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. 2001 Aug 15;535(Pt 1):33-45.
doi: 10.1111/j.1469-7793.2001.00033.x.

Changes in extracellular Ca2+ can affect the pattern of discharge in rat thalamic neurons

A Formenti  1 , A De Simoni, E Arrigoni, M Martina
Affiliations

Changes in extracellular Ca2+ can affect the pattern of discharge in rat thalamic neurons

A Formenti et al. J Physiol. .

Abstract

1. The aim of this study was to investigate some of the cellular mechanisms involved in the effects caused by changes in extracellular Ca2+ concentration ([Ca2+](o)). 2. Current- and voltage-clamp experiments were carried out on acutely isolated thalamic neurons of rats. 3. Increasing [Ca2+](o) alone induced a transition of the discharge from single spike to burst mode in isolated current-clamped neurons. 4. Increasing [Ca(2+)](o) caused the voltage-dependent characteristics of the low voltage-activated (LVA) transient Ca2+ currents to shift towards positive values on the voltage axis. Changing [Ca2+](o) from 0.5 to 5 mM caused the inactivation curve to shift by 21 mV. 5. Extracellular Ca2+ blocked a steady cationic current. This current reversed at -35 mV, was scarcely affected by Mg2+ and was completely blocked by the non-selective cation channel inhibitor gadolinium (10 microM). The effect of [Ca2+](o) was mimicked by 500 microM spermine, a polyamine which acts as an agonist for the Ca(2+)-sensing receptor, and was modulated by intracellular GTP-gamma-S. 6. At the resting potential, both the voltage shift and the block of the inward current removed the inactivation of LVA calcium channels and, together with the increase in the Ca2+ driving force, favoured a rise in the low threshold Ca2+ spikes, causing the thalamic firing to change to the oscillatory mode. 7. Our data indicate that [Ca2+](o) is involved in multiple mechanisms of control of the thalamic relay and pacemaker activity. These findings shed light on the correlation between hypercalcaemia, low frequency EEG activity and symptoms such as sleepiness and lethargy described in many clinical papers.

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Figures

Figure 1
Figure 1. Single spikes and burst mode discharge in isolated thalamic neurons in vitro as a function of [Ca2+]o
A, in a current-clamped cell, a depolarising current stimulus (upper trace) evoked cell firing. Altering [Ca2+]o from 1 to 5 mm caused the discharge to turn into burst mode (oscillatory mode), which returned to the single spike discharge (relay mode) when the [Ca2+]o was returned to 1 mm. Note the hyperpolarisation of the resting potential induced by the increase in [Ca2+]o. B, scaled current traces from a neuron with an ‘anode-break’ stimulus (duration, 200 ms; amplitude,-30 pA) from a resting potential near −60 mV, during superfusion with [Ca2+]o at the values indicated beside each trace. Note the appearance of a low threshold calcium spike on changing from 1.5 to 2 mm Ca2+ (arrow). C, the profile of the electrical potential across the cell membrane. According to the surface potential hypothesis, the potential difference between the bulk solutions (Vm) recorded in the whole-cell patch-clamp configuration is different from the potential between the two membrane surfaces (Vs), which is sensed by the channels.
Figure 3
Figure 3. Effects of changes in [Ca2+]o on the membrane resistance and on the resting potential in whole cell current-clamp experiments
A, increasing extracellular Mg2+ from 0.5 to 4.5 mm caused a weak hyperpolarisation compared to that caused by the same change in Ca2+ (Mg2+,-6.6 ± 0.7 mV; Ca2+,-22.8 ± 2.5 mV; n = 4; Int 2 and Ext 11/12 in Table 1). The membrane hyperpolarisation was accompanied by a proportional increase in membrane resistance, tested by hyperpolarising current steps (amplitude, 500 pA; duration, 50 ms). B, the divalent cations in all the extracellular solutions were balanced to 5 mm by adding Mg2+, to minimise the induction of a voltage shift of the voltage-dependent characteristics of the channels. Raising [Ca2+]o from 0.5 to 4.5 mm increased membrane resistance from 78.5 ± 0.3 to 82 ± 0.7 MΩ, n = 11, when evaluated between -80 and −60 mV. This effect was accompanied by persistent hyperpolarisation when the resting membrane potential was maintained below −35 mV (Int 1 and Ext 13 in Table 1). In A and B the bars at the top indicate the external solution changes. C, the resistance and voltage changes seen on raising [Ca2+]o from 0.5 mm (○) to 4.5 mm (□). Same cell as shown in B; each arrow represents one trial. D, the amplitude of the membrane hyperpolarisation on raising [Ca2+] from 0.5 to 4.5 mm as a function of the resting potential in 0.5 mm Ca2+. Each point represents the mean of 11 recordings from different cells.
Figure 2
Figure 2. Voltage-dependent characteristics of LVA Ca2+ currents measured at different [Ca2+]o
The effects were tested by whole-cell voltage-clamp recordings in acutely dissociated thalamic neurons. A, lower panel, the current-voltage relationship (I-V) of LVA Ca2+ currents was obtained using voltage ramp stimuli from a holding potential of −140 mV to +20 mV (inset) at different [Ca2+]o; the concentration (mm) is indicated on each curve. The activation curves (upper panel) were calculated from the experimental I-V traces: relative conductance = ICa/(VmECa), normalised to 100 % (where ECa is the Ca2+ equilibrium potential). An increase in [Ca2+]o caused an augmentation of the peak Ca2+ current amplitude and shifted the current activation and current peak towards positive values. B, the inactivation curve measured at different [Ca2+]o. LVA Ca2+ currents (not shown) were obtained using a voltage ramp stimulus repeated after conditioning potentials scaled between -140 and −30 mV (inset). The ramp stimulus was adopted in order to identify and measure the peak LVA calcium current and to distinguish it clearly from other currents during the voltage changes induced by different [Ca2+]o. For each concentration and each cell recorded, peak currents were normalised as a percentage of the maximum current. The means ± s.e.m. from six cells were plotted in the figure as a function of the conditioning potential. The values were fitted with Boltzmann curves: 1/[1 + exp(VV0.5)/k] where V0.5, the midpoint, changed from -84 to-78,-71 and −63 mV,when the [Ca2+]o was 0.5, 1, 2 and 5 mm, respectively; k (8 mV) is the slope factor.
Figure 4
Figure 4. External Ca2+ and Mg2+ had similar effects on the surface potential
To study the effect of changes in [Ca2+]o on membrane current, avoiding the voltage shift due to the non-specific screening effect of Ca2+ ions, the solutions were balanced with Mg2+. This figure shows that the voltage at the peak of the Na+ current evoked by a voltage ramp (0.66 mV ms−1; inset) was not affected by changing the external solution from 4.5 mm Ca2+ and 0.5 mm Mg2+ (trace a) to 0.5 mm Ca2+ and 4.5 mm Mg2+ (trace b; Int 1 and Ext 13 in Table 1). The voltage at the peak was -43 ± 0.5 and -45 ± 3.4 mV with 4.5 mm Ca2+ and 4.5 mm Mg2+, respectively (n = 15). The difference was not significant (P > 0.05). This indicates that Ca2+ and Mg2+ ions exert the same screening effect on the negative fixed charges at the mouth of the Na+ channels.
Figure 5
Figure 5. Effects of external Ca2+ on the total persistent ionic currents
The effects of different [Ca2+]o on the total persistent ionic currents were investigated in whole-cell voltage-clamp experiments using solutions balanced with Mg2+ (Ext 4, Int 2 in Table 1). A, to study the effects of [Ca2+]o (top bar) on the whole-cell currents (traces below) a voltage-clamp protocol was used, consisting of a 100 ms depolarising ramp from -80 to −20 mV (to check and inactivate the transient current components), followed by 10 s scaled step potentials from -20 to −80 mV. Removing the extracellular Ca2+ increased the ionic currents. B, the current-voltage relationship shows the fraction of the currents activated by changing from 4.5 to 0 mm Ca2+, as a function of the test potential (○, ± s.e.m.). Note that the reversal potential is nearly the same as in current-clamp recordings (Fig. 3D). C, the percentage inhibition of the current as a function of [Ca2+]o (Int 2 and Ext 5 in Table 1). Data were fitted with a saturation binding curve: y = [Ca2+]/Kd+[Ca2+], where Kd, the equilibrium dissociation constant, is 0.91 ± 0.15 mm (n = 19). All measurements were taken at −80 mV. The number of observations is indicated beside each data point in B and C.
Figure 6
Figure 6. Effects of the external concentration of Na+ and K+ on the selectivity of the Ca2+-sensitive channels
The current activated by decreasing the [Ca2+]o to zero (top bar) was completely abolished when the external Na+ and K+ (Ext 4 in Table 1) were replaced with choline (A, Ext 6 in Table 1). The Ca2+-sensitive current was partially preserved in the presence of external K+ at 144 and 20 mm (B, Ext 8 and 7 in Table 1) or 144 and 20 mm Na+ (C, Ext 10 and 9 in Table 1). Note that Na+ is more permeant than K+. All the recordings were taken at −80 mV. C, control before changing to 0 Ca2+; R, recovery on return to 4.5 mm Ca2+.
Figure 7
Figure 7. Gadolinium was a powerful blocker of the Ca2+-sensitive cation current
Gd3+ (10 μm) added to the external solutions completely abolished the current activated by lowering [Ca2+]o from 4.5 to 0 mm (top bar), recorded at −80 mV (Int 2 and Ext 4 in Table 1). The effect persisted even when the [Ca2+]o was raised again to 4.5 mm. This suggests that it blocks a residual Ca2+-sensitive current present at that Ca2+ concentration. C, control before changing to 0 Ca2+; R, recovery on return to 4.5 mm Ca2+.
Figure 8
Figure 8. The Ca2+-sensitive current is modulated by GTP-γ-S
GTP-γ-S (500 μm) added to the intracellular solution in the recording pipette had a biphasic effect. It increased the Ca2+-sensitive current, which reached a peak about 6-7 min after the establishment of the whole-cell configuration. The Ca2+-sensitive current then showed rapid decay until it was almost completely abolished in all the neurons (B). The current traces shown in A relate to the labelled points in B. All the recordings were taken at −80 mV (Int 2 and Ext 4 in Table 1).
Figure 9
Figure 9. Spermine mimics Ca2+ in blocking the cationic current
The effects of spermine (500 μm; top bar) on the total persistent ionic currents (traces below) were tested in whole-cell voltage-clamp experiments. The stimulus protocol was similar to that shown in Fig. 5. The potential at which each current was recorded is indicated (Ext 4 with 0.5 mm Ca2+, Int 2 in Table 1). Spermine perfused extracellularly partially inhibited the persistent ionic currents. The effect was reversible. The graph below shows the spermine-sensitive current plotted against the membrane potential (○, ± s.e.m.). The current reversed at about −28 mV.
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