Data were collected from a total of 1,272 cells from 104 animals. Every experimental and control group in this study contains data from at least four animals. Each of these cells were determined to be within the SCN by directly visualizing the cell’s location with IR-DIC videomicroscopy before any data were collected. In most cases, the IR-DIC video images were sufficient to label a cell as being in VL or DM regions of the SCN. In addition, some neurons were filled with biocytin through the patch electrode and histologically processed. Slices containing these labeled cells were counter-stained with a Nissl stain to confirm the neuron’s location within the SCN. All of the cells, which had been visually determined to be in the dorsal (6/6) or ventral (6/6) regions of the SCN, also demonstrated this localization with biocytin fills and Nissl stain.
sEPSCs recorded in VL and DM SCN
The mean frequency and the mean amplitude of sEPSCs recorded at a holding potential of −70 mV were 0.26 ± 0.04 (SE) events/s (n = 51; range: 1.29–0.01 events/s) and −17.5 ± 0.8 pA (range: 37–7 pA), respectively. The time to rise and the time to decay (latency of the inward current peak from the baseline) were 1.5 ± 0.04 ms (range: 3.6–1.4 ms) and 2.2 ± 0.1 ms (range: 4.6–1 ms), respectively. These sEPSCs did not appear to be driven by action potentials as neither the amplitude nor the frequency of the currents was significantly altered by the application of the sodium (Na+) channel blocker tetrodotoxin (TTX, n = 6). The sEPSCs were completely abolished with AMPA/KA GluR antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 25 µM, 5 of 5 neurons tested), indicating that they are mediated by AMPA/KA GluRs (, top). Interestingly, these sEPSCs could be recorded from both VL and DM SCN, and there were no significant differences between the currents recorded in each region. In the DM SCN, the mean frequency and amplitude of sEPSCs were 0.30 ± 0.07 events/s and −18.9 ± 1.4 pA (n = 23), respectively. Whereas in VL SCN, the mean frequency and amplitude of sEPSCs were 0.23 ± 0.03 events/s and −16.3 ± 1.0 pA (n = 28), respectively. Thus sEPSCs are not restricted to the retinal recipient VL region but instead are a general feature of cells within the SCN.
FIG. 1 Example of spontaneous excitatory postsynaptic currents (sEPSCs) recorded in the day (top) in the suprachiasmatic nucleus (SCN). The sEPSCs were blocked by the bath application of CNQX (25 µM; middle). The sEPSCs were completely abolished with (more ...)
Spontaneous EPSCs frequency and amplitude did not vary between day and night in the SCN
The next experiment was designed to determine whether sEPSCs recorded in SCN neurons varied between day and night. These experiments were performed with brain slices taken from animals during their day and compared with data obtained from brain slices from animals during their night. Rats were killed 2–3 h after lights-on for the “day” group or immediately before lights-off for the “night” group. Other than the time that the animals are killed, all conditions between the day and night groups remained constant. The data were collected between zeitgeber time (ZT) 4–8 and ZT 14–16 and were pooled to form “day” and “night” groups, respectively. There were no significant differences in the amplitude, frequency, rise, or fall times of the sEPSCs (). During the day, sEPSCs had a mean amplitude of 16.5 ± 1.0 pA with a mean frequency of 0.27 ± 0.05 Hz (range: 1.2–0.05 Hz; n = 29). Similarly, during the night, the mean amplitude of the sEPSCs was found to be 18.8 ± 1.3 pA with a frequency of 0.25 ± 0.06 Hz (range: 0.65–0.01 Hz; n = 22). These data indicate that the presynaptic release as well as postsynaptic sensitivity to glutamate is fairly constant throughout the daily cycle.
