Prolonged Intracellular Na+ Dynamics Govern Electrical Activity in Accessory Olfactory Bulb Mitral Cells

Abstract

Persistent activity has been reported in many brain areas and is hypothesized to mediate working memory and emotional brain states and to rely upon network or biophysical feedback. Here, we demonstrate a novel mechanism by which persistent neuronal activity can be generated without feedback, relying instead on the slow removal of Na+ from neurons following bursts of activity. We show that mitral cells in the accessory olfactory bulb (AOB), which plays a major role in mammalian social behavior, may respond to a brief sensory stimulation with persistent firing. By combining electrical recordings, Ca2+ and Na+ imaging, and realistic computational modeling, we explored the mechanisms underlying the persistent activity in AOB mitral cells. We found that the exceptionally slow inward current that underlies this activity is governed by prolonged dynamics of intracellular Na+ ([Na+]i), which affects neuronal electrical activity via several pathways. Specifically, elevated dendritic [Na+]i reverses the Na+-Ca2+ exchanger activity, thus modifying the [Ca2+]i set-point. This process, which relies on ubiquitous membrane mechanisms, is likely to play a role in other neuronal types in various brain regions.

Fig 1. AOB mitral cells are capable of responding with persistent firing to transient stimuli both in vivo and in vitro.

(A) Top: raster plots showing a single unit response to ten repetitions of VNO stimulation by female saliva (red), urine (green), and vaginal secretion (blue). Bottom: Peristimulus time histogram (PSTH) of the changes in firing frequency. Vertical black lines denote the times of application of the solutions to the nasal cavity and the activation of the sympathetic nerve. (B) Change in the mean firing frequency, measured during 20 s following stimulus flush (highlighted interval in A) compared with the initial firing rate for the same unit and stimulus. Error bars denote standard error of the mean (SEM). (C) Top: in vivo response of a single unit in the AOB to a brief train of electrical stimuli delivered to the vomeronasal nerve fibers (red bar). Bottom: PSTH showing the mean change in firing frequency over seven repetitions of the same stimulation protocol. (D) Top: the response of an AOB mitral cell (upper panel), recorded in vitro using whole cell patch technique, following a 30 Hz spike train evoked by pulse current injections (lower panel). Spikes are truncated (at −10 mV) to show the underlying plateau potential. Bottom: a PSTH showing the mean change in firing frequency of three cells (ten repetitions in each, same time scale and stimulus as in the top panel). Thick black line: mean PSTH of the three cells. Red bar denotes 4 s of 30 Hz stimulation. Shaded areas denote SEM. See also S1 Fig.

Fig 2. The complex current response following a spike train is comprised of a Na⁺-dependent outward current and a prolonged Ca²⁺-dependent inward current.

(A) Mean voltage response (n = 4 cells) to a 30 Hz spike train. A hyperpolarizing current was injected throughout the recording to prevent firing before and after the stimulus train. (B) Mean current responses (n = 6–10 cells per trace) following a 4 s long spike train at multiple frequencies (denoted by different shades). Arrows point to the peak of the filtered signals. (C) Magnified view of the initial 3 s of the currents shown in (B), revealing the initial transient phase. (D) The charge transferred (current-time integral) as a function of stimulus frequency. Gray curve: the charge transfer between t = 0.25 s and t = 1.5 s post stimulation (transient phase, up to the vertical dashed line in B). Black curve: the charge transferred between t = 1.5 s and t = 42 s post stimulation (persistent phase). Error bars denote SEM. (E) The mean current (n = 11 cells) following an evoked 4 s long spike train at 30 Hz before (blue) and after removal of Ca²⁺ from the bath solution (green). The difference between the two traces is shown in black, presumably reflecting the prolonged Ca²⁺-dependent current per se. Shaded areas denote SEM. (F) The outward current recorded after Ca²⁺ removal at a holding voltage of −80 mV (dark green) and −50 mV (light green). (G) The mean inward current (n = 5 cells) following an evoked 4 s long spike train at 30 Hz before (blue) and after application of the Na⁺-K⁺ pump blocker ouabain (green). The difference between the two traces is shown in black. Low opacity traces show the average responses of individual cells.

