GABAergic signaling induces divergent neuronal Ca2+ responses in the suprachiasmatic nucleus network

Abstract

Intercellular communication between gamma-aminobutyric acid (GABA)ergic suprachiasmatic nucleus (SCN) neurons facilitates light-induced phase changes and synchronization of individual neural oscillators within the SCN network. We used ratiometric Ca(2+) imaging techniques to record changes in the intracellular calcium concentration ([Ca(2+)](i)) to study the role of GABA in interneuronal communication and the response of the SCN neuronal network to optic nerve stimulations that mimic entraining light signals. Stimulation of the retinohypothalamic tract (RHT) evoked divergent Ca(2+) responses in neurons that varied regionally within the SCN with a pattern that correlated with those evoked by pharmacological GABA applications. GABA(A) and GABA(B) receptor agonists and antagonists were used to evaluate components of the GABA-induced changes in [Ca(2+)](i). Application of the GABA(A) receptor antagonist gabazine induced changes in baseline [Ca(2+)](i) in a direction opposite to that evoked by GABA, and similarly altered the RHT stimulation-induced Ca(2+) response. GABA application induced Ca(2+) responses varied in time and region within the SCN network. The NKCC1 cotransporter blocker, bumetanide, and L-type calcium channel blocker, nimodipine, attenuated the GABA-induced rise of [Ca(2+)](i). These results suggest that physiological GABA induces opposing effects on [Ca(2+)](i) based on the chloride equilibrium potential, and may play an important role in neuronal Ca(2+) balance, synchronization and modulation of light input signaling in the SCN network.

Fig. 1. Measurement of intracellular Ca²⁺ in the suprachiasmatic nucleus (SCN)

(A) Example of the recording setup showing a coronal hypothalamic slice containing the SCN and 3^rd ventricle (3V) with a bipolar stimulating electrode (SE) used to stimulate the retinohypothalamic tract (RHT) positioned in the optic chiasm (OC). (B) Higher power fluorescent pseudo-color image of fura-2 AM-loaded SCN neurons located within the rectangle in (A). (C) N-methyl-d-aspartate (NMDA; 200 μm, 5 s) induced a transient increase in the Ca²⁺ ratio. Note the range of baseline Ca²⁺ ratios in these SCN neurons during the day (Zeitgeber time = 5 h). (D) Example showing postsynaptic Ca²⁺ in two SCN neurons with similar responses to NMDA (200 μm, 5 s), but divergent responses to stimulation of the RHT (100 pulses, 200 μs at 20 Hz) via a bipolar electrode placed in the OC (A) that paralleled the response to application of g-aminobutyric acid (GABA; 200 μm, 10 s).

Fig. 2. SCN neuronal baseline Ca²⁺ is dependent on the time of day

(A) The mean baseline Ca²⁺ ratio varied with the time of the day. Each point represents the mean ± SEM of the Ca²⁺ ratio (total 1172 neurons, mean n = 90 and range 8–236 neurons) at the indicated times. The darkened box indicates the night phase. (B) Distribution of the baseline Ca²⁺ ratios in SCN neurons recorded during the day (n = 861) and night (n = 311), respectively. The scale bars indicate the number of cells in each bin. (C) A box plot of the data in (B) showing the range (vertical line), 25^th and 75^th percentiles (box) and median (horizontal line) for day and night SCN neurons. The variance in Ca²⁺ ratios significantly differed between the day and night (equality of variances F_860,310 = 1.65, ***P < 0.0001), and were shifted higher during the day compared with night (Kolmogorov-Smirnov test χ²₂ = 104.8, ***P < 0.0001). ZT, Zeitgeber time.

Fig. 3. The [Ca²⁺]_i in SCN neurons varied with membrane potential and the spontaneous action potential firing frequency

(A) A SCN neuron recorded during the day with a microelectrode filled with an internal solution containing the Ca²⁺-sensitive probe bis-fura-2 and external ACSF containing picrotoxin (50 μm) was hyperpolarized to inhibit spontaneous action potentials (not shown) followed by small current steps (bottom trace) to further hyperpolarize or depolarize the membrane potential (middle trace). A reduction of membrane potential did not lower the estimated [Ca²⁺]_i, while depolarizing steps produced a rapid increase in spontaneous action potential firing with a concomitant elevation of [Ca²⁺]_i (top trace). (B) Summary of the neuron in (A) showing a sigmoid relationship between the voltage reached from each hyperpolarizing and depolarizing current step and the corresponding change in somatic [Ca²⁺]_i. During action potential firing membrane voltage was estimated by a line halfway between the action potential threshold and peak of the afterhyperpolarization. Estimated [Ca²⁺]_i was calculated as previously described (Irwin & Allen, 2007).

