Role of Ca2+ ions in nicotinic facilitation of GABA release in mouse thalamus

Abstract

Presynaptic nicotinic acetylcholine receptors (nAChRs) are present in many regions of the brain and potentially serve as targets for the pharmacological action of nicotine in vivo. To investigate their mechanism of action, we performed patch-clamp recordings in relay neurons from slices of thalamus sensory nuclei. In these nuclei, nAChR activation facilitated the release of the inhibitory neurotransmitter GABA. Micromolar concentrations of nicotinic agonists increased the frequency of miniature GABAergic synaptic currents and decreased the failure rate of evoked synaptic currents. These actions of nicotinic agonists were not observed in knock-out mice lacking the beta 2 nAChR subunit gene. Nicotinic effects were dependent on extracellular calcium ions, and they persisted when calcium was replaced by strontium or barium but not by magnesium. Furthermore, in high extracellular calcium concentrations, nicotinic agonists evoked an increase in spontaneous release lasting for minutes after removal of the agonist. This supports the view that presynaptic nAChRs facilitate the release of neurotransmitter by increasing the calcium concentrations in presynaptic nerve endings. With use of cadmium and nickel ions as selective blockers, it was found that in different sensory nuclei the presynaptic influx of calcium could result either from the activation of voltage-dependent calcium channels or from a direct influx through nAChR channels. Finally, we propose that the nicotinic facilitation of GABAergic transmission may contribute to the increase of signal-to-noise ratio observed in the thalamus in vivo during arousal.

Fig. 1.

DMPP (10 μm) increases the frequency of miniature GABAergic IPSCs without altering their amplitude distribution in the thalamic relay neurons. A(top), Current trace from a neuron in the VB. The nicotinic agonist DMPP elicits an inward current, an increase in baseline noise, and an acceleration of the frequency of miniature IPSCs; (bottom) frequency plot of miniature IPSCs corresponding to the current trace above; bin is 4 sec.B, Cumulative probability plot corresponding totrace A (2 min of control condition and 30 sec in the presence of DMPP). The amplitude distribution was unchanged (p > 0.1) from control conditions in the presence of DMPP, whereas the frequency was increased significantly (p < 0.001). C, Part oftrace A at an expanded time-scale in control condition (three top traces) and in DMPP condition (bottom trace). The short bars indicate the detection of an IPSC by the analysis program. D, Same asA in the presence of the nicotinic antagonist DHβE: all of the effects of DMPP are abolished. E, Same astrace A in the presence of the GABA_Aantagonist Gabazine (Gbz) all of the IPSCs are blocked, but not the postsynaptic response to DMPP.

Fig. 2.

DMPP reduces the number of failures in the GABAergic transmission in the VB. A, Current traces of 10 successive evoked GABAergic synaptic currents (top) and spontaneous activity recorded between the electrical stimulations (bottom) in control condition. The failure rate has been increased by raising the extracellular magnesium concentration to 4 mm. B, Same as in A in the presence of 10 μm DMPP. Note the dramatic reduction in the number of failures in evoked synaptic currents. Calibration is the same as in A. C, Scheme of the preparation. The VB is isolated from the reticularisthalamus GABAergic neurons with two cuts (cut1,cut2) at its borders with a razor blade. The stimulation with a bipolar tungsten electrode (S) is performed at random positions in the nucleus. The perfusion pipette (P) is placed in front of the recording electrode (R). D, Amplitude of evoked synaptic currents (top: one point is one event; the amplitude is plotted downward) and frequency of spontaneous IPSCs (bottom: bin = 1 sec) during the same experiment. Failures are rare in the presence of DMPP. There is no dramatic change in the amplitude distribution of the successfully evoked IPSCs. (A, B, and D are from the same cell); E, decrease of the failure rate of evoked IPSCs before and during the application of DMPP (n= 6). Matched t test indicated a significance probability of p = 0.0004. The contribution of spontaneous IPSCs that would occur together or in place of evoked IPSCs was evaluated by using the protocol used for detecting evoked IPSCs between the stimulations instead of during the stimulation episodes. In 2/6 cells we found that we may have overestimated by 10–15% the number of successfully evoked IPSCs because of the high frequency of spontaneous IPSCs, whereas in 4/6 cells we found a possible 2–5% overestimation. When corrected for these biases, however, the decrease in failure rate was still statistically significant (p = 0.0008).

Fig. 3.

