Analysis of Neuronal Microcircuits in L2/3 of Mouse Barrel Cortex

Neocortical microcircuits are thought to contribute importantly to the sophisticated processing abilities of the mammalian brain. Neurons within the neocortex communicate with each other through releasing the excitatory neurotransmitter glutamate or the inhibitory neurotransmitter GABA. In order to begin to understand the functional operation of the neocortex, it will clearly be important to establish the basic organizing principles of synaptic connectivity between different classes of cortical neurons. Here I specifically investigate excitatory and GABAergic neuronal networks within layer 2/3 of mouse somatosensory cortex in vitro. In order to analyze synaptic connectivity I made multiple simultaneous whole-cell recordings from GFP expressing GABAergic neurons and from GFP-negative pyramidal neurons targeted by two-photon microscopy. Additionally, I express channelrhodopsin-2 (ChR2), a light-activated cation channel, in genetically defined populations of excitatory neurons which allows optically control of neuronal networks with millisecond precision. I show that optically evoked, near synchronous action potentials in excitatory neurons are sufficient to drive rapid and reliable disynaptic local inhibition onto neighboring pyramidal neurons. Local inhibition was mediated by parvalbumin expressing, fast-spiking (FS) GABAergic neurons, which responded to excitatory network input with significantly larger amplitude depolarizing PSPs compared to both non-fast-spiking (NFS) GABAergic and excitatory pyramidal neurons. The strong excitatory drive onto FS neurons can partially be explained with the finding that pyramidal neurons in cortical layer 2/3 synaptically connect to FS neurons with a higher probabilities and larger unitary EPSPs. Furthermore, FS neurons provided strong unitary inhibitory feedback onto pyramidal neurons and other FS neurons in layer 2/3. These results suggest an important role of FS GABAergic neurons in mediating strong local inhibition to control the extent of excitatory layer 2/3 network activity.

In vivo measurement of excitatory synaptic transmission between identified neurons in layer 2/3 mouse barrel cortex

Aurélie Pala

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

Optogenetic Probing of Cortical Synaptic Circuits Measured Through in Vivo Whole-Cell Recordings in the Mouse Barrel Cortex

Céline Matéo

Although neocortex underlies higher-order brain functions, little is known about the synaptic interactions that drive neocortical microcircuit function in vivo. The neocortex is spontaneously active in vivo so I explored how such spontaneous network activity affects in vivo the interactions between excitatory and inhibitory neurons in the barrel cortex. To this aim, I have focused on the layer 2/3 microcircuit of the C2 barrel column driving sensory processing for the C2 whisker, and combined in vivo whole-cell patch-clamp recordings in anesthetised and awake head-restrained mice with optogenetic stimulation, that allows controlling the activity of genetically defined neurons with light. I expressed channelrhodopsin-2 (ChR2), a light-gated cation channel to evoke action potentials in excitatory neurons with brief light pulses. The impact of this optogenetic stimulus was measured on the membrane potential dynamics of their postsynaptic target neurons. The optogenetic stimulus drove reliable action potentials in ChR2-expressing excitatory neurons, independent of the state of ongoing spontaneous cortical activity. In contrast, I found a marked modulation of light-evoked postsynaptic responses in ChR2-non-expressing excitatory neurons by cortical activity. During spontaneously hyperpolarised periods (DOWN state), optogenetic stimulation resulted in depolarisations of postsynaptic excitatory neurons, while during depolarised periods (UP state), the response was significantly smaller and shorter. The optogenetic stimulus drove the membrane potential of postsynaptic excitatory layer 2/3 neurons towards reversal potentials, which were hyperpolarised relative to action potential threshold, leading to a reduction in firing rates. Targeting whole-cell recordings to GFP-labelled GABAergic layer 2/3 neurons with two-photon microscopy revealed that both fast-spiking and non-fast-spiking inhibitory postsynaptic neurons were rapidly driven to fire action potentials, preferentially from UP states. Such rapid activation of inhibitory neurons in the UP state is likely to be responsible for decreasing firing rate in excitatory neurons. As a result, optogenetic stimulation of a given set of excitatory neurons results in a state-dependent recruitment of inhibitory neurons that limit the spread of excitation within the layer 2/3 microcircuit. The data presented here indicates that a powerful inhibitory loop within the cortical microcircuit rapidly balances excitation with inhibition and enforces sparse coding of layer 2/3 excitatory neurons.

EPFL2011

In Vivo Optogenetic Stimulation of Neocortical Excitatory Neurons Drives Brain-State-Dependent Inhibition

Michael Avermann, Céline Matéo, Carl Petersen

Background: Synaptic interactions between excitatory and inhibitory neocortical neurons are important for mammalian sensory perception. Synaptic transmission between identified neurons within neocortical microcircuits has mainly been studied in brain slice preparations in vitro. Here, we investigate brain-state-dependent neocortical synaptic interactions in vivo by combining the specificity of optogenetic stimulation with the precision of whole-cell recordings from postsynaptic excitatory glutamatergic neurons and GFP-labeled inhibitory GABAergic neurons targeted through two-photon microscopy.