Global dendritic calcium spikes in mouse layer 5 low threshold spiking interneurones: implications for control of pyramidal cell bursting

Abstract

Interneuronal networks in neocortex underlie feedforward and feedback inhibition and control the temporal organization of pyramidal cell activity. We previously found that lower layer neocortical interneurones can reach action potential threshold in response to the stimulation of a single presynaptic cell. To better understand this phenomenon and the circuit roles of lower layer neocortical interneurones, we combined two-photon calcium imaging with whole cell recordings and anatomical reconstructions of low threshold spiking (LTS) interneurones from mouse neocortex. In both visual and somatosensory cortex, LTS interneurones are somatostatin-positive, concentrated in layer 5 and possess dense axonal innervation to layer 1. Due to the LTS properties, these neurones operate in burst and tonic modes. In burst mode, dendritic T-type calcium channels boosted small synaptic inputs and triggered low threshold calcium spikes, while in tonic mode, sodium-based APs evoked smaller calcium influxes. In both modes, the entire dendritic tree of LTS interneurones behaved as a 'global' single spiking unit. This, together with the fact that synaptic inputs to layer 5 LTS cells are facilitating, and that their axons target the dendritic region of the pyramidal neurones where bursts are generated, make these neurones ideally suited to detect and control burst generation of individual lower layer pyramidal neurones.

Figure 1. Low threshold spiking somatostatin-positive layer 5 cells

A, left, voltage response (top) of a representative somatostatin-positive layer 5 LTS interneurone cell to hyperpolarizing current injection (bottom). Note the rebound LTS burst after release from hyperpolarization. Firing pattern was generated with cell held at −70 mV, middle, and −55 mV, right. Note that the bursting firing pattern at −70 mV is absent at −55 mV. B, left, delivery of transient (4 ms) depolarizing current (bottom), at −70 mV resulted in an LTS burst (top), which outlasted the duration of the current injection. Right, the same current injection at −55 mV evoked a single spike. C, somatostatin-positive cells from layers 4–5 of the visual cortex of a somatostatin–GFP transgenic mouse. Scale bar = 20 μm. D, biocytin reconstruction of a layer 5 LTS Martinotti cell. Note the local dendritic arborization (dark), and the axons ascending to layer 1 (light).

Figure 2. Low threshold spikes dominate dendritic calcium dynamics

A, anatomical reconstruction of layer 5 LTS cell. B, basal dendritic arborization from reconstructed in A (top), and z-projection of the identical cellular region acquired during fluorescence imaging (bottom). Arrows indicate dendritic sites where line scans were performed at an additional 10× zoom. Scale bar = 10 μm. C, voltage responses (top) to 4 ms somatic current injections (bottom). Note that the envelope of the low threshold spike recruited at the hyperpolarized potential (−75 mV) outlasted the duration of the current injection. Calcium transients during the stimulation regimes outlined in C acquired at the soma (D), and 20 μm, 60 μm, and 110 μm from the soma (E–G), respectively. The LTS-associated calcium signal evoked in burst mode (−80 mV) dominated over single APs or AP trains evoked in tonic mode (−55 mV). The number of APs evoked in a train was matched to the number of APs generated during the LTS burst, and ranged from 2 to 4 (n = 18). Note the different time scales in B and C–F. H, pooled data, demonstrating the dominance of the dendritic calcium signal during the LTS. *P < 0.005; **P < 0.001.

Figure 3. T-type calcium channels trigger regeneratively propagated dendritic spikes and cause calcium influx in distal dendrites

A, low concentrations of nickel (20 μm) significantly reduced dendritic calcium influx and the size and duration of the LTS. Calcium transients (top) during the LTS (middle) evoked during 4 ms somatic current injections (bottom) in control (left) and after addition of 20 μm nickel (right). B, data plotted as in A in the presence of TTX. Both transients were acquired from distal dendrites (> 80 μm). C, 20 μm Ni²⁺ (n = 3) significantly reduced dendritic calcium transients, while blockade of sodium channels with 1μm TTX (n = 3) did not.

Figure 4. Global propagation of LTS evoked by local synaptic stimulation

A, z-projection of an LTS interneurone from layer 5 of primary somatosensory cortex. S marks the location of the stimulation electrode, which was always placed adjacent to terminal dendritic branches. Coloured boxes indicate the dendritic domains selected for line scan imaging at an additional 10× zoom (not shown). Scale bar = 20 μm. We systematically tested dendritic calcium responses throughout the dendritic tree. Ba and Ca, voltage response (top) to 5 ms somatic current injection (bottom) to a cell held at −78 mV (B), and −55 mV (C). Bb and Cb, calcium transients at the soma, and at three dendritic locations, as colour-coded in A. Da and Ea, synaptic response to a train of 3 stimulations (40 Hz) delivered at −78 mV (D) and −55 mV (E). Note how the LTS was triggered by the third stimulation when the cell was hyperpolarized. Db and Eb, calcium transients plotted as in Bb and Cb during synaptic stimulations. Dot markers indicate the timing of the synaptic stimulations. Note that the calcium influx was time-locked to the LTS in D, and to the AP in E. Also note that the magnitude of the synaptically evoked LTS was comparable to the somatically evoked LTS throughout the dendritic tree.

Figure 5. Subthreshold EPSP trains do not evoke calcium signals

Top, ΔF/F transient taken from distal dendrite immediately adjacent to the stimulation electrode. Bottom, synaptic response to a train of shocks (3 delivered at 40 Hz). Cell was held at −78 mV.

Figure 6. AMPA receptor driven depolarization recruits low threshold spikes to trigger global synaptic calcium events

Calcium transients during local synaptic stimulation are plotted above synaptically evoked EPSPs in control (black) conditions and in the presence of 100 μm d-APV (A), 30 μm CPA (B), 20 μm nickel (C), and DNQX (D). E, data pooled from d-APV (n = 4), nickel (n = 3), CPA (n = 3) and DNQX (n = 3) experiments. Calcium transients were from the distal (> 80 μm) dendrites adjacent to the stimulation electrode.