Fig. 4. γ-Aminobutyric acid (GABA) induced divergent Ca²⁺ responses in SCN neurons

(A) SCN neurons imaged during the day, and treated with N-methyl-d-aspartate (NMDA; 200 μm, 5 s), which induced an increase in the Ca²⁺ ratio in all neurons. GABA (200 μm, 10 s) application induced divergent Ca²⁺ responses. The box within the SCN indicates the region recorded. Note the range of baseline Ca²⁺ concentrations. (B) Example of a GABA-induced rise in Ca²⁺ ratio and membrane depolarization in a SCN neuron recorded during the night using a loose-seal recording electrode. (C) The Ca²⁺ response to GABA (200 μm, 10 s) application varied with the baseline Ca²⁺ ratio. Ca²⁺ responses were separated into three groups: a transient elevation in the Ca²⁺ ratio [GABA(Ca+)]; a decrease in the Ca²⁺ ratio [GABA(Ca-)]; or no change in the Ca²⁺ ratio [GABA(Ca0)]. Data are the percentage of neurons in each response group based on the baseline Ca²⁺ ratio (range 0.5-1.0, n = 257 neurons; 1.0-1.5, n = 395; 1.5-2.0, n = 306; 2.0-2.5, n = 56). Note that the percentages of GABA(Ca-) and GABA(Ca0) neurons appeared reversed between low and high baseline Ca²⁺ ratios. (D) The percentage in the SCN neuronal response to GABA (200 μm, 10 s) varied between the day (n = 760) and night (n = 292). Note the change in the GABA(Ca+) and GABA(Ca-) groups, but not in the percentage of GABA(Ca0) neurons responding. (E) The percentage of SCN neurons with GABA-induced changes in the Ca²⁺ ratio varied between the dorsomedial (DM; n = 226 day and 145 night neurons) and ventrolateral (VL; n = 534 day and 147 night) regions of the SCN. The percentage of GABA(Ca+) neurons was higher in the DM during both the day and night. Conversely, the percentage of GABA(Ca-) and GABA(Ca0) neurons was higher in the VL in both day and night. 3V, 3^rd ventricle; OC, optic chiasm.

Fig. 5. γ-Aminobutyric acid (GABA)-induced Ca²⁺ responses of SCN neurons varied regionally

Regional and time of day variation of GABA-induced Ca²⁺ responses in SCN neurons (see Fig. 4D and E). The position for each neuron is superimposed on a representative drawing of the SCN, with the 3^rd ventricle (3V) on the left and optic chiasm (OC) at the bottom. Note that while the number of cells in the day and night were not equal, the relative proportions varied between the day and the night.

Fig. 6. Blocking the chloride cotransporter NKCC1 attenuated the γ-aminobutyric acid (GABA)-induced elevation of Ca²⁺

(A) Example of the Ca²⁺ response in a SCN neuron loaded with fura-2 AM and treated with GABA (200 μm, 10 s) during the day before and after bumetanide (10 μm, > 300 s) application. (B) The maximal bumetanide attenuation of the GABA-induced Ca²⁺ transient required > 300 s of treatment. The y-axis represents the area under the Ca²⁺ ratio curve [AUC (Ca²⁺ ratio/s), mean ± SEM] during the first 10 s of GABA treatment in 13 GABA(Ca+) neurons. (C) Bumetanide attenuated GABA-induced Ca²⁺ transients in GABA(Ca+) neurons and further reduced the Ca²⁺ ratio in GABA(Ca-) neurons. Data are the mean ± SEM of the AUC (0-10 s) of three different SCN slices for GABA(Ca+) (n = 39, t₃₈ = 5.80, ****P < 0.00001) and GABA(Ca-) (n = 29, t₂₈ = 2.89, **P = 0.007) SCN neurons before and after treatment with bumetanide (> 300 s).

Fig. 7. Blocking L-type voltage-dependent Ca²⁺ channels attenuated γ-aminobutyric acid (GABA)-induced increases of [Ca²⁺]_i

(A) Ca²⁺ responses induced by GABA (200 μm, 10 s) or muscimol (50 μm, 10 s) in a GABA(Ca+) neuron during the day before and after nimodipine (20 μM) application. Both GABA- and muscimol-induced Ca²⁺ transients were attenuated. (B) Nimodipine attenuated GABA- and muscimol-induced Ca²⁺ transients in GABA(Ca+) neurons. Data are the mean ± SEM of the area under the curve (AUC; Ca²⁺ ratio/s over 0-10 s of GABA treatment) for GABA(Ca+) (n = 30, t₂₉ = 6.03 GABA, t₂₉ = 10.6 muscimol, ***P < 0.00001) SCN neurons.