Fig 3. The dynamics of the prolonged inward current is directly related to the Ca²⁺ concentration in the dendritic tuft, enabling stimulus integration over extended time scales.

(A) Fluorescence signal recorded from the dendritic tuft during repeated stimulation by spike trains (4 s long at 5, 10, 15, and 20 Hz, red bars). Inset: fluorescence microscope image of the mitral cell filled with OGB-1, showing the dendritic tuft from which the signals were recorded (red circle). Scale bar is 50 μm. The fluorescence level recorded before the first train (dashed line) was used as F_min to calculate dF/F values ([F-F_min] / F_min). (B) Current traces recorded simultaneously along with the fluorescence signals shown in (A). (C) A scatter plot of the recorded current (B), calculated relative to the current recorded before the first train, versus the simultaneously recorded dendritic tuft fluorescent signal (A), color coded as in (A) and (B). The data of the first 7 s following each spike train were discarded. A sigmoid curve was fitted to the data (dashed line). (D) Same as (C), with somatic fluorescence signals. (E) The mean fluorescence signal in the dendritic tuft during responses to 15 Hz (blue) and 30 Hz spike trains (green). Both signals can be modeled as a sum of two exponential functions (gray lines), one having a short time constant (~2 s) and the other a very long one (~200 s). See also S3 Fig.

Fig 4. A simple abstract model, based on the interactions between ionic extrusion mechanisms, reproduces the long-term voltage behavior observed in mitral cells.

(A) A schematic description of the abstract model—the “voltage” is the sum of an external stimulation, inward [Ca²⁺]_i-dependent current (I_CAN) and outward [Na⁺]_i- dependent current (I_pump). [Ca²⁺]_i and [Na⁺]_i influx rates are determined linearly by the “voltage” once it crosses a threshold. [Na⁺]_i exponentially decays to zero, while [Ca²⁺]_i decays to a level set by [Na⁺]_i. I_CAN and [Ca²⁺]_i have a sigmoidal relationship, while I_pump and [Na⁺]_i have a logarithmic one. (B)–(D), the dynamics of: (B) “voltage;” (C) [Ca²⁺]_i (orange) and [Na⁺]_i (red); (D) I_CAN (orange) and I_pump (red) when running the model with transient external stimulation (black bar). All time and quantity units are arbitrary. (E) A description of the detailed conductance-based model, showing the compartmental distribution of the channels, pumps, and exchangers overlaid on the reconstruction of the mitral cell used for the model. g_leak: leak conductance, composed from separate passive Na⁺ and K⁺ conductances; g_nat: transient voltage-gated Na⁺ conductance; g_k-fast/g_k-slow: fast and slow voltage-gated K⁺ channels; g_Ca: voltage-gated Ca²⁺ conductance; g_CAN: Ca²⁺-dependent non-specific cation conductance.

Fig 5. The passive and firing properties of an AOB mitral cell are reproduced by a conductance-based model of a reconstructed cell.

(A)–(G) Comparison between the experimental observation (blue) and the model prediction (orange) of the: (A) Mean voltage response to a hyperpolarizing current steps (30 and 60 pA). (B) Mean spike trajectory. (C)–(G) Firing responses to step current injections of 30, 100, 150, 200, and 350 pA, respectively. (H) I-f curve of the real (blue) and model (orange) cells. See also S4 and S5 Figs.

Fig 6. The slow tuft fluorescence signal and the prolonged inward current are reproduced by a conductance-based model of a reconstructed mitral cell, which is validated by producing persistent firing and predicting Na⁺ imaging results.

(A) Tuft fluorescence signal during a train of four spikes evoked at 1 Hz in the real (light blue) and model (red) cells. (B) Tuft fluorescence signal during and after a 4 s long spike train (green bar) evoked at 15 Hz (blue) and 30 Hz (green) compared to the corresponding simulation results (orange and red lines, respectively). (C) Same as (B), for the current following the stimulation. (D) The model voltage response to a 30 Hz spike train evoked by pulse current injections. Gaussian white noise (σ = 20 pA) was injected during the simulation. (E) A PSTH showing the mean firing frequency in the model cell of ten trials such as the one shown in (D). Shaded area denotes SEM. (F) The Z score (based on pre-stimulus standard deviation) of the Na⁺ indicator SBFI fluorescence signal in the apical dendrite of five cells (ten repetitions in each) in control conditions (green lines) and two cells in the presence of TTX (light blue lines) while a 30Hz train stimulus was applied (black bar). Upward direction denotes decreased fluorescence. Thick lines denote multi-cell average. The red line denotes the tuft [Na⁺]_i predicted by the model, scaled in the Y direction. See also S7 Fig.

