Periodic Report Summary - NEUGLIANET (Role of astrocytes in neuronal network function in visual cortex)
The project entitled NEUGLIANET (Grant Agreement No. 253635) was started on 1 August 2010 at Massachusetts Institute of Technology (MIT) (United States).
During the outgoing phase at Dr Mriganka Sur lab (Picower Institute for Learning and Memory, MIT), Dr Gertruids Perea has got access to use the state-of-the-art techniques for real-time physiological recordings of neuronal and astrocyte activities in visual cortical regions (V1). She has successfully implemented in the Sur lab the use of channelrhodopsin-2 (ChR2) in astrocytes using viral vectors that was an essential issue to accomplish the current objectives of the project. The use of optogenetic tools to manipulate selectively the activity of astrocytes was critical to evaluate its consequences on neuronal network activity. The results obtained during the outgoing phase of NEUGLIANET project have been sent to publication; the paper 'Astrocyte activation modulates excitatory / inhibitory balance and response selectivity of visual cortex neurons in vivo' is currently under review in the journal 'Science'.
Using two-photon microscopy for calcium imaging and electrophysiological recordings to monitor neuronal electrical activity of single cells or population cells in vitro and in vivo, this project utilises cutting-edge tools and represents an important conceptual advance.
In particular, the main findings of the project are:
1. Astrocytes were selectively targeted with adeno-associated viral vector with light-sensitive ChR2 under glial fibrillary acidic protein (GFAP) promoter and neuronal activity was recorded from layer 2 / 3 neurons of V1. The selective expression of ChR2 in cortical astrocytes was confirmed by GFAP staining, and the ability of this construct to evoke Ca2+ responses was measured in V1 cortical astrocytes where transduced cells showed robust increases in intracellular Ca2+ in response to 473 nm light stimulation.
2. The analysis of neuronal activity showed that optogenetic activation of astrocytes evoked a transient potentiation of both spontaneous excitatory and inhibitory synaptic transmission, increasing the frequency of postsynaptic currents without modifying their amplitudes.
3. The increase of synaptic activity was prevented in the presence of MCPG, an antagonist of metabotropic glutamate receptor (mGluR)s, suggesting that AAV-GFAP-ChR2-expressed astrocytes can release glutamate that activates mGluRs at presynaptic terminals.
4. The effect of astrocyte activity was cell specific showing that excitatory pyramidal cells and inhibitory gamma-aminobutyric acid (GABA)-ergic cells (parvoalbumin-positive neurons (PV+)) displayed an increase in the excitatory drive through mGluRs activation.
5. To confirm the ability of ChR2-expressed astrocytes to release glutamate, I recorded slow inward currents (SICs), i.e. N-methyl-D-aspartate (NMDA)-mediated currents induced by glutamate released from astrocytes. To optimise SIC recordings magnesium was removed from the artificial cerebrospinal fluid (ACSF), and tetrodotoxin (TTX) was added to block action potential-mediated neuronal activity. In those conditions, optogenetic astrocyte stimulation evoked both increase of SIC frequency and potentiation of miniature excitatory synaptic currents. SICs were abolished by antagonists of NMDA glutamate receptors.
6. Combining two-photon microscopy and single cell-attached electrophysiological recordings in vivo, I showed that selective photostimulation of astrocytes in vivo enhances spontaneous firing rate of inhibitory PV+ neurons and excitatory neurons in visual cortex.
7. Astrocytes impact key visual response features of visual cortex neurons. Astrocyte activation selectively increases the baseline visual response level of PV+ neurons and reduces their orientation selectivity, whereas excitatory neurons show either increased or decreased baseline visual responses together with complementary changes in visual response features.
8. Astrocytes influence inhibitory transmission in cortical networks through the stimulation of particular subsets of inhibitory neurons, such as PV+ neurons rather than somatostatin-positive (SOM+) neurons.