AMPA-evoked currents recorded in SCN neurons
Whole cell patch-clamp recording techniques were used to directly measure currents evoked by a submaximal dose of AMPA (25 µM) in SCN neurons. In these experiments, the current required to hold the cell’s membrane potential at −70 mV was monitored. In addition, the voltage dependence of the AMPA-evoked currents was measured by moving the cell through either a ramp of voltages (from −70 to 40 then back to −90 mV) or a series of voltage-steps (from −120 to 40 mV) before, during, and after treatment with AMPA in the bath. Because activation of voltage-dependent Na+, Ca2+, and K+ currents could distort measurement of AMPA-evoked currents, cesium (Cs+, 125 mM) was used in the patch pipette while tetraethyl-ammonium chloride (TEA, 10 mM), cadmium (Cd2+, 25 µM), and TTX (1 µM) were in the bath. , top, shows the current-voltage relationship of an AMPA current recorded using this protocol. Most SCN neurons (91%; 78 of 86 neurons tested) exhibited AMPA-evoked currents. These inward currents exhibited a linear voltage dependency with reversal potentials around 0 mV. AMPA-evoked currents were eliminated by the addition of the AMPA/KA GluR antagonist CNQX (25 µM; n = 16).
FIG. 2 AMPA currents in SCN neurons. Whole cell patch-clamp recording techniques were used to directly measure currents evoked by AMPA in SCN neurons. The voltage dependence of the AMPA-evoked currents was measured by moving the membrane potential of the cell (more ...)
AMPA-evoked currents could be recorded from both VL and DM SCN, and there were no significant differences between the currents recorded in each region. In the DM SCN, the bath application of AMPA (25 µM) produced a normalized peak current of −4.1 ± 0.5 pA/pF (range: 0.5 to −21.6 pA/pF; n = 66) while in the VL SCN the same treatment produced a peak current of −3.2 ± 0.7 pA/pF (range: 1.2 to −9.9 pA/pF; n = 16). The next experiment was designed to determine whether these inward currents evoked by bath application of AMPA varied between day and night in SCN neurons. As described in the preceding text, these experiments were performed with brain slices taken from animals during their day and compared with data obtained from brain slices from animals during their night. Under these conditions, there was no day/night difference in AMPA-evoked inward currents. During the day, AMPA (25 µM, 120–300 s) produced an average peak inward current of −3.7 ± 0.5 pA/pF (range: 0.8 to −16.2 pA/pF; n = 52), whereas during the night, this same treatment produced an average peak current of −3.6 ± 0.6 pA/pF (0.3 to −14.8 pA/pF; n = 33). These data provide additional support for the conclusions that AMPA-sensitive neurons are found throughout the SCN and that the sensitivity of these cells to AMPA stimulation does not vary with time of day.
AMPA regulation of Ca2+ transients in SCN neurons
A bulk loading procedure was used to load cells with a membrane-permeable form of the Ca2+ indicator dye fura2. This procedure loads many cells in SCN slices from young animals (10- to 15-day-old rats were used in the current study). Cells that exhibited uneven loading due to dye sequestration were not included in the data set. Small cell types including glia were easily identified and were not included in the data set. Bath application of AMPA (1–100 µM, 60 s, day) caused Ca2+ transients in SCN cells in the brain slice (). For example, AMPA (25 µM, 60 s) produced an average increase in Ca2+ of 45 ± 1% or increased estimated free [Ca2+ ]i 20.6 ± 2 nM (range 144 to −12 nM; n = 1027). This response was wide-spread within the SCN with 93% of cells (960/1027) examined showing a Ca2+ increase of 5% or greater. AMPA-evoked Ca2+ transients could be recorded from both VL and DM SCN, and there were no significant differences between the responses recorded in each region. In the DM SCN, the bath application of AMPA (25 µM) produced a peak Ca2+ increase of 43 ± 3% (mean: 20 ± 1 nM; range: 161 to −3 nM; n = 300). In the VL SCN, the same treatment produced a peak Ca2+ increase of 47 ± 5% (mean: 21 ± 2 nM; range: 125 to −0.5 nM; n = 139). These AMPA-induced Ca2+ transients were blocked by treatment with the AMPA/KA GluR antagonist CNQX (AMPA + CNQX: 1.3 ± 0.2%, n = 50).
