Interneurons with soma residing in the str. lacunosum moleculare have wide spread dendritic and axonal arbors. Dendrites extend horizontally through the str. lacunosum moleculare and ventrally into the str. lucidum (Ascoli et al., 2009
) receiving MF input on both the dorsal and ventral dendritic arbors (Cosgrove et al., 2009
). Axonal arbors of L-Mi are complex and extend throughout the str. radiatum (Ascoli et al., 2009
), presumably contacting CA3 pyramidal cells along the apical dendrite, providing feedforward inhibition (Lacaille and Schwartzkroin, 1988
; Williams et al., 1994
). To determine whether MF input to L-Mi is regulated by group III mGluRs, recordings were made from visually identified L-Mi and the mossy fiber was stimulated both within the str. lucidum (MFSL
), and at the suprapyramidal blade of the dentate gyrus (MFSDG
; ; Cosgrove et al., 2009
; Galvan et al., 2008
; Galvan et al., 2010
). The origin of EPSCs was confirmed to be MF by application of the group II mGluRs agonist DCG-IV (Kamiya et al., 1996
). While DGC-IV inhibition of MF transmission in pyramidal cells is consistently complete (≥90%; Kamiya et al., 1996
), it is partial in interneurons. For example, Alle et al. (2001)
observed a reduction of about 70% for both the composite and unitary postsynaptic responses obtained from paired recordings. Thus, EPSCs in L-Mi were considered of MF origin if the percent decrease from DCG-IV application was greater than 50%, a measure that has been used previously (Cosgrove et al., 2009
; Galvan et al., 2008
; Galvan et al., 2010
; Lawrence et al., 2004
; Toth et al., 2000
High affinity group III mGluRs are present on MF terminals contacting L-Mi
Receptors belonging to group III mGluRs are mGluRs 4, 6, 7 and 8 and are activated by the agonist L-AP4 (Conn and Pin, 1997
). Of those receptors, mGluRs 4, 7 and 8 are expressed in the hippocampus (Saugstad et al., 1994
; Shigemoto et al., 1997
; Tanabe et al., 1993
). Previous reports indicate that MF input to str. lucidum interneurons is modulated by mGluR 7, the low affinity group III mGluRs (Pelkey et al., 2005
) which binds glutamate in the 1 mM range and L-AP4 at 160 to 500 μM (Conn and Pin, 1997
). To determine whether this receptor is also present on MF terminals contacting L-Mi, we performed a dose-response curve using the group III mGluR agonist L-AP4 (). Surprisingly, we found no evidence for the presence of mGluR 7, but did find evidence for the presence of high affinity group III mGluRs 4 and 8, which have an affinity for glutamate near 3–38 μM, and for L-AP4 in the 1 – 2 μM range (Conn and Pin, 1997
). Specifically, the dose response curve shows that L-AP4 at 400 μM produced no further decrease in EPSC amplitude compared to L-AP4 at 20 μM, a saturating dose for mGluRs 4/ 8 that is insufficient to activate mGluR 7 (400 μM: 31.93 ± 7.13% of control, N = 5; 20 μM: 32.30 ± 6.47% of control, N = 9; p = 0.971; ). Though these data do not conclusively exclude the presence of mGluR 7, they do reveal the novel presence of high affinity group III mGluRs 4/8 at a MF to interneuron synapse.
