Strategies for mapping synaptic inputs on dendrites in vivo by combining two-photon microscopy, sharp intracellular recording, and pharmacology

Abstract

Uncovering the functional properties of individual synaptic inputs on single neurons is critical for understanding the computational role of synapses and dendrites. Previous studies combined whole-cell patch recording to load neurons with a fluorescent calcium indicator and two-photon imaging to map subcellular changes in fluorescence upon sensory stimulation. By hyperpolarizing the neuron below spike threshold, the patch electrode ensured that changes in fluorescence associated with synaptic events were isolated from those caused by back-propagating action potentials. This technique holds promise for determining whether the existence of unique cortical feature maps across different species may be associated with distinct wiring diagrams. However, the use of whole-cell patch for mapping inputs on dendrites is challenging in large mammals, due to brain pulsations and the accumulation of fluorescent dye in the extracellular milieu. Alternatively, sharp intracellular electrodes have been used to label neurons with fluorescent dyes, but the current passing capabilities of these high impedance electrodes may be insufficient to prevent spiking. In this study, we tested whether sharp electrode recording is suitable for mapping functional inputs on dendrites in the cat visual cortex. We compared three different strategies for suppressing visually evoked spikes: (1) hyperpolarization by intracellular current injection, (2) pharmacological blockade of voltage-gated sodium channels by intracellular QX-314, and (3) GABA iontophoresis from a perisomatic electrode glued to the intracellular electrode. We found that functional inputs on dendrites could be successfully imaged using all three strategies. However, the best method for preventing spikes was GABA iontophoresis with low currents (5–10 nA), which minimally affected the local circuit. Our methods advance the possibility of determining functional connectivity in preparations where whole-cell patch may be impractical.

Functional clustering of dendritic activity during decision-making

“…Both types of conductances are modulated by synaptic currents and postsynaptic electrogenesis, complicating the interpretation of dendritic calcium signals. Synaptic signals can be isolated during dendritic spine imaging by abolishing bAPs, using invasive approaches that hyperpolarize (Jia et al, 2010; Levy et al, 2012) or depolarize (Mainen et al, 1999) the neuron. These manipulations, however, are difficult to apply in behaving animals, especially in chronic imaging preparations, and could trigger plasticity (Wigström et al, 1986).…”

Interpreting in vivo calcium signals from neuronal cell bodies, axons, and dendrites: a review

“…To mitigate this issue, synaptic calcium signals may be imaged in the absence of spiking by hyperpolarizing the cell body or usingpharmacology. 77 , 97 This would prevent bAP, although dendritic regenerative events may still occur in the distal compartment due to the space-clamp problem. Another caveat is that calcium enters dendritic spines predominantly through NMDAR, so we would report only those synaptic activations that are sufficiently strong to relieve the magnesium block.…”

Optimal Pipette Resistance, Seal Resistance, and Zero-Current Membrane Potential for Loose Patch or Breakthrough Whole-Cell Recording in vivo

“…However, this study has some limitations. First, the recording time can be influenced by other factors (craniotomy quality (Lee et al, 2014 ), animal movement (Lee and Lee, 2017 ), brain pulsation (Levy et al, 2012 ), and cleanliness of the recording electrode (Hamill et al, 1981 ; Stett et al, 2003 ). These other factors cannot be controlled based on identical standards and may cause a biased result.…”