Abstract
Although we know a great deal about monosynaptic connectivity, transmission and integration in the mammalian nervous system from in vitro studies, very little is known in vivo. This is partly because it is technically difficult to evoke action potentials and simultaneously record small amplitude subthreshold responses in closely (<150 μm) located pairs of neurons. To address this, we have developed in vivo two-photon targeted multiple (2–4) whole-cell patch clamp recordings of nearby neurons in superficial cortical layers 1–3. Here, we describe a step-by-step guide to this approach in the anesthetized mouse primary somatosensory cortex, including: the design of the setup, surgery, preparation of pipettes, targeting and acquisition of multiple whole-cell recordings, as well as in vivo and post hoc histology. The procedure takes ~4 h from start of surgery to end of recording and allows examinations both into the electrophysiological features of unitary excitatory and inhibitory monosynaptic inputs during different brain states as well as the synaptic mechanisms of correlated neuronal activity.
Highlights
Monosynaptic transmission underpins action potential generation and the flow of information within neural circuits
In vivo approaches to identify connected pairs of neurons in the mammalian nervous system have typically performed electrophysiological recordings of multiple single neurons and examined the average response of one neuron to spontaneously occurring action potentials in another neuron. ‘‘Spike triggered averaging’’ of cortical neurons has been performed both with multiple extracellular recordings (Reid and Alonso, 1995; Csicsvari et al, 1998; Swadlow and Gusev, 2002; Barthó et al, 2004; Fujisawa et al, 2008; English et al, 2017), a combination of extracellular and intracellular Vm recordings (Matsumura et al, 1996; Bruno and Sakmann, 2006; London et al, 2010; Yu and Ferster, 2013) and dual Vm recordings (Crochet et al, 2005)
Because it is Multiple Whole-Cell Recordings in vivo not yet possible to record the activity of all neurons presynaptic to the cells of interest and cortical neurons can fire simultaneously, it is difficult to confirm whether correlated activity is the result of a direct synaptic connection between the two recorded neurons or input from a third, unrecorded neuron with similar firing dynamics
Summary
Monosynaptic transmission underpins action potential generation and the flow of information within neural circuits. ‘‘Spike triggered averaging’’ of cortical neurons has been performed both with multiple extracellular recordings (Reid and Alonso, 1995; Csicsvari et al, 1998; Swadlow and Gusev, 2002; Barthó et al, 2004; Fujisawa et al, 2008; English et al, 2017), a combination of extracellular and intracellular Vm recordings (Matsumura et al, 1996; Bruno and Sakmann, 2006; London et al, 2010; Yu and Ferster, 2013) and dual Vm recordings (Crochet et al, 2005) Because it is Multiple Whole-Cell Recordings in vivo not yet possible to record the activity of all neurons presynaptic to the cells of interest and cortical neurons can fire simultaneously, it is difficult to confirm whether correlated activity is the result of a direct synaptic connection between the two recorded neurons or input from a third, unrecorded neuron with similar firing dynamics. While care has to be taken in concluding that any synaptic response is the result of a monosynaptic rather than polysynaptic input (Berry and Pentreath, 1976; Parker, 2010), this approach has been used in vivo to characterize the wiring and functional properties of synaptic connections in a number of non-mammalian species (Burrows, 1996; Parker, 2003; Poulet and Hedwig, 2006; Roberts et al, 2010) as well as in a more limited number of studies in mammals (Crochet et al, 2005; Jouhanneau et al, 2015, 2018; Pala and Petersen, 2015, 2018)
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