In the next series of experiments, we applied 10 μM L-AP4 to ensure that we were selectively activating mGluRs 4/8, and also stimulated the RC pathway of CA3, which has been shown to be relatively insensitive to mGluRs agonists (Kamiya et al., 1996
; Toth and McBain, 1998
; but see Doherty and Dingledine, 1998
). EPSCs evoked by MF stimulation decreased by 45.65 ± 5.47% (N = 12, p < 0.001) in the presence of 10 μM L-AP4 and was fully reversible (93.38 ± 4.34% of control, p = 0.183; , blue bars). For this sample of cells, the group II mGluR agonist DCG-IV (1 μM) decreased EPSCs by 67.75 ± 4.34% (N = 12, p < 0.001), which is similar to the effect of DCG-IV on MF-evoked responses in other interneurons (Alle et al., 2001
; Lawrence et al., 2004
; Toth et al., 2000
). In contrast, EPSCs evoked by RC stimulation did not change in response to either L-AP4 or DCG-IV application (L-AP4: 2.71 ± 5.92% decrease, p = 0.666; DCG-IV: 4.45 ± 5.14% decrease, p = 0.426; N = 6; , red bars). Though RC and MF axons cross in the str. lucidum of CA3, it is unlikely that RC inputs were activated by the MFSL
stimulation location since both the MFSL
stimulation locations had the same DCG-IV sensitivity (). Interestingly, the decrease in EPSC amplitude with 10 μM L-AP4 was similar to that previously reported at the MF to pyramidal cell synapse of the guinea pig (Manzoni et al., 1995
; Yoshino et al., 1996
) and at the granule cell input to hilar border interneurons (Doherty and Dingledine, 1998
MFSDG and MFSL input to L-Mi are not different
In a subset of these cells, the control recordings had a failure rate of at least 10%. For these cells, we analyzed the failure rate, paired-pulse ratio and coefficient of variation before and after the application of 10 μM L-AP4. As would be expected from a presynaptically mediated depression, application of L-AP4 increased the failure rate (21.0 ± 6.23% control to 66.45 ± 11.47% L-AP4; p < 0.01; N = 6), paired-pulse ratio (1.04 ± .05 control to 1.54 ± .14 L-AP4; p < 0.01; N = 5) and coefficient of variation (0.535 ± 0.08 control to 0.857 ± 0.208; p < 0.05; N = 6) indicating that activation of mGluRs 4/8 decreased the probability of transmitter release ().
Since MFs are pharmacologically identified through the application of the group II mGluRs agonist DCG-IV (Kamiya et al., 1996
), and both group II and group III mGluRs have been shown to inhibit adenylyl cyclase (Cartmell and Schoepp, 2000
; Conn and Pin, 1997
), we wanted to know whether activation of mGluRs 4/8 on MF terminals by L-AP4 occluded the actions of DCG-IV on group II mGluRs. In a separate group of cells, we first applied L-AP4 and then DCG-IV without a washout period. In these experiments, L-AP4 did not occlude the actions of DCG-IV. Following application of 10 μM L-AP4 (38.28 ± 4.2% decrease, N = 10), DCG-IV caused a further decrease of 66.08 ± 5.32% in EPSC amplitude (p < 0.001; N = 10; ). Additionally, since 10 μM L-AP4 was not a maximal concentration (see ), the effect of DCG-IV after L-AP4 application was compared to the effect of DCG-IV alone (previous experiment). There was no significant difference in the inhibition produced by DCG-IV alone (67.75 ± 4.34%; N = 12) vs. DCG-IV after L-AP4 (66.08 ± 5.32%; N = 10; p = 0.967; ).
