Release of endocannabinoids by DCN cartwheel cells
Previous studies have shown that depolarization of cartwheel cells evokes release of endogenous cannabinoids that transiently suppress synaptic strength at their parallel-fiber synaptic inputs (Tzounopoulos et al., 2007
; Zhao et al., 2009
). To further investigate the role of endocannabinoids at parallel fiber-CWC synapses and determine the calcium dependence of depolarization-induced suppression of excitation (DSE), whole cell voltage clamp recordings were obtained from CWCs and their identity was confirmed by visualizing the morphology of the cells with fluorescence microscopy (). The protocol for evoking DSE is shown in . To quantify DSE, the average amplitudes of the three EPSCs preceding (“pre
”) and immediately following (“post
”) a voltage step were measured, and DSE was defined as 1-(post
). DSE evoked by 5-second voltage steps under control conditions was 48.7±3.0% (; n=8). This suppression was abolished following bath application of the CB1 receptor antagonist AM251 (; DSE = −0.6±1.3%; n=9). Maximum levels of DSE were obtained with 1-second voltage steps and no additional suppression was observed with more prolonged depolarization of cartwheel cells ().
Figure 1 Depolarization of cartwheel cells induces endocannabinoid-mediated suppression of excitatory synaptic transmission at parallel fiber to cartwheel cell synapses. A. A two-photon laser scanning microscope image of a cartwheel cell loaded with Alexa Fluor (more ...)
Calcium dependence of DSE
High levels of dendritic calcium are required for DSE in cerebellar Purkinje cells (Brenowitz and Regehr, 2003
; Maejima et al., 2005
), but the calcium requirement for DSE in cartwheel cells is not known. We therefore combined synaptic recordings with calcium imaging to determine the relationship between postsynaptic calcium transients and suppression of parallel fiber EPSCs. To measure dendritic calcium, cartwheel cells were loaded through the patch pipette with the low-affinity green fluorescent calcium indicator Fluo-4FF (500 μM, KD
=8.1 μM). Since this indicator has very low fluorescence at resting calcium levels, neurons were also loaded with the red fluorophore Alexa-594 (20 μM) to permit visualization of dendrite and spine morphology (). We used two-photon laser scanning microscopy (2PLSM) to measure calcium transients evoked in CWC dendrites and spines by voltage steps. Both fluorophores were efficiently excited with a Ti-sapphire laser tuned to 810 nm, and fluorescence emission was separated with a dichroic mirror to allow simultaneous collection of green and red fluorescence. Line scans were performed at 500 Hz across a spine and its neighboring dendrite during voltage step trials (), and fluorescence signals were converted to calcium concentrations using ratiometric equations as described in the methods.
Figure 2 Calcium requirements for cartwheel cell DSE. A. Left, Image of the cartwheel cell loaded with Alexa Fluor 594. Right, Enlarged views of proximal and distal dendritic regions of interest (ROIs) indicated in the left panel by 1 and 2, respectively. Line (more ...)
Our strategy for measuring the calcium dependence of DSE was to determine the relationship between peak levels of dendritic calcium evoked by voltage steps and the amount of suppression of PF EPSCs. However, it was not possible to determine the precise dendritic location of the parallel fiber synapses because extracellular stimulation activated a population of fibers that synapse over a region of the CWC dendritic tree. Calcium levels may reach different levels in different dendritic regions as a result of spatial gradients of ion channel density, changes in dendrite diameter and the inability to voltage clamp electrotonically distant regions of the CWC dendrite. Therefore, we measured calcium transients in both proximal and distal CWC dendrites and spines to determine the range of calcium levels reached throughout the dendritic tree during voltage steps (). Proximal regions were selected by locating spines that appeared on proximal dendrites closest to the soma (range: 22–27 μm from the center of the soma, n=5). Distal sites were chosen along the same dendritic branch at tips of dendrites near the pial surface (range: 80–116 μm from the center of the soma measured along the dendritic path, n=5). An example of such an experiment is shown in in which calcium transients were measured in a proximal and a distal dendritic location in response to 1 second depolarizations. In a proximal dendrite 25 μm from the soma, calcium reached a plateau of 6.7 μM during the voltage step. In a subsequent trial in the same neuron at a distal site 96 μm from the soma, dendritic calcium reached 6.5 μM (6% lower than at the proximal site). During voltage steps ranging from 0.1 to 5 seconds, calcium at distal sites reached levels that were 9.4±3.3% lower than at proximal sites (n=5 neurons, data not shown). In addition, peak levels of calcium in dendritic spines and the neighboring shaft were not significantly different, so in subsequent voltage clamp experiments we focused on measuring calcium transients in proximal dendritic shafts of CWCs.
To determine the dependence of the amount of DSE on the peak dendritic calcium during voltage steps, we combined synaptic recordings with dendritic calcium imaging in the same neurons. Data from a representative CWC is shown in . In this neuron, a voltage step of 0.1 second elevated calcium to 2.0 μM in the proximal dendrite but the EPSC was not suppressed. When the voltage step was extended to 0.5 second, peak dendritic calcium reached 5.6 μM and EPSCs were suppressed by 52%. Prolonging the voltage step to 1 and 5 seconds further elevated calcium and increased DSE to 61% and 65%, respectively. Results from a group of 5 neurons are summarized in . Maximal suppression of EPSCs was 53.4±4.8% and was obtained with voltage steps of 1–5 seconds that produced peak calcium transients of 7.4±1.0 μM in proximal dendrites and 6.1±1.1μM in distal dendrites (, ).
