In Vivo Measurement of Cell-Type-Specific Synaptic Connectivity and Synaptic Transmission in Layer 2/3 Mouse Barrel Cortex

Aurélie Pala, Carl Petersen
Cell Press, 2015
Article

Résumé

Intracellular recordings of membrane potential in vitro have defined fundamental properties of synaptic communication. Much less is known about the properties of synaptic connectivity and synaptic transmission in vivo. Here, we combined single-cell opto-genetics with whole-cell recordings to investigate glutamatergic synaptic transmission in vivo from single identified excitatory neurons onto two genetically defined subtypes of inhibitory GABAergic neurons in layer 2/3 mouse barrel cortex. We found that parvalbumin-expressing (PV) GABAergic neurons received unitary glutamatergic synaptic input with higher probability than somatostatin-expressing (Sst) GABAergic neurons. Unitary excitatory postsynaptic potentials onto PV neurons were also faster and more reliable than inputs onto Sst neurons. Excitatory synapses targeting Sst neurons displayed strong short-term facilitation, while those targeting PV neurons showed little short-term dynamics. Our results largely agree with in vitro measurements. We therefore demonstrate the technical feasibility of assessing functional cell-type-specific synaptic connectivity in vivo, allowing future investigations into context-dependent modulation of synaptic transmission.

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Publications associées (18)

The neocortex is the most distinctive feature of the mammalian brain and it is considered to be the substrate of high-order cognitive functions. The nature and the arrangement of the diverse neuronal elements constituting its multiple areas have received longstanding attention. Progressively such anatomical and functional investigations are undertaken in the context of an intact living being. It offers the possibility of examining the role of particular areas, networks or even individual neurons during various behavioral states. Synaptic connectivity and synaptic transmission have been traditionally investigated in reduced preparations. Typically, electrophysiological and optical techniques have been used to control and record the propagation of electrical activity between two or more neurons in acute brain slices in vitro. The purpose of this thesis is the investigation of synaptic connectivity and synaptic transmission within the intact neocortex of the living mouse. Here I took advantage of the recent development of optogenetics, in combination with electrophysiology and two-photon microscopy to systematically and directly record synaptic transmission between a single excitatory neuron and two main types of GABAergic neurons in layer 2/3 of the mouse barrel cortex in vivo. Overall, I discovered stronger excitatory connections onto GABAergic neurons than onto excitatory neurons, irrespective of the absolute or relative locations of the pre- and postsynaptic neurons somas. I further revealed that parvalbumin-expressing (PV) and somatostatin-expressing (Sst) GABAergic neurons received excitatory inputs that were similar in magnitude, but were more reliable and faster in PV neurons than in Sst neurons. Exploring postsynaptic responses to multiple presynaptic action potentials elicited at high frequency, I found a strong short-term facilitation accompanied by significant input summation in Sst neurons, but little short-term dynamics with no summation in PV neurons. Lastly, I compared the amplitude of single action potential-evoked postsynaptic responses as a function of neocortical activity level and found that it was unchanged in both neuron types. Overall, the results of this thesis provide new insights into the functioning of microcircuits in vivo while confirming many findings from reduced preparations. In the future, it will be interesting to extend these initial in vivo measurements to other neuron and synapse types, particularly in awake animals engaged in different behavioral states.

EPFL2014

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Combined voltage and calcium epifluorescence imaging in vitro and in vivo reveals subthreshold and suprathreshold dynamics of mouse barrel cortex

Thomas Berger, Sylvain Crochet, Sandrine Lefort, Carl Petersen

Cortical dynamics can be imaged at high spatiotemporal resolution with voltage-sensitive dyes (VSDs) and calcium-sensitive dyes (CaSDs). We combined these two imaging techniques using epifluorescence optics together with whole cell recordings to measure the spatiotemporal dynamics of activity in the mouse somatosensory barrel cortex in vitro and in the supragranular layers in vivo. The two optical signals reported distinct aspects of cortical function. VSD fluorescence varied linearly with membrane potential and was dominated by subthreshold postsynaptic potentials, whereas the CaSD signal predominantly reflected local action potential firing. Combining VSDs and CaSDs allowed us to monitor the synaptic drive and the spiking activity of a given area at the same time in the same preparation. The spatial extent of the two dye signals was different, with VSD signals spreading further than CaSD signals, reflecting broad subthreshold and narrow suprathreshold receptive fields. Importantly, the signals from the dyes were differentially affected by pharmacological manipulations, stimulation strength, and depth of isoflurane anesthesia. Combined VSD and CaSD measurements can therefore be used to specify the temporal and spatial relationships between subthreshold and suprathreshold activity of the neocortex.

2007

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Microcircuits of excitatory and inhibitory neurons in layer 2/3 of mouse barrel cortex

Michael Avermann, Wulfram Gerstner, Céline Matéo, Carl Petersen, Christian Tomm

Avermann M, Tomm C, Mateo C, Gerstner W, Petersen CC. Microcircuits of excitatory and inhibitory neurons in layer 2/3 of mouse barrel cortex. J Neurophysiol 107: 3116-3134, 2012. First published March 7, 2012; doi:10.1152/jn.00917.2011.-Synaptic interactions between nearby excitatory and inhibitory neurons in the neocortex are thought to play fundamental roles in sensory processing. Here, we have combined optogenetic stimulation, whole cell recordings, and computational modeling to define key functional microcircuits within layer 2/3 of mouse primary somatosensory barrel cortex. In vitro optogenetic stimulation of excitatory layer 2/3 neurons expressing channelrhodopsin-2 evoked a rapid sequence of excitation followed by inhibition. Fast-spiking (FS) GABAergic neurons received large-amplitude, fast-rising depolarizing postsynaptic potentials, often driving action potentials. In contrast, the same optogenetic stimulus evoked small-amplitude, subthreshold postsynaptic potentials in excitatory and non-fast-spiking (NFS) GABAergic neurons. To understand the synaptic mechanisms underlying this network activity, we investigated unitary synaptic connectivity through multiple simultaneous whole cell recordings. FS GABAergic neurons received unitary excitatory postsynaptic potentials with higher probability, larger amplitudes, and faster kinetics compared with NFS GABAergic neurons and other excitatory neurons. Both FS and NFS GABAergic neurons evoked robust inhibition on postsynaptic layer 2/3 neurons. A simple computational model based on the experimentally determined electrophysiological properties of the different classes of layer 2/3 neurons and their unitary synaptic connectivity accounted for key aspects of the network activity evoked by optogenetic stimulation, including the strong recruitment of FS GABAergic neurons acting to suppress firing of excitatory neurons. We conclude that FS GABAergic neurons play an important role in neocortical microcircuit function through their strong local synaptic connectivity, which might contribute to driving sparse coding in excitatory layer 2/3 neurons of mouse barrel cortex in vivo.

American Physiological Society2012

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Microcircuits of excitatory and inhibitory neurons in layer 2/3 of mouse barrel cortex

Michael Avermann, Wulfram Gerstner, Céline Matéo, Carl Petersen, Christian Tomm

American Physiological Society2012

EPFL2014