Both the increase in failure rate and PPR as a result of L-AP4 application suggests a presynaptic localization of mGluRs 4/8 at the MF – L-Mi synapse, an interpretation supported by anatomical studies (Bradley et al., 1999
; Shigemoto et al., 1997
; but see Bradley et al., 1996
). To obtain direct confirmation of the presynaptic localization of the receptor, we bath applied Sr2+
(see Methods) to de-synchronize the evoked release into putative single quanta (Bekkers and Clements, 1999
; Goda and Stevens, 1994
; Lawrence et al., 2004
). The amplitude distribution of asynchronous EPSCs (aESPCs; see Methods) revealed a quantal amplitude of 7.88 ± 0.29 pA (range: 6.70 pA to 9.59 pA; N = 10; Supplemental Fig. 1D
; ), a value similarly found at other central synapses (Bekkers and Clements, 1999
; Jonas et al., 1993
) but significantly smaller than the quantal amplitude for the MF synapse on str. lucidum interneurons (mean: 24.5 pA; Lawrence et al., 2004
). To confirm that activation of mGluRs 4/8 had a presynaptic effect, we assessed the effect of L-AP4 (10 μM) on the amplitude and frequency of aEPSCs (Price et al., 2005
) in a separate group of cells. In the presence of 3 mM Sr2+
, L-AP4 application decreased the frequency of aEPSCs following stimulation (3.72 ± 0.72 Hz in control vs. 2.30 ± 0.30 Hz after L-AP4; p < 0.05; N = 7) without changing the amplitude (7.09 ± 0.40 pA control vs. 7.73 ± 0.61 pA L-AP4; p = 0.389; N = 7; ). In addition to comparing the mean amplitudes, the distribution of aEPSC amplitudes within each cell was compared before and after application of L-AP4 to ensure that no change in the distributions of aEPSC amplitudes was observed (; p = 0.198). None of the cells tested showed any significant difference in the distribution of aEPSC amplitudes (p ≥ 0.198 for all cells), confirming the lack of effect of L-AP4 on aEPSC amplitude. The effect on frequency was fully reversible (3.84 ± 0.33 Hz after washout). The lack of change in aEPSC amplitude combined with a significant decrease in frequency indicates that activation of mGluRs 4/8 decreases the probability of release without affecting the postsynaptic cell (Price et al., 2005
L-AP4 decreases frequency but not amplitude of aEPSCs
Because PP synaptic responses are sensitive to both group II and group III mGluRs, it could be argued that electrical stimulation applied to MFSDG
could also evoke PP EPSCs. This is unlikely for the following reasons. First, the medial and lateral PPs are modulated by different mGluRs, the medial PP by group II mGluRs and the lateral PP by group III mGluRs, but not both (Macek et al., 1996
). The postsynaptic responses that were evoked from MFSDG
stimulation were highly sensitive to agonists of both group II and group III mGluRs. Additionally, when the MF was stimulated within the str. lucidum (MFSL
) activating synapses far from the str. lacunosum-moleculare (Cosgrove et al., 2009
), the sensitivity to L-AP4 was identical to stimulation at the MFSDG
(). Together, these data support the claim that glutamate release from MF onto L-Mi is modulated by mGluRs 4/8. This novel finding indicates the presence of high affinity group III mGluRs at MF terminals targeting a specific feedforward inhibitory interneuron, suggesting that L-Mi serve unique functional roles in the CA3 neural network.
The N-type calcium channel is a target of high affinity group III mGluRs at the MF to L-Mi synapse
Presynaptic receptors commonly regulate transmitter release by modulating the voltage-gated calcium channels (VGCCs) linked to release (Ferraguti and Shigemoto, 2006
; Takahashi et al., 1996
). Group III mGluRs inhibit adenylyl cyclase, decreasing the activity of protein kinase A (PKA) which has been shown to target VGCCs (Anwyl, 1999
; de Jong and Verhage, 2009
). In contrast, group II mGluRs are frequently found to inhibit transmitter release through VGCC-independent mechanisms (Anwyl, 1999
; Capogna, 2004
; Glitsch, 2006
; Knöpfel and Uusisaari, 2008
). Since application of L-AP4 did not occlude the action of DCG-IV at the MF to L-Mi synapse, we hypothesized that mGluRs 4/8 activation results in inhibition of VGCCs, whereas activation of group II mGluRs would not, further confirming the independence of their intracellular signaling cascades. To determine whether VGCCs are a downstream target of mGluRs 4/8 or mGluRs 2/3 activation at the MF to L-Mi synapse, we first determined the complement of VGCCs linked to release of glutamate at MF to L-Mi synapses. Using specific toxins to isolate the P/Q- and N-type calcium channels, we found that glutamate release at the MF to L-Mi connection is largely dependent on P/Q-type VGCCs, as previously reported for the MF to pyramidal cell, and MF to str. lucidum interneuron connections (Breustedt et al., 2003
; Castillo et al., 1994
; Li et al., 2007
; Miyazaki et al., 2005
; Pelkey et al., 2006
). Specifically, we found that glutamate release was predominantly linked to P/Q-type channels (ω-agatoxin IVA decreased EPSC amplitude by 83.1 ± 2.4%; N = 13; p < 0.0001) with a smaller but significant contribution from N-type VGCCs (ω-conotoxin GVIA decreased EPSC amplitude by 27.29 ± 5.5%, N = 18; p < 0.001; ).