To obtain a measure of the calcium dependence of DSE in CWCs, we plotted the amount of DSE for each trial against peak dendritic calcium measured in proximal regions of CWC dendrites (). A fit of the data points to the Hill equation indicated that the dendritic calcium concentration producing half-maximal DSE was 3.9 μM and the Hill coefficient was 5.7. Because calcium levels in distal regions of CWC dendrites are slightly lower, our results demonstrate a high calcium requirement for DSE in the range of 3.6–3.9 μM. Also, because of the steep cooperativity, brief calcium transients with peaks less than ~2 μM are not effective in evoking endocannabinoid release.
Dendritic calcium transients during spontaneous CWC activity
Because CWCs fire spontaneously in vivo
(Davis and Young, 1997
; Ding and Voigt, 1997
; Portfors and Roberts, 2007
) and in brain slices (Zhang and Oertel, 1993
; Kim and Trussell, 2007
) we investigated whether action potential backpropagation during sustained firing of CWCs could elevate dendritic calcium and evoke endocannabinoid release. Spontaneous firing rates of CWCs in slices measured with cell-attached recordings (, top) ranged from 12 to 26 Hz (mean 18±2, n=10). In whole-cell current clamp recordings, CWCs held with zero bias current were spontaneously active at 21.3±0.8 Hz (range: 19.5–23.9 Hz, n=6, , bottom). Although in some cases complex spikes were observed, in subsequent experiments we focused on the role of simple sodium spikes in producing dendritic calcium transients and retrograde inhibition, and cells firing complex spikes at rates above 1 Hz were excluded from our analysis.
Figure 3 Sodium action potentials backpropagate into the cartwheel cell dendrites and elevate intracellular calcium. A. Cartwheel cells are spontaneously active. Top, cell-attached recording of action potentials in tonic firing mode, performed in voltage-clamp (more ...)
Action potentials in CWCs can backpropagate and evoke dendritic calcium transients (Molitor and Manis, 2003
; Roberts et al., 2008
). We measured calcium transients in CWC dendrites and spines at proximal and distal locations to confirm that calcium transients propagate throughout the dendritic tree. In response to single action potentials triggered with brief current injections (2 ms, 1–2 nA), calcium transients were always observed in both proximal and distal dendrites and spines of CWCs (), indicating that calcium levels throughout CWC dendrites become elevated during ongoing spontaneous firing. To determine the relationship between CWC firing rates and dendritic calcium levels during spike trains, we measured dendritic calcium during spike trains of different frequencies that were obtained by varying the amplitude of current injections. Results from a representative CWC are shown in (left
). In response to a series of current steps that ranged from 100–500 pA, dendritic calcium levels reached a plateau level that was dependent on the CWC firing frequency. Data from multiple trials from 6 CWCs were binned by spike rate, indicating that dendritic calcium levels progressively increase with firing rates in CWCs (). In addition, the differences in calcium levels between spines and neighboring dendritic shafts following single spikes in proximal dendritic regions () were not observed during trains of action potentials. These results indicate that during spontaneous firing, dendritic calcium increases to a plateau level of several hundred nanomolar, and suggest that prolonged spiking could evoke endocannabinoid release from CWC dendrites and produce retrograde suppression of parallel fiber inputs.
Repetitive firing evokes endocannabinoid release and suppression of parallel fiber synapses
To test whether sustained firing by CWCs evokes endocannabinoid release, we measured parallel fiber EPSPs before and after spike trains of 15 second duration. Current injection amplitudes were adjusted to generate sustained firing of simple spikes throughout the 15 second trial, and PF EPSPs were evoked at 0.5 Hz before and after the spike train. A representative trial is shown in in which the CWC fired for 15 seconds at a mean rate of 34.5 Hz. Suppression of EPSPs (calculated as the ratio of the mean of 3 EPSPs evoked after vs. before the spike train) was 79%, and returned to baseline levels over about 20 seconds (). We next examined the effects of varying the duration of CWC firing on parallel fiber suppression by monitoring EPSP amplitudes before and after action potential trains of 1, 5 and 15 seconds (). For the 5 neurons tested, mean firing rates during the spike train were 42±2 Hz. After 1 second of firing, no suppression of PF EPSP amplitudes was observed (p>0.05, t test). Following 5 and 15-second spike trains, the amplitudes of EPSPs evoked 1 second after the spike train were suppressed by 38.1±8.8% (p<0.01, t test) and 79.6±3.5% (p<0.01, t test), respectively.
Figure 4 Suppression of parallel fiber synapses following sustained firing of cartwheel cells requires endocannabinoid release. A. Top, Schematic of the experimental protocol. Parallel fiber EPSPs were monitored with 0.5 Hz stimulation in CWCs before and after (more ...)
To test the role of endocannabinoid release in PF suppression, we repeated the 15 second spike trains in the presence of the CB1 receptor antagonist AM251 (). As demonstrated in the group data (), EPSP suppression by the 15-second spike train was reduced from 75.5±2.8% (control, n=4) to 9.6±3.7% (AM251, n=4). The amplitudes of EPSPs evoked 3 or more seconds after the end of a 15 second spike train were not significantly different from the pre-stimulus baseline (p>0.05, t test, n=4). However, the EPSP evoked 1 second after the termination of action potential firing was suppressed by 22±7% (p<0.05, t test, n=4) relative to EPSP amplitudes before the spike train. Suppression of the first EPSP in the presence of AM251 could arise from a variety of mechanisms, including incomplete blockade of CB1 receptors, release of an additional retrograde messenger, or alteration in dendritic filtering following 15-second action potential trains. In contrast, under control conditions, PF suppression was substantially greater and the amplitudes of EPSPs remained significantly suppressed for more than 20 seconds after the spike train. The results indicate that sustained firing by cartwheel cells evokes endocannabinoid release that causes prolonged suppression of PF inputs.
Endocannabinoid production can be greatly facilitated by synergistic interactions between postsynaptic calcium and metabotropic receptors coupled to Gq/11
proteