Functionally independent columns of rat somatosensory barrel cortex revealed with voltage-sensitive dye imaging

Whisker movement is somatotopically represented in rodent neocortex by electrical activity in clearly defined barrels, which can be visualized in living brain slices. The functional architecture of this part of the cortex can thus be mapped in vitro with respect to its physiological input and compared with its anatomical architecture. The spatial extent of excitation was measured at high temporal resolution by imaging optical signals from voltage-sensitive dye evoked by stimulation of individual barrels in layer 4. The optical signals correlated closely with subthreshold EPSPs recorded simultaneously from excitatory neurons in layer 4 and layer 2/3, respectively. Excitation was initially (3 msec) spread in a columnar manner into layer 2/3 and then subsided in both layers after approximately 50 msec. The lateral extent of the response was limited to the cortical column defined structurally by the barrel in layer 4. Two experimental interventions increased the spread of excitation. First, blocking GABA(A) receptor-mediated synaptic inhibition caused excitation to spread laterally throughout wide regions of layer 2/3 and layer 5 but not into neighboring barrels, suggesting that the local excitatory connections within layer 4 are restricted to single barrels and that inhibitory neurons control spread in supragranular and infragranular layers. Second, NMDA receptor-dependent increase of the spread of excitation was induced by pairing repetitive stimulation of a barrel column with coincident stimulation of layer 2/3 in a neighboring column. Such plasticity in the spatial extent of excitation in a barrel column could underlie changes in cortical map structure induced by alterations of sensory experience.

The excitatory neuronal network of rat layer 4 barrel cortex

Carl Petersen

Sensory whiskers are mapped to rodent layer 4 somatosensory cortex as discrete units termed barrels, which can be visualized at high resolution in living brain slices. Both anatomical and physiological properties of the layer 4 neuronal network can thus be investigated in the context of the functional boundaries of this sensory map. Large-scale confinement of neuronal arbors to single barrels was suggested by restricted lateral diffusion of DiI across septa between barrels. Morphological analysis of dendritic and axonal arborizations of individual excitatory neurons showed that neuronal processes remain within the barrel of origin through polarization toward the center of the barrel. Functionally, the large-scale properties of the neuronal network were investigated through mapping the spatial extent of field EPSPs, which were found to attenuate at barrel borders. This ensemble property of a layer 4 barrel was further investigated by analyzing the connectivity of pairs of excitatory neurons with respect to the locations of the somata. Approximately one-third of the excitatory neurons within the same barrel were synaptically coupled. At the septum between adjacent barrels the connectivity dropped rapidly, and very few connections were found between neurons located in adjacent barrels. Each layer 4 barrel is thus composed of an excitatory neuronal network, which to a first order approximation, acts independently of its neighbors.

2000

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

Michael Avermann

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.

EPFL2011

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

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

Michael Avermann

EPFL2011

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

Céline Matéo

EPFL2011