Group III mGluR activation targets glutamate release linked to N-type VGCCs
Next, we assessed whether N-type or P/Q-type VGCCs were downstream targets of mGluRs 4/8 or mGluRs 2/3 activation by applying L-AP4 or DCG-IV, respectively, after selectively blocking the N- or P/Q-type VGCCs. DCG-IV decreased EPSC amplitude by 63.75 ± 7.14% (N = 7) in the presence of ω-CgTx and 63.35 ± 5.94% (N = 6) in the presence of ω-AgaTx, indicating that neither VGCC blocker occluded the effect of DCG-IV (). Furthermore, in comparing the effect of DCG-IV in the presence of the selective VGCC blockers to the effect of DCG-IV alone (decrease of 67.75 ± 4.34%, N = 11, value from previous experiment, see ), there was no significant difference in the percent decrease (p = 0.812, ), indicating that activation of mGluRs 2/3 does not involve N- or P/Q-type VGCCs as a downstream target.
In contrast to what was seen with DCG-IV, application of L-AP4 in the continued presence of the selective VGCC blockers did indicate that the N-type VGCC is a downstream target of mGluRs 4/8 activation. L-AP4 decreased EPSC amplitude by 28.05 ± 4.90% (N = 10) in the presence of ω-CgTx, but caused a decrease of 62.0 ± 11.69% (N = 7; p < 0.01; ) in the presence of ω-AgaTx. These data indicate that activation of mGluRs 4/8 results in selective inhibition of glutamate release linked to N-type VGCCs, which is a similar to what has been reported at other synapses (Rusakov et al., 2004
). Additionally, when these data are compared to the effect of 10 μM L-AP4 in the absence of a VGCC blocker, the effect of L-AP4 after blockade of N-type VGCCs is significantly smaller, indicating that ω-CgTx partially occludes the effects of L-AP4 (L-AP4 after CgTx: 28.05 ± 4.90% decrease; N = 10 vs. L-AP4 alone: 45.65 ± 5.47% decrease; N = 11; p < 0.05; value from previous experiment, see ; ).
Since there was large variability in the effect of ω-CgTx at MF to L-Mi synapses, determining whether the effect of L-AP4 was occluded by blocking N-type VGCCs was difficult because in some cells N-type VGCCs were not linked to release, as the effect of ω-CgTx was very small (). If mGluRs 4/8 selectively target glutamate release linked to N-type over P/Q-type VGCCs, we hypothesized that there would be a correlation between the effects of ω-CgTx and L-AP4 in the continued presence of ω-CgTx. To investigate this, the percent decrease in EPSC amplitude following L-AP4 application in the continued presence of ω-CgTx was plotted vs. the percent decrease in EPSC amplitude produced by the application of ω-CgTx alone. As shown in , we obtained a significant correlation (slope: −42.75 ± 4.44; N = 10; R = −0.832; p < 0.01) such that when ω-CgTx had its largest effect (% decrease near 50%), L-AP4 application resulted in no further decrease in EPSC amplitude (% decrease near 10%; , square symbol and traces).