Fig 7. In the model, the prolonged elevation in tuft Ca²⁺ is due to a new quasi-stable state of Ca²⁺, dictated by the tuft [Na⁺]_i.

(A) [Na⁺]_i in the axon initial segment (dashed lines) and the dendritic tuft (solid lines) during and after 15 Hz (orange) and 30 Hz (red) stimulations. (B) The tuft [Ca²⁺]_i (solid lines, truncated) during and after 15 Hz (orange) and 30 Hz (red) stimulations, along with the quasi-stable state [Ca²⁺]_i (dashed lines) calculated according to the relationship between [Na⁺]_i and the quasi-stable state [Ca²⁺]_i (inset, inferred by running the simulation to relaxation at a range of fixed [Na⁺]_i). (C) The simulated Ca²⁺ indicator fluorescence (top) and the Ca²⁺ currents (bottom) during and after a 30 Hz stimulation. The Na⁺-Ca²⁺ exchanger Ca²⁺ current (green) and the Ca²⁺ pump current (orange) are summed to almost zero (blue) soon after the stimulus, creating a new quasi-stable state. (D) Schematic representations of a section in the dendritic tuft, showing the model mechanisms dictating [Na⁺]_i and [Ca²⁺]_i during the spike train (left), during the initial decay when both the exchanger and the pump extrude Ca²⁺ (middle), and during the quasi-stable state when the two processes oppose each other (right). Background colors correspond to the colors in (C).

Fig 8. Eliminating the Na⁺-Ca²⁺ exchanger activity by substituting Na⁺ with Li⁺ abolishes the prolonged elevation of tuft [Ca²⁺]_i and, hence, the prolonged I_CAN.

(A) The model prediction for the normalized tuft fluorescence following a 30 Hz stimulation under control conditions (orange) and when the Na⁺-Ca²⁺ exchanger is blocked (red). Inset: magnified view of the initial 1 s of decay. (B) The model prediction for the current measured following a 30 Hz stimulation under control conditions (orange) and when the Na⁺-K⁺ pump is fully blocked and the Na⁺-Ca²⁺ exchanger is 60% blocked (simulating the Na⁺-Li⁺ substitution, red). (C) Normalized mean tuft fluorescence signal in a single cell during and after 30 Hz stimulation in normal bath solution (blue) and after substituting Na⁺ with Li⁺ (green). (D) The mean net inward current following an evoked 4 s long spike train at 30 Hz under control conditions (blue) and after substituting Na⁺ with Li⁺ (green). Low opacity traces showing average responses of individual cells (n = 5). The abbreviation “a.u.” stands for arbitrary units.

Fig 9. Transitions from transient to persistent firing are predicted by simulating the inputs of a mitral cell during natural stimuli.

(A) An example of predicted firing frequency in a VNO sensory neuron (red) based on ligand-receptor interaction during a brief stimulus presentation (red bar), and a corresponding random spike time series (black). (B) Schematic representation of local network simulation, incorporating the VNO sensory inputs to a single mitral cell. (C) Unitary excitatory post-synaptic current (EPSC,blue) recorded at mitral cell soma (a in inset) following stimulation by a bipolar theta electrode (b in inset) located close to the dendritic tuft (c in inset). The synaptic current was represented by double-exponential approximation (red). (D) A transient response of the simulated cell to a weak (short duration and high dilution) stimulus, that was insufficient to trigger persistent firing. (E) A persistent response of the simulated cell to a strong (long duration and low dilution) stimulus. (F) Mean duration of simulated firing in a range of ligand dilution factors (y-axis) and stimulus durations (x-axis). (G) Relative frequencies of different firing durations on multiple runs with the stimulus concentration and duration ranges as in (F).