Odor-evoked calcium signals in dendrites of rat mitral cells

Abstract

Mitral cell dendrites do more than passively integrate and convey synaptic potentials to the soma, they release transmitter onto local interneurones to mediate recurrent and lateral inhibition. Several mechanisms may control the level of dendritic intracellular calcium ([Ca(2+)]) and define timing for dendritic release. Here we investigated in vivo, how odor controls calcium dynamics in mitral cell dendrites by combining intracellular recording and two-photon microscopy imaging of [Ca(2+)]. During odor stimulation, two types of [Ca(2+)] changes accompany membrane potential oscillations that are phase-locked with the respiratory cycle: (i) one is graded and parallels the membrane potential, even below the threshold for action potential firing; (ii) a second is transient, triggered by sodium action potentials that invade the entire dendritic tree. These results indicate that mitral cell dendritic compartments are synchronized by action potentials and suggest that the efficacy of dendritic synapses is finely tuned by odor-evoked graded changes in [Ca(2+)].

Figure 1

Sodium action potentials synchronize [Ca²⁺] transients in all dendritic compartments of mitral cells in the olfactory bulb of anesthetized rats. (A) Fast sodium action potentials were observed with sharp electrode recordings from apical secondary dendrites and from the soma. Shown is a schematic diagram of mitral cell synaptic connections and responses from four different mitral cells to depolarizing current pulses. Sodium action potentials were seen at all sites. Note that the resting membrane potential and the injected currents varied slightly at each site. (B) Examples of mitral and tufted cells that were filled with Ca²⁺-Green-1 and imaged using two-photon microscopy (see Methods for recording and imaging techniques). Both pictures are two-dimensional projections of image stacks (up to 200 frames each separated by a 2-μm step in depth) obtained at the end of the recording session (scale bars = 50 μm). Electrophysiological recordings were obtained from the soma (Left, mitral cell, length of the apical dendrite, 210 μm) and at the origin of a secondary dendrite (Right, tufted cell, length of the apical dendrite, 130 μm). (C) Spontaneous action potentials induce a [Ca²⁺] rise in the tuft. (Left) Fluorescence signals indicative of [Ca²⁺] changes were integrated in the region indicated by a white rectangle (same tufted cell as in B; the ** indicates a spike doublet). The Inset Illustrates the firing of the cell for which hyperpolarizing pulses activated an I_A-type current (arrow). (D) Action potentials propagate backward in vivo. (Left) A recording micropipette was placed in the secondary dendrite (arrow) of a mitral cell with a long apical dendrite (230 μm, scale bar = 50 μm), and fluorescence signals were measured in two tuft branchlets (double arrow) by using a line scan. (Right) Three single spikes, evoked with depolarizing current pulses (1.5 nA, 5 ms) injected in the secondary dendrite, induce fast [Ca²⁺] transients in the tuft. Because of the location of the current injection site distal to the soma, it is most likely that sodium action potentials were initiated in the secondary dendrite or possibly in the soma and propagated backward to the tuft (i.e., they did not initiate in the tuft dendrite). Note the rapid decay of the transient elicited by a single action potential.

Figure 2

Odor stimulation evokes two types of [Ca²⁺] changes in mitral cell dendrites. (A–C) Fluorescence measurements from the distal tuft, the apical dendrite at the edge of the glomerulus (200 μm from the soma), and the soma of a cell. The microelectrode was located in the apical dendrite 75 μm distal to the soma. (A Left) During odor stimulation (3 s isoamyl acetate), transient [Ca²⁺] increases were observed, phase-locked with bursts of action potentials that occurred during each respiratory cycle. [Ca²⁺] increases recovered substantially between breaths. (B) Subthreshold depolarizations evoke [Ca²⁺] changes. Hyperpolarization of membrane potential below the threshold for spike generation revealed underlying cyclical depolarizations during odor presentation. In both the apical tuft and dendrite, subthreshold slow depolarizations were co-incident with increases in [Ca²⁺]. Dashed line below voltage trace highlights the sustained depolarization after odor offset. (C) Increased [Ca²⁺] during odor-evoked subthreshold depolarization was also seen in the soma (same cell, apical dendrite projecting toward left and top). Vertical arrow indicates an increase in [Ca²⁺], which begins with the first subthreshold depolarization (fluorescence changes expressed as ΔF/F%). (D–G) Voltage dependency of depolarization- and action potential-evoked [Ca²⁺] increases in fine tuft dendrites. The microelectrode was located in the soma of another cell. (D) The mixed response to application of the odor was initially inhibitory overall but became excitatory toward the end of application with one subthreshold depolarization inducing a slow [Ca²⁺] increase (see below). (E) Expanded portion of records in D as indicated shows the rapid recovery kinetics of Δ[Ca²⁺] provoked by action potentials (∗) compared with subthreshold voltage depolarizations (∗∗). (F) Another odor application in the same cell. (G) A large hyperpolarization blocks all [Ca²⁺] changes. Hyperpolarizing current (middle trace) was progressively injected to oppose the increasing odor-induced excitation. [Ca²⁺] increases in phase with respiration-linked slow depolarizations were no longer observed. Instead, a reduction in the steady-state [Ca²⁺] level occurred.

Figure 3

The steady [Ca²⁺] level is modulated by membrane potential below rest. Top traces in each panel are ΔF/F%, middle are membrane potential, and bottom are dc current. (A) Depolarization of the mitral cell from approximately −73mV to below the action potential threshold is accompanied by an increase in the steady [Ca²⁺]. The current injection site was located in a secondary dendrite, and [Ca²⁺] was measured in the tuft. The action potentials are truncated (B and C); hyperpolarization from threshold by 5–8 mV reduced the steady [Ca²⁺] level in both the secondary dendrite (B) (near the recording electrode) and the tuft (C) of another cell. Dashed line indicates resting fluorescence levels before changing the membrane potential, and scale bar in B applies to C.