Interestingly, however, these data also indicate a second, N-type VGCC-independent mechanism through which mGluRs 4/8 inhibit transmitter release at the MF to L-Mi synapse. In a subset of cells that were relatively ω-CgTx resistant (% decrease ≤ 20%, , triangle symbol and trace) indicating that N-type VGCCs were not linked to glutamate release, the effect of L-AP4 was similar to the effect of L-AP4 in the absence of VGCCs blockers (effect of L-AP4 in ω-CgTx resistant cells: −39.08 ± 4.11%, N=4 vs. effect of L-AP4 alone from previous experiment: −45.65 ± 5.47%, N =11; p = 0.504). Thus, it can be concluded that in terminals where N-type VGCCs are linked to transmitter release at the MF to L-Mi connection, the N-type VGCC is a downstream target of mGluRs 4/8 activation (e.g. , square symbol and traces). If N-type VGCCs are not linked to glutamate release however, mGluRs 4/8 activation is equally effective at decreasing glutamate release (e.g. 4F, triangle symbol and traces), possibly through a direct action on the release machinery, as proposed by others (Anwyl, 1999
; Scanziani et al., 1995
; Schoppa and Westbrook, 1997
; Woodhall et al., 2007
Low frequency activity is insufficient to activate high affinity group III mGluRs
Application of the agonist L-AP4 to synapses with high affinity group III mGluRs frequently overestimates the endogenous impact of this receptor on the system (Billups et al., 2005
; Lorez et al., 2003
; von Gersdorff et al., 1997
). Thus, to determine the functional impact of this receptor at the MF to L-Mi connection, we used the competitive antagonist of group III mGluRs, MSOP (100 μM), to prevent endogenous activation (Rusakov et al., 2004
; Semyanov and Kullmann, 2000
; Thomas et al., 1996
). To assess the pattern of activity that might physiologically activate the receptor, we investigated whether the receptor was tonically active and whether it could be activated by low or high frequency activity.
To assess tonic activity, MSOP (100 μM) was applied and the effect on the amplitude of single postsynaptic responses was determined. No effect on the initial amplitude of the postsynaptic response was seen using a test frequency of 0.2 Hz (23.94 ± 2.69 pA control; 23.83 ± 3.14 pA MSOP; p = 0.950; N = 9; ). This was also true for responses recorded in current clamp (amplitude: 1.59 ± 0.57 mV control; 1.48 ± 0.68 mV MSOP; N = 5; p = 0.279; ). These data indicate that the group III mGluRs are not tonically active on MF terminals contacting L-Mi, suggesting that ambient extracellular glutamate concentrations are insufficient to activate the receptor.
Group III mGluRs are neither tonically active nor activated by low frequency activity
Since mGluRs 4/8 have a high affinity for glutamate, these receptors could be activated by low frequency MF discharge. At other targets, the MF input exhibits frequency facilitation, an increase in the amplitude of the postsynaptic response as the stimulation frequency increases from very low (0.05 Hz) to more moderate (1 – 4 Hz; Henze et al., 2000
; Salin et al., 1996
). MF input to pyramidal cells undergoes robust facilitation at low and moderate frequencies that can exceed 600% at 4 Hz vs. 0.05 Hz (Henze et al., 2000
; Salin et al., 1996
; Scanziani et al., 1998
; Toth et al., 2000
). Similarly, it has been reported that mossy fiber input to str. lucidum interneurons exhibits strong frequency facilitation (Toth et al., 2000
). To investigate whether glutamate release by low to moderate frequencies of MF activity were sufficient to activate mGluRs 4/8 at the MF to L-Mi synapse, we recorded MF-evoked EPSCs in L-Mi at stimulation frequencies from 0.05 to 4Hz (Salin et al., 1996
). In contrast to MF responses in pyramidal cells and str. lucidum interneurons, we did not detect a significant increase in EPSC amplitudes at 4 Hz compared to the 0.05 Hz control (1.09 ± 0.12 normalized to 0.05 Hz control; p = 0.460; N = 8; ).