Fig. 8. γ-Aminobutyric acid (GABA)_A and GABA_B receptor-mediated components of the GABA-induced Ca²⁺ response

(A) GABA (200 μm, 10 s) induced Ca²⁺ responses before and during treatment with gabazine (10 μm) followed by gabazine together with CGP55485 (3 μm) in a GABA(Ca+) (top) and GABA(Ca-) (bottom) neuron. Note that the addition of gabazine in this GABA(Ca-) neuron did not attenuate the GABA-induced reduction in Ca²⁺. (B) Gabazine inhibited the GABA-induced rise of Ca²⁺ but did not alter the GABA-induced reduction of Ca²⁺. Simultaneous inhibition of GABA_A and GABA_B receptors eliminated the GABA-induced Ca²⁺ changes. Data are the mean ± SEM of the area under the curve (AUC; Ca²⁺ ratio/s over 0-10 s of GABA treatment) of neurons (GABA(Ca+) (n = 19, F_2,36 = 28.91, P < 0.0001, power = 1.00); GABA(Ca-) (n = 12, F_2,22 = 6.56, P = 0.0058, power 0.875); GABA(Ca0) (n = 4, F_2,6 = 0.198, P = 0.826, power = 0.069) from three different SCN slices with repeated-measures anova analysis and post hoc Bonferroni t-test adjusted for three comparisons [(GABA(Ca+) ***P < 0.0001, Gz vs. Gz + CGP55485 P = 0.46; GABA(Ca-) control vs. Gz P = 0.49, control vs. Gz + CGP55485 P = 0.13, *P = 0.0048). (C) Example showing GABA-induced Ca²⁺ response during CGP55485 application followed by CGP55485 together with gabazine in GABA(Ca+) (top) and GABA(Ca-) (bottom) neurons. Note that the addition of gabazine in this GABA(Ca-) neuron eliminated the GABA-induced reduction in Ca²⁺. (D) Effects of baclofen (10 μm), muscimol (50 μm) and GABA (200 μM) on GABA(Ca+) neurons. The top trace is an example of Ca²⁺ responses in a GABA(Ca+) neuron. Below is the mean ± SEM of the AUC in 69 GABA(Ca+) neurons. (E) Effects of baclofen, muscimol and GABA as in (D), but with GABA(Ca-) neurons. Below is the mean ± SEM of the Ca²⁺ change in 21 GABA(Ca-) neurons. (F) The top traces show GABA- and gabazine-induced changes in the baseline Ca²⁺ ratio in two SCN neurons during the night. Gabazine induced a reduction in the baseline Ca²⁺ ratio in the GABA(Ca+) neuron (dashes) and an elevation of the baseline Ca²⁺ ratio in the GABA(Ca-) neuron (solid). The bottom trace shows an example of gabazine-induced hyperpolarization in a GABA(Ca+) SCN neuron during the night recorded using a loose-seal recording electrode.

Fig. 9. Inhibiting endogenous γ-aminobutyric acid (GABA)_A alters [Ca²⁺]_i in SCN neurons

(Top) Regional variation of gabazine-induced (10 μm) changes in baseline Ca²⁺ ratio in SCN neurons. Responses were separated into three groups: a decrease of the Ca²⁺ ratio [gabazine(Ca-), top-left]; an increase of the Ca²⁺ ratio [gabazine(Ca+), top-middle]; and a non-responsive group Ca²⁺ [gabazine(Ca0), top-right]. The position for each neuron was superimposed on a representative drawing of the SCN, with the 3^rd ventricle (3V) on the left and optic chiasm (OC) at the bottom. (Bottom left) Regional localization of individual SCN neurons with a GABA (200 μm)-induced increase in Ca²⁺ and a gabazine-induced decrease in Ca²⁺ (see Figs 5 and 8F). (Bottom middle) Regional variation of individual SCN neurons demonstrating both a GABA-induced decease and a gabazine-induced increase of [Ca²⁺]_i. (Bottom right) The mean change in the baseline Ca²⁺ ratio produced by gabazine was in an opposite direction to the Ca²⁺ response evoked by GABA. Data are the mean ± SEM of the Ca²⁺ ratio change in gabazine-responsive neurons for GABA(Ca+) (n = 125), GABA(Ca-) (n = 77) and GABA(Ca0) (n = 65) neurons (F_2,264 = 14.74, P < 0.0001, power = 1.00) by anova with Bonferroni t-tests adjusted for three comparisons, ***P < 0.0001. Note that GABA(Ca-) and GABA(Ca0) respond similarly (P = 1).

