Investigation of dendritic integration in spiny stellate cells of barrel cortex with 2-photon uncaging. E Chaigneau1 and RA Silver1 1. University College London, Department of Neuroscience, Physiology & Pharmacology, Gower street, London WC1E 6BT, United Kingdom Corresponding author: [email protected] Keywords: 2-photon microscopy, 2-photon photolysis, cortex Excitatory spiny neurons in layer 4 of somato-sensory exert a strong influence on transmission of information from the thalamus to the cortex [1] [2]. Their hyperpolarized resting potential [3] and relatively weak synaptic input [4] recorded in vivo raises the question of how they reach spike threshold. We have investigated how spiny stellate cells in barrel cortex integrate synaptic input by applying 2-photon uncaging in acute thalamocortical slices from P22-28 mice. To do this we patch loaded the cells with Alexa-594 and visualized the dendritic tree with 2-photon imaging. Layer 4 excitatory cells were identified on the basis of their location, anatomy and regular spiking firing properties. Different patterns of synaptic input onto the dendritic tree was mimicked at high spatiotemporal resolution by uncaging MNI-glutamate close to individual spines, while recording the membrane voltage through the somatic patch pipette. Under our experimental conditions, the resting potential of spiny stellate cells was -77 ± 7 mV (n = 52 cells). To ensure that the level of activation of synaptic glutamate receptors with photolysis was comparable to that during synaptic transmission, we first measured the quantal size of synaptic events which was -10 ± 2 pA (n = 4 cells). We then adjusted the photolysis laser power and duration so that the current evoked by uncaging MNI-glutamate on spines close to the cell soma, where dendritic filtering is minimized, matched the miniature current amplitude,. Last we adjusted the laser power to account for attenuation due to scattering/aberration using an experimental mean of path of 77 ± 11 μm (n = 4 mice) [5] to ensure the level of activation of synaptic glutamate receptors was similar throughout the cells dendritic tree which is highly three dimensional. We have examined the spatio-temporal properties of synaptic integration by comparing the synaptic potentials evoked by the near simultaneous activation of a group of spines to the arithmetic sum of synaptic potential evoked by photolysis on individual spines at distinct time points. We found that the synaptic potentials evoked by the near simultaneous activation of a cluster of spines from - 80 mV equaled the arithmetic sum of the individual synaptic potentials (paired t-test p = 0.3, n = 8). We are currently investigating the nature of the conductances involved. Acknowledgements [6]. References [1] Lubke, J. and D. Feldmeyer, Excitatory signal flow and connectivity in a cortical column: focus on barrel cortex. Brain Struct.Funct., 2007. 212(1): p. 3-17. [2] Lefort, S., et al., The excitatory neuronal network of the C2 barrel column in mouse primary somatosensory cortex. Neuron, 2009. 61(2): p. 301-16. [3] Brecht, M. and B. Sakmann, Dynamic representation of whisker deflection by synaptic potentials in spiny stellate and pyramidal cells in the barrels and septa of layer 4 rat somatosensory cortex. J.Physiol, 2002. 543(Pt 1): p. 49-70. [4] Bruno, R.M. and B. Sakmann, Cortex is driven by weak but synchronously active thalamocortical synapses. Science, 2006. 312(5780): p. 1622-1627. [5] Chaigneau, E., et al., Impact of wavefront distortion and scattering on 2-photon microscopy in mammalian brain tissue. Opt. Express, 2011. 19(23): p. 22755-22774. [6] The authors acknowledge the MRC, the EMBO and the Wellcome trust .