Although we did not observe frequency facilitation in the control condition, it is possible that application of MSOP (100 μM) might reveal an underlying facilitation at moderate frequencies (Scanziani et al., 1997
; Toth et al., 2000
). To test this hypothesis, we stimulated the MF with alternate periods of 0.05 Hz and 2 Hz (Scanziani et al., 1997
), a frequency that shows strong facilitation at both the MF to pyramidal cell and MF to str. lucidum interneuron connections (Toth et al., 2000
). Application of MSOP did not rescue frequency facilitation at the MF – L-Mi synapse tested at 2Hz (normalized to 0.05 Hz: 1.21 ± 0.10 control; 1.11 ± 0.20 MSOP; N = 5; p = 0.677; ). Furthermore, amplitudes at 2 Hz were not significantly different than amplitudes at 0.05 Hz for either the control or MSOP condition (control: 28.71 ± 3.91 pA at 0.05 Hz; 34.79 ± 5.47 pA at 2 Hz; p = 0.108; N = 5; MSOP: 29.82 ± 5.46 pA at 0.05 Hz; 29.70 ± 2.83 pA at 2Hz; p = 0.972; N = 5) indicating that low and moderate frequencies are insufficient to activate this receptor.
Activation of high affinity group III mGluRs by high frequency MF activity delays the onset of L-Mi firing
Having demonstrated that mGluRs 4/8 are not tonically active at the MF to L-Mi connection, nor activated by low and moderate frequencies of activity, we were interested in determining the effect of the receptor at high frequencies of activity. Previous reports have demonstrated that short trains of high frequency activity are sufficient to activate high affinity group III mGluRs (Chen et al., 2002
; Losonczy et al., 2003
). To determine whether short trains of high frequency activity were sufficient to activate mGluRs 4/8 at MF input to L-Mi, we recorded trains of five MF-evoked EPSCs at 20 and 40 Hz, frequencies that fall within the range of granule cell firing as a rat traverses a place field (Henze et al., 2002
; Jung and McNaughton, 1993
), before and after application of MSOP.
shows the data from those experiments, demonstrating the facilitation ratio (EPSCN / EPSC1) of the postsynaptic response for each stimulus in the train before and after application of MSOP. MSOP revealed an average enhancement of facilitation at 20 Hz, and though there was a trend at 40 Hz, the effect was not significant for most test stimuli (stimuli 2 – 5) when the data from each condition were averaged. In part, the lack of significance at all points in the 40 Hz train for these data is due to the high variability of facilitation in the control condition, as well as in the MSOP condition, as demonstrated in the averaged traces in . For example, in some cells the largest effect of MSOP was at stimulus #3, whereas for other cells, stimulus #5 showed the largest effect (see averaged traces in ). Because of the variability in the timing of the facilitation resulting from MSOP application, the averaged population data did not accurately represent the effect of MSOP on short term facilitation at the MF to L-Mi connection. Therefore, the data were aligned to the stimulus number at which the greatest difference was observed after application of MSOP. Using this method, enhanced facilitation is observed following application of 100 μM MSOP at both 20 (EPSCN / EPSC1: control, 1.32 ± 0.18; MSOP, 1.79 ± 0.24; N = 8; p < 0.001; ) and 40 Hz (EPSCN / EPSC1: control, 1.49 ± 0.17; MSOP, 2.05 ± 0.24; N = 7; p < 0.05; ).