FIG. 3 Examples of Ca2+ transients measured from SCN cells in a brain slice loaded with the Ca2+ indicator dye fura2. Top: SCN cells show an increase in Ca2+ concentration in response to bath application of AMPA (25 µM, 60 s). Each line represents data (more ...)
influx can be mediated directly by Ca2+
-permeable AMPA receptors and indirectly by depolarization-induced activation of voltage-gated Ca2+
channels. In addition, in some preparations, reverse Na+
exchange contributes to glutamate-induced Ca2+
influx (e.g., Hoyt et al. 1997
; Schroeder et al. 1999
). To investigate the extracellular source of Ca2+
influx, we examined the effect of blocking voltage-gated Ca2+
channels with Cd2+
and the reverse mode of the Na+
exchange with the selective inhibitor 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea (KB-R7943) on the AMPA-induced Ca2+
transients. By itself, KB-R7943 (10–25 µM) did not have any effect on resting Ca2+
(0.9 ± 0.4%). When AMPA was applied in the presence of KB-R7943, there was no significant effect on the magnitude of the AMPA-induced Ca2+
transients. For example, AMPA (25 µM) in the presence of KB-R7943 increased estimated free [Ca2+
by 29.9 nM (range: 168 to −7 nM, n
= 88), whereas the same cells treated with AMPA alone increased Ca2+
by 28.9 nM (range: 155 to −10 nM, n
= 88). In contrast, the application of the Ca2+
channel blocker Cd2+
(25 µM) produced a major reduction in the magnitude of the AMPA-evoked response (AMPA alone: 45 ± 1%, n
= 1027; Cd2+
+ AMPA: 4 ± 1%, n
= 119; ). In the presence of Cd2+
, bath application of AMPA (25 µM) still produced significant Ca2+
< 0.05). However, these responses were restricted to only a few cells (10 of 119 SCN cells exhibited a more than 5% increase in Ca2+
) with most SCN neurons not exhibiting a detectable Ca2+
transient in the presence of Cd2+
. Overall, the majority of the AMPA-induced Ca2+
influx is sensitive to Cd2+
, and it can be assumed to result from the activation of voltage-sensitive Ca2+
channels by AMPA.
Circadian rhythm in the magnitude of AMPA-induced Ca2+ transients
The next experiment was designed to determine whether Ca2+ transients evoked by bath application of AMPA in SCN neurons varied between day and night. As described in the preceding text, these experiments were performed with brain slices taken from animals during their day and compared with data obtained from brain slices from animals during their night. There was a daily rhythm in Ca2+ transients with peak AMPA responses significantly higher during the night than during the day (P < 0.001; ). During the day, bath application of AMPA (25 µM) produced Ca2+ transients with an average peak value 31 ± 26% above baseline having increased estimated [Ca2+ ]i 14 ± 0.7 nM (range: 78 to −12 nM; n = 418). The same treatment during the night, caused an average increase of 55 ± 2% having increased estimated [Ca2+ ]i 25 ± 0.8 nM (range: 144 to −1 nM; n = 609). An examination of the distribution of the Ca2+ transients (, bottom) indicates that the larger mean responses at night was due to a combination of more cells responding to AMPA as well as a larger average response per cell. For example, if analysis is limited to those cells that showed at least a 10% increase in Ca2+, only about half of the cells tested (212/419) reached this threshold compared with 80% at night (473/609). In these responding cells, a diurnal rhythm is still present with AMPA causing an average of 40% change during the day and a 61% change at night. In contrast, treatment with a solution high in potassium (K+, 50 mM, 15 s) produced Ca2+ transients that were not different from day to night (day: 101 ± 7 nM increase, n = 83; night: 102 ± 6 nM increase, n = 83).
FIG. 4 Diurnal rhythm in AMPA-evoked Ca2+ transients in SCN cells. In these experiments, AMPA-evoked Ca2+ transients were measured in SCN neurons in brain slices from animals during their day and compared with data obtained from brain slices from animals during (more ...)
