Elevation of intracellular Na+ induced by hyperpolarization at the dendrites of pyramidal neurones of mouse hippocampus

Abstract

1. Whole-cell recordings were made from CA1 pyramidal cells in mouse hippocampal slices with patch pipettes containing the sodium indicator dye SBFI (sodium binding benzofuran isophthalate). Using a high-speed imaging system, we investigated changes in intracellular sodium concentration, [Na+]i, in response to hyperpolarizing pulses applied to the soma. 2. In current-clamp recordings, we detected increases in [Na+]i during negative current injection. Hyperpolarization-induced [Na+]i elevation was more prominent in the middle apical dendrites than in the soma. 3. In the voltage-clamp mode, hyperpolarization induced rapid increases in [Na+]i at the apical dendrites that were significantly faster than those at the soma. The signals were not affected by bath application of 1 microM TTX, but were reduced by 5 mM CsCl. 4. Changes in membrane potential recorded from the apical dendrites in response to negative currents were significantly smaller than those recorded from the soma. In the presence of 5 mM CsCl, the I-V relationships measured at the soma and the dendrites became almost identical, indicating that CsCl-sensitive components are predominantly in the apical dendrites. 5. These results suggest that hyperpolarization-induced [Na+]i elevations reflect Na+ influx through the non-selective cation channel (Ih channel), and that this channel is distributed predominantly in the apical dendrites. The non-uniform Na+ influx may contribute to integrative functions of the dendrites.

Figure 1. Changes in SBFI signals in response to hyperpolarizing and depolarizing pulses

Somatic whole-cell current-clamp recording. A, inset, grey-scale picture of the fluorescence image of the recorded cell. Regions from which Δ(F₃₅₀/F₃₉₀) was measured are indicated by black (soma), red (40 μm from the soma), and blue (100 μm from the soma) squares. Middle and lower panels, membrane voltage response induced by current injection (-400 pA, 1 s, followed by 400 pA, 1 s) and average changes in the ratio of fluorescence (Δ(F₃₅₀/F₃₉₀)) of 4 consecutive trials. Total sampling time was 3450 ms. Black, red and blue lines represent Δ(F₃₅₀/F₃₉₀) measured in the corresponding regions in the inset. Vertical dashed and interrupted lines indicate periods of hyperpolarization and depolarization, respectively. B, the same graph as shown in A, with the ordinate enlarged to show changes during hyperpolarization.

Figure 2. Spatial distribution of the change in SBFI signals during hyperpolarization

Mean changes in the ratio of fluorescence (Δ(F₃₅₀/F₃₉₀)) at the soma and at three different regions in the apical dendrites (20-80, 80-140 and 140-200 μm from the soma). Each plot represents the mean ±s.d. obtained from 12 neurones. Vertical dashed lines indicate periods of hyperpolarization (-400 pA, 1 s). Asterisks indicate significant difference between the soma and the dendrites at the end of the 1 s hyperpolarizing pulse (P < 0.001, Student's paired t test).

Figure 3. Changes in SBFI signals at the soma and the dendrites in response to hyperpolarization

Somatic whole-cell voltage-clamp recording. A, fluorescence image of the recorded cell. Regions of interest are shown by a black square (soma) and a red square (dendrite) on a grey-scale picture. B, current responses during a -60 mV hyperpolarizing step from -60 mV (holding potential). The control trace, the trace in the presence of 5 mM CsCl, and the trace during washing are superimposed. C, changes in Δ(F₃₅₀/F₃₉₀) in response to hyperpolarization from -60 mV to -120 mV. Black lines indicate the signal at the soma, and red lines indicate the signal at the dendrites. Mean of 8 control trials, mean of 8 trials during 5 mM CsCl application, and mean of 4 trials during washing (interrupted lines). Responses in each region are superimposed. Total sampling time was 8 s. Vertical dashed lines indicate the period of hyperpolarization.

Figure 4. Pharmacological properties of hyperpolarization-induced changes in SBFI signals

Averaged changes in SBFI signals were measured at 1 s after the beginning of hyperpolarization in normal bathing solution (control, ▪), in the presence of 200 μM BaCl₂ () and in the presence of 5 mM CsCl (□). Data were obtained from the soma and the apical dendrites (20-80, 80-140 and 140-200 μm from the soma) of six neurones. Asterisks indicate significant difference from the data in the normal solution (control) (P < 0.001, Student's paired t test).

Figure 5. Time course of mean changes in CsCl-sensitive components of SBFI signals

Somatic (▪) and dendritic (□) changes are shown. Each pair of columns indicates the mean increase ±s.d. in consecutive 500 ms periods during a 4 s hyperpolarization. Data were obtained from six cells. Regions of interest on the dendrites were 50-150 μm away from the soma. Asterisks indicate significant difference between the soma and the dendrites (P < 0.01, Student's paired t test).

Figure 6. Perforated patch recordings from the soma and dendrites

A, representative traces of membrane potential in response to negative current injections (-0.4 nA and -0.8 nA, 500 ms) at the soma and at the apical dendrite 100 μm away from the soma. Small regular waves on each trace are electrical noise due to the high access resistances of the perforated patch electrodes (30-60 MΩ). Upper traces are control, and lower traces are responses in the presence of 5 mM CsCl. B, changes in membrane potential (ΔV_m) at the soma (○) and at the dendrite (•) plotted against amplitude of current commands, in control and in the presence of 5 mM CsCl. ΔV_m of each response was obtained by subtracting the mean membrane potential 50-100 ms before current injection from the mean membrane potential 380-420 ms after the beginning of current injections. Data were obtained from 11 dendritic recordings (80-120 μm from the soma) and 10 somatic recordings. Asterisks indicate significant difference between the soma and the dendrites (P < 0.001, Student's paired t test).