Relief of inhibition mediated by mGluRs 4/8 using the selective receptor antagonist MSOP reveals enhanced facilitation during short, high frequency trains
Though the EPSC train data demonstrate that mGluRs 4/8 are activated by synaptically released glutamate during a physiologic pattern of high frequency MF activity, we were primarily interested in determining the effect of mGluRs 4/8 activation on the CA3 network, and specifically the ability of MF-evoked synaptic activity to elicit action potentials in the L-Mi. In vivo
data show that short trains of high frequency MF activity can elicit action potentials in CA3 interneurons (Henze et al., 2002
). Consequently, we determined the effect of MSOP on spike transmission at the MF – L-Mi connection. Based on the EPSC data in , we hypothesized that activation of mGluRs 4/8 during high frequency MF activity would modulate the MF to L-Mi connection such that activation of the receptor would result in delayed spike transmission between the MF to the L-Mi. To test this, we applied trains of ten stimuli to the MF at 20 and 40Hz before and after the application of MSOP. We then determined whether the group III receptor had an effect on the probability of action potential generation, and whether it changed the latency to the first action potential in response to the train. In these experiments, L-Mi were recorded in current clamp conditions, and the MF input was stimulated only in the MFSDG
location, as the MFSDG
stimulation location was less likely to recruit additional glutamatergic inputs throughout the train of stimuli (observation from the previous experiment). Once a stable EPSP amplitude was obtained, the Vh
was adjusted between −60 mV and −55 mV to allow for a low probability of firing (P(AP) = ~0.1) in response to 10 stimuli at 20 Hz. 30 trials of 10 stimuli at 20 and 40 Hz, delivered at 20 second intervals were then collected before and after the application of MSOP (100 μM). shows several overlapping, consecutive sweeps from a representative cell, and depicts the raster plot of action potentials during the train before and after MSOP application. From these data, the probability of firing in response to each stimulus in the train was calculated and the summary data plotted in . We found that the overall probability of action potential firing in response to both frequencies was significantly higher in the presence of MSOP (20 Hz: 0.08 ± 0.02 control to 0.20 ± 0.05 MSOP, N = 5, p < 0.05; 40Hz: 0.16 ± 0.02 control to 0.21 ± 0.03 MSOP; N = 5; p < 0.05; ). At the end of the experiment, DCG-IV (2.5 μM) was applied to confirm MF origin (71.20 ± 8.37% decrease, N = 5, p < 0.001).
Activation of presynaptic group III mGluRs decreases the probability of AP firing in L-Mi in response to trains of MF input
More importantly, the time to first action potential was shorter in the presence of MSOP, indicating that activation of the group III mGluRs delays the firing of the postsynaptic L-M interneuron in response to MF input. This was determined by calculating the latency to the first action potential in response to the train of MF input for each trial before and after MSOP (). These data were then binned by stimulus number and plotted as a cumulative scatter plot, which was fit with a Boltzmann function. MSOP application significantly decreased the time to first action potential in response to stimuli at both 20 Hz (50% control: 192.24 ± 12.0 ms; 50% MSOP: 102.02 ± 13.7 ms; p < 0.05; ) and 40 Hz (50% control: 79.71 ± 3.32 ms; 50% MSOP: 51.71 ± 1.46ms; p < 0.05; ). It is important to note that in some of the trials, the L-Mi never fired and so these numbers are an underestimate of the shift in the timing of L-Mi firing. Of equal importance is that these changes occurred without any significant change in action potential threshold (−38.26 ± 1.36 mV control; −39.28 ± 1.73 mV MSOP; N = 5; p = 0.134; ).
Activation of group III mGluRs delays L-Mi firing in response to a train of MF stimulation
Because we were concerned that intracellular dialysis during whole-cell recordings may be affecting the threshold for action potential elicitation, we repeated this experiment using the cell-attached configuration in voltage clamp (seal resistance ~100 to 500 MΩ; Perkins, 2006
). Nearly identical results were obtained in cell attached for both 20 Hz (probability of AP: 0.12 ± 0.01 to 0.34 ± 0.05; p < 0.001; N = 7; time to first AP: 150.67 ± 88.6 ms to 93.98 ± 8.93 ms; p < 0.05; N = 7) and 40 Hz (probability of AP: 0.25 ± 0.06 to 0.44 ± .06; p < 0.05; N = 7; time to first AP: 174.55 ± 5.49 ms to 47.34 ± 3.32 ms; p < 0.05; N = 7) confirming that this effect is due to activation of the group III mGluRs, and not heavily influenced by the recording conditions ( and ).