Fig. 10. Retinohypothalamic tract (RHT) stimulation induces a range of postsynaptic Ca²⁺ responses

Stimulation of the RHT (100 pulses, 200 μs at 20 Hz) via a bipolar electrode placed in the optic chiasm (Fig. 1A) induced a transient elevation in Ca²⁺ [RHT(Ca+)], a transient reduction in Ca²⁺ [RHT(Ca-)] or no alteration in the Ca²⁺ ratio [RHT(Ca0)]. (A) The Ca²⁺ response to RHT stimulation and g-aminobutyric acid (GABA; 200 μm, 10 s) in two SCN neurons recorded during the day. CGP55845 (3 μm) application did not alter the RHT-induced Ca²⁺ signal in either cell, but the addition of gabazine (10 μm) eliminated the RHT-induced Ca²⁺ transient in the GABA(Ca+) neuron and induced a RHT-evoked Ca²⁺ response and a small elevation in the baseline Ca²⁺ ratio in the GABA(Ca0) neuron. Tetrodotoxin (TTX; 0.5 μm) eliminated the RHT-induced Ca²⁺ transients in both neurons. Note that GABA induced a biphasic Ca²⁺ response in the neuron with the higher baseline Ca²⁺ ratio (top trace), and the GABA_B blocker reduced the duration of the later phase. Blocking both GABA_A and GABA_B eliminated both phases, and TTX reduced the baseline Ca²⁺ ratio but did not eliminate the rise in Ca²⁺ induced by GABA. (B) Example of a RHT(Ca-) response in a GABA(Ca-) SCN neuron during the day. Gabazine elevated the baseline Ca²⁺ and changed the direction of the RHT-induced Ca²⁺ response [RHT-Gz(Ca+)]. (C) Example showing the Ca²⁺ response following RHT stimulation in GABA(Ca+) and GABA(Ca-) neurons recorded during the night. Gabazine lowered the baseline Ca²⁺ in the GABA(Ca+) neuron and eliminated the RHT-induced transient elevation of Ca²⁺ [RHT-Gz(Ca-)]. In contrast, gabazine increased the RHT-induced Ca²⁺ elevation in a GABA(Ca-) neuron. NMDA, N-methyl-d-aspartate.

Fig. 11. Regional variation of retinohypothalamic tract (RHT) stimulation induced Ca²⁺ transients in the SCN

(Top) Regional variation in Ca²⁺ responses following optic chiasm (OC) stimulation (100 pulses at 20 Hz). Responses were separated into three groups: a transient elevation in Ca²⁺ [RHT(Ca+)]; a reduction [RHT(Ca-)]; and no alteration in the Ca²⁺ ratio [RHT(Ca0)]. The position for each neuron was superimposed on a representative drawing of the SCN, with the 3^rd ventricle (3V) on the left and OC at the bottom. The distribution appears similar to that of the Ca²⁺ response to GABA (Fig. 5). (Bottom left) Regional variation of individual SCN neurons that showed both a RHT stimulation-induced and GABA-induced (200 μm) increase in Ca²⁺. (Bottom middle) Regional variation of individual SCN neurons with a RHT- and GABA-induced decease of Ca²⁺. (Bottom right) The percentage of GABA(Ca+) (day n = 94, night n = 71) and GABA(Ca-) (day n = 76, night n = 8) SCN neurons also responding to RHT stimulation.

Fig. 12. Theoretical model of glutamatergic light input signaling to the g-aminobutyric acid (GABA)ergic suprachiasmatic nucleus (SCN) network

The effect of GABA on membrane potential and [Ca²⁺]_i is a balance, with excitatory and inhibitory effects of GABA neurotransmission within the network driven by the chloride gradient that is under control by the cotransporters, NKCC1 and KCC2. (A) Depolarization induced by external light input signaling to glutamate receptors (GluR) via the retinohypothalamic tract (RHT) is facilitated by GABA release from other SCN neurons (dashed circle) within the network to a SCN neuron (solid circle) with a high intercellular chloride concentration ([Cl^-]_i), where opening of GABA_A receptors (GABA_AR) moves Cl^- out of the neuron, further depolarizing the membrane potential (V_m), opening voltage-dependent calcium channels (VDCC) and increasing [Ca²⁺]_i. (B) In neurons with low baseline [Cl^-]_i, GABA released from the SCN network opens GABA_AR and moves Cl^- into the neuron hyperpolarizing the cell beyond the depolarizing effect of RHT signaling and decreasing [Ca²⁺]_i. A balance may also occur between the depolarizing action of RHT light input signaling with a hyperpolarizing effect from GABA release within the network, and no change in membrane potential, firing frequency or [Ca²⁺]_i occurs.