Tonic activation of AMPA/KA GluRs may contribute to resting Ca2+ concentration and tonic firing rate of some SCN neurons
The next experiment was designed to determine if AMPA-mediated currents tonically influence the firing rate of SCN neurons (, top). To address this question, the firing rate of SCN neurons was monitored using the cell-attached recording technique. With this configuration, the patch electrode forms a giga-ohm seal but the membrane is not ruptured. The frequency of spontaneous action potential generation was monitored before and after application of the AMPA/KA GluR antagonist CNQX. Again, CNQX did not have a significant effect (P > 0.05) on frequency of firing of the SCN neurons sampled (n = 16). However, a subset of SCN neurons (6/16 or 37% of cells examined) did show a marked decrease in firing rate after application of CNQX. These six cells exhibit an average decrease of 49%. To examine the possibility of tonic glutamatergic contribution to the resting Ca2+ concentration, SCN cells were treated with CNQX (25 µM) alone during the day (, bottom). Overall, there was no significant effect (P > 0.05) of CNQX on resting Ca2+ concentration; however, many cells (28/73 or 38% of cells examined) did show a reduction of ≥5%. On these cells, application of CNQX produced a modest reduction of 8 ± 0.4%. Together, these results raise the possibility that tonic excitatory drive may influence the activity of at least some cells within the SCN.
FIG. 5 Application of AMPA/KA GluR antagonist CNQX decreases the firing rate and resting calcium levels of a subset of SCN neurons. Top: example of SCN neuron before and after treatment with CNQX (25 µM). The firing rate of this SCN neuron was monitored (more ...)
AMPA receptors are expressed in SCN in both VL and DM subdivisions
To examine the pattern of expression of receptors likely involved in mediating the AMPA-evoked currents, an antibody raised against an AMPA preferring GluR subunit, GluR1, was utilized (). Rats (age 18–21 days) were perfused at ZT 2–4, and sections containing the SCN were examined immunohistochemically. The GluR1 immunoreactivity was clear in the SCN but more diffuse compared with staining in cortex or hippocampus. Most cell bodies were not clearly stained and those that were stained showed “halo” of immunoreactivity around the soma that may represent staining of cell membrane but not cytoplasm. Staining was most robust in two regions of the SCN including the ventral lateral portions as well as a dorsal region of SCN near the 3rd ventricle. This dorsal region contained the most labeled cell bodies, whereas the staining in the ventral region was mostly limited to the neuropil. There was a core region in the center of the SCN that did not exhibit any clear staining. This general pattern was seen throughout the rostral to caudal extent of the SCN. To look for possible day-night differences in immunoreactivity, staining in tissue collected from ZT 2–4 was compared with those collected between ZT 13–15. Qualitatively, there were no obvious differences between these two time points.
FIG. 6 Photomicrographs showing immunoreactivity for GluR1 and GluR2/3 in the suprachiasmatic nucleus. 3rdV, third ventricle; OC, optic chiasm. Left panel: The GluR1 immunoreactivity was most robust in 2 regions of the SCN including the ventral lateral portions (more ...)
An antibody raised against other AMPA preferring GluR subunits, GluR2/3, was also used (). The GluR2/3 immunolabeling in the SCN was quite different that the GluR1. In general GluR2/3 immunoreactivity was seen on cell bodies and in the neuropil throughout the SCN. The staining was most robust in the ventral region of the SCN near the optic chiasm. This was particularly true in the more rostral regions of the SCN as the staining became more diffuse in the caudal regions. Similar to the GluR1 immunoreactivity, most cell bodies stained showed “halo” of immunoreactivity around the soma. Qualitatively, the staining in tissue collected from ZT 2–4 was similar to that observed from tissue collected between ZT 13–15. Overall, the immunocytochemistry analysis clearly indicates the presence of AMPA preferring GluR subunits within the SCN. These receptors are rather broadly expressed and certainly not restricted to the retino-recipient ventral SCN regions.