9. In summary, these results show that astrocytes in vivo modulate excitation / inhibition balance in cortical circuits, through the dual control of excitatory drive and of PV+ inhibitory drive, and thus influence integrative response features of neurons critical for sensory information processing.
During the outgoing phase at Dr Mriganka Sur lab (Picower Institute for Learning and Memory, MIT), Dr Gertruids Perea has got access to use the state-of-the-art techniques for real-time physiological recordings of neuronal and astrocyte activities in visual cortical regions (V1). She has successfully implemented in the Sur lab the use of channelrhodopsin-2 (ChR2) in astrocytes using viral vectors that was an essential issue to accomplish the current objectives of the project. The use of optogenetic tools to manipulate selectively the activity of astrocytes was critical to evaluate its consequences on neuronal network activity. The results obtained during the outgoing phase of NEUGLIANET project have been sent to publication; the paper 'Astrocyte activation modulates excitatory / inhibitory balance and response selectivity of visual cortex neurons in vivo' is currently under review in the journal 'Science'.
Using two-photon microscopy for calcium imaging and electrophysiological recordings to monitor neuronal electrical activity of single cells or population cells in vitro and in vivo, this project utilises cutting-edge tools and represents an important conceptual advance.
In particular, the main findings of the project are:
1. Astrocytes were selectively targeted with adeno-associated viral vector with light-sensitive ChR2 under glial fibrillary acidic protein (GFAP) promoter and neuronal activity was recorded from layer 2 / 3 neurons of V1. The selective expression of ChR2 in cortical astrocytes was confirmed by GFAP staining, and the ability of this construct to evoke Ca2+ responses was measured in V1 cortical astrocytes where transduced cells showed robust increases in intracellular Ca2+ in response to 473 nm light stimulation.
2. The analysis of neuronal activity showed that optogenetic activation of astrocytes evoked a transient potentiation of both spontaneous excitatory and inhibitory synaptic transmission, increasing the frequency of postsynaptic currents without modifying their amplitudes.
3. The increase of synaptic activity was prevented in the presence of MCPG, an antagonist of metabotropic glutamate receptor (mGluR)s, suggesting that AAV-GFAP-ChR2-expressed astrocytes can release glutamate that activates mGluRs at presynaptic terminals.
4. The effect of astrocyte activity was cell specific showing that excitatory pyramidal cells and inhibitory gamma-aminobutyric acid (GABA)-ergic cells (parvoalbumin-positive neurons (PV+)) displayed an increase in the excitatory drive through mGluRs activation.
5. To confirm the ability of ChR2-expressed astrocytes to release glutamate, I recorded slow inward currents (SICs), i.e. N-methyl-D-aspartate (NMDA)-mediated currents induced by glutamate released from astrocytes. To optimise SIC recordings magnesium was removed from the artificial cerebrospinal fluid (ACSF), and tetrodotoxin (TTX) was added to block action potential-mediated neuronal activity. In those conditions, optogenetic astrocyte stimulation evoked both increase of SIC frequency and potentiation of miniature excitatory synaptic currents. SICs were abolished by antagonists of NMDA glutamate receptors.
6. Combining two-photon microscopy and single cell-attached electrophysiological recordings in vivo, I showed that selective photostimulation of astrocytes in vivo enhances spontaneous firing rate of inhibitory PV+ neurons and excitatory neurons in visual cortex.
7. Astrocytes impact key visual response features of visual cortex neurons. Astrocyte activation selectively increases the baseline visual response level of PV+ neurons and reduces their orientation selectivity, whereas excitatory neurons show either increased or decreased baseline visual responses together with complementary changes in visual response features.
8. Astrocytes influence inhibitory transmission in cortical networks through the stimulation of particular subsets of inhibitory neurons, such as PV+ neurons rather than somatostatin-positive (SOM+) neurons.
9. In summary, these results show that astrocytes in vivo modulate excitation / inhibition balance in cortical circuits, through the dual control of excitatory drive and of PV+ inhibitory drive, and thus influence integrative response features of neurons critical for sensory information processing.