The presynaptic effect is dependent on extracellular calcium. A, Control application of DMPP and frequency plot. B, Application after replacement of 90% of extracellular calcium by magnesium. DMPP does not elicit any increase in frequency of miniature IPSCs in 200 μmCa²⁺ and 2.8 mm Mg²⁺. The amplitude of the postsynaptic response is unchanged from A toB (A and B are from the same cell). C, D, Same as in A but 90% of calcium is replaced by barium or strontium; the control applications are not shown. DMPP is still able to elicit a presynaptic effect in 200 μm Ca²⁺, 1 mm Mg²⁺, and 1.8 mm Ba²⁺ or Sr²⁺.C and D are from different cells.

Fig. 4.

Quantification of the remaining presynaptic effect of DMPP and KCl after treatment with low [Ca²⁺], Cd²⁺, and Ni²⁺ in the VB and DLG. Low [Ca²⁺] solutions block the increase of frequency of IPSCs produced by DMPP in the VB and the DLG, or by KCl in the DLG; 50 μm Cd²⁺ blocks the effect of potassium in the DLG and of DMPP in the VB but not in the DLG; 50 μmNi²⁺ has no significant effect on any of the conditions tested. The remaining presynaptic effect after the treatments was evaluated with the formula (X2 − 1)/(X1 − 1) where X1 andX2 are the frequency of IPSCs on DMPP application normalized to the frequency before the application, in control conditions and in treatment conditions. In most cases,X1 was the average of the effect in the control applications performed before and after the treatment application. A value of 100% means that the treatment did not affect the increase in frequency, whereas a value of 0% corresponds to its complete blockage. The large deviation in the results corresponds to the variability in the responses of the cells rather than a variable effect of the treatments. Similar values of the deviation were obtained by comparing successive control applications in the cells. There is a continuous distribution of the values around the mean value and no evidence for the existence of subsets. Statistical differences were tested by a nonparametric paired comparison test (Wilcoxon) between the pairs (X1, X2). The differences in remaining presynaptic effect of DMPP in the VB and DLG neurons in the presence of Cd²⁺ was tested with a nonparametric test (Mann–Whitney) and yielded a value of p = 0.01. The plot represents mean ± SEM (*p = 0.03, **p < 0.01). The number of points represented in each bar of the histogram is (left toright) 4,6,5, 5,11,10, 4,7,5.

Fig. 5.

DMPP causes an increase in frequency of miniature IPSCs lasting longer than the postsynaptic current in the DLG. A, Samples of current trace from a single cell. Note the presynaptic effect of DMPP in 1 mm, 2 mm, and 4 mm extracellular calcium.B, Running average of the frequency in 1 mm(n = 5), 2 mm (n = 7), and 4 mm (n = 5) calcium. The traces were normalized to the basal frequency in 2 mmextracellular calcium before averaging. Most cells were recorded from 2-week-old animals that had a larger presynaptic effect but also a large postsynaptic current that hindered an accurate measure of the miniature IPSCs frequency during the application. Therefore, the frequency during the application is not plotted in this figure.

Fig. 6.

The nicotinic postsynaptic currents in thereticularis thalamus are insensitive to the replacement of calcium by magnesium or by Cd²⁺ or Ni²⁺treatment. A, Examples of current traces fromreticularis thalamus neurons; 10 μm DMPP is applied in control condition, after the replacement of calcium by magnesium, or after the addition of Cd²⁺ or Ni²⁺. B, Average of the responses to DMPP after the different treatments and normalization to the control responses. The number of points averaged is (from theleft to the right bar) 4, 6, 3.

Fig. 7.

The presynaptic effect of DMPP is blocked by Cd²⁺ in the VB. A, B, Control application of DMPP; C, D, application in the presence of Cd²⁺ in the same cell. A, Current traces and frequency plot (bin = 4 sec). B, Traces fromA at expanded time scale in control condition (top) and during application (bottom). The occurrence of the synaptic currents is indicated by abar. C, Same as in A in the presence of Cd²⁺. D, Traces fromC at an expanded time scale in the presence of Cd²⁺ during control (top) and application (bottom).

Fig. 8.

The presynaptic effect of DMPP is unchanged by Cd²⁺ in the DLG. A, Control application of DMPP; B, application in the presence of Ni²⁺; C, application in the presence of Cd²⁺. Same vertical organization as in Figure 7.

Fig. 9.

The increase in frequency of IPSCs induced by 25 mm potassium is calcium-dependent and blocked by Cd²⁺. A, Samples of current traces in control conditions (top), in 25 mm potassium (middle), and in 200 μm Ca²⁺, 2.8 mm Mg²⁺, and 25 mm potassium (bottom). B, Current traces in (fromtop to bottom) control condition, 25 mmpotassium, 25 mm potassium + 50 μmCd²⁺ or Ni²⁺. A andB are from different cells.