons can regulate the position of an entire subcellular structure according to their ongoing levels and patterns of electrical activity. This novel form of activity-dependent plasticity may fine-tune neuronal excitability during development.

Synaptic gain control and homeostasis.

Chronic changes in activity can induce neurons to alter the strength of all their synapses in unison. Although the specific changes that occur appear to vary depending on the experimental preparation, their net effect is to counter the experimentally induced modification of activity. Such adaptive, cell-wide changes in synaptic strength serve to stabilize neuronal activity and are collectively referred to as homeostatic synaptic plasticity. Recent studies have shed light on what triggers homeostatic synaptic plasticity, whether or not it is distinct from other forms of synaptic plasticity and whether or not it occurs in the intact brain.

Activity-dependent regulation of inhibitory synaptic transmission in hippocampal neurons

Neural activity regulates the number and properties of GABAergic synapses in the brain, but the mechanisms underlying these changes are unclear. We found that blocking spike activity globally in developing hippocampal neurons from rats reduced the density of GABAergic terminals as well as the frequency and amplitude of miniature inhibitory postsynaptic currents (mIPSCs). Chronic inactivity later in development led to a reduction in the mIPSC amplitude, without any change in GABAergic synapse density. By contrast, hyperpolarizing or abolishing spike activity in single neurons did not alter GABAergic synaptic inputs. Suppressing activity in individual presynaptic GABAergic neurons also failed to decrease synaptic output. Our results indicate that GABAergic synapses are regulated by the level of activity in surrounding neurons. Notably, we found that the expression of GABAergic plasticity involves changes in the amount of neurotransmitter in individual vesicles.

Multi-site optical excitation using ChR2 and micro-LED array.

Studying neuronal processes such as synaptic summation, dendritic physiology and neural network dynamics requires complex spatiotemporal control over neuronal activities. The recent development of neural photosensitization tools, such as channelrhodopsin-2 (ChR2), offers new opportunities for non-invasive, flexible and cell-specific neuronal stimulation. Previously, complex spatiotemporal control of photosensitized neurons has been limited by the lack of appropriate optical devices which can provide 2D stimulation with sufficient irradiance. Here we present a simple and powerful solution that is based on an array of high-power micro light-emitting diodes (micro-LEDs) that can generate arbitrary optical excitation patterns on a neuronal sample with micrometre and millisecond resolution. We first describe the design and fabrication of the system and characterize its capabilities. We then demonstrate its capacity to elicit precise electrophysiological responses in cultured and slice neurons expressing ChR2.

Synaptic vesicle recycling studied in transgenic mice expressing synaptopHluorin

Synaptic vesicles are recycled locally within presynaptic specializations. We examined how vesicles are reused after endocytosis, using transgenic mice expressing the genetically encoded fluorescent indicator synaptopHluorin in subsets of neurons. At both excitatory and inhibitory synapses in cultured hippocampal neurons, newly endocytosed vesicles did not preferentially enter the releasable pool of vesicles. Rather, they entered the reserve pool first and subsequently the readily releasable pool over a period of several minutes. All vesicles in the recycling pool could be accessed by spaced stimuli, arguing against preferential local reuse of the readily releasable vesicles. Interestingly, nearly half the vesicles at excitatory synapses, and a third at inhibitory synapses, could not be recruited for release even by sustained stimuli. We conclude that, at presynaptic terminals in the hippocampus, most vesicles vacate release sites after exocytosis and are replaced by existing vesicles from the reserve pool, placing constraints on kiss-and-run recycling.

Photocycles of Channelrhodopsin-2

Recent developments have used light‐activated channels or transporters to modulate neuronal activity. One such genetically‐encoded modulator of activity, channelrhodopsin‐2 (ChR2), depolarizes neurons in response to blue light. In this work, we first conducted electrophysiological studies of the photokinetics of hippocampal cells expressing ChR2, for various light stimulations. These and other experimental results were then used for systematic investigation of the previously proposed three‐state and four‐state models of the ChR2 photocycle. We show the limitations of the previously suggested three‐state models and identify a four‐state model that accurately follows the ChR2 photocurrents. We find that ChR2 currents decay biexponentially, a fact that can be explained by the four‐state model. The model is composed of two closed (C1 and C2) and two open (O1 and O2) states, and our simulation results suggest that they might represent the dark‐adapted (C1‐O1) and light‐adapted (C2‐O2) branches. The crucial insight provided by the analysis of the new model is that it reveals an adaptation mechanism of the ChR2 molecule. Hence very simple organisms expressing ChR2 can use this form of light adaptation.

Optobionic vision?a new genetically enhanced light on retinal prosthesis

The recent discovery that neurons can be photostimulated via genetic incorporation of artificial opsins is creating a revolution in the field of neural stimulation. In this paper we show its potential in the field of retinal prosthesis. We show that we need typically 100 mW cm(-2) in instantaneous light intensity on the neuron in order to stimulate action potentials. We also show how this can be reduced down to safe levels in order to negate thermal and photochromic damage to the eye. We also describe a gallium nitride LED light source which is also able to generate patterns of the required intensity in order to transfer reliable images.

Optical control of muscle function by transplantation of stem cell-derived motor neurons in mice.

Optogenetics Applied to Motorneuron Control Nerves damaged by disease or injury do not always regenerate. In such cases, therapies involving transplanted stem cells show some promise. However, the new neurons derived from transplanted cells cannot communicate with the central control systems that would normally regulate movement. To avoid the need for such communication, in a proof-of-principle study, Bryson et al. (p. 94; see the Perspective by Iyer and Delp) added optogenetic control to differentiation and transplantation of motor neurons. In the mouse, these engineered neurons were able to reestablish connections within a damaged sciatic nerve and, when activated by localized light stimulation, could drive muscle contractions. Transplanted neurons controlled by light can drive muscle function in damaged mouse sciatic nerves. [Also see Perspective by Iyer and Delp] Damage to the central nervous system caused by traumatic injury or neurological disorders can lead to permanent loss of voluntary motor function and muscle paralysis. Here, we describe an approach that circumvents central motor circuit pathology to restore specific skeletal muscle function. We generated murine embryonic stem cell–derived motor neurons that express the light-sensitive ion channel channelrhodopsin-2, which we then engrafted into partially denervated branches of the sciatic nerve of adult mice. These engrafted motor neurons not only reinnervated lower hind-limb muscles but also enabled their function to be restored in a controllable manner using optogenetic stimulation. This synthesis of regenerative medicine and optogenetics may be a successful strategy to restore muscle function after traumatic injury or disease.

Studying vesicle cycling in presynaptic terminals using the genetically encoded probe synaptopHluorin

Genetically encoded fluorescent probes have become indispensable tools in the biological sciences. Studies of synaptic vesicle recycling have been facilitated by a group of GFP-derived probes called pHluorins. These probes exploit changes in pH that accompany exocytosis and recapture of synaptic vesicles. Here we describe how these synaptic tracers can be used in rodent hippocampal neurons to monitor the synaptic vesicle cycle in real time and to obtain mechanistic insights about it. Synapses can be observed in living samples using a wide-field fluorescence microscope and a cooled charge-coupled device camera. A simple specimen chamber allows electrical stimulation of synapses to evoke exocytosis in a precisely controlled manner. We present protocols to measure various parameters of the synaptic vesicle cycle. This technique can be easily adapted to study different classes of synapses from wild-type and mutant mice. Once cultured neurons expressing synaptopHluorin are available, the whole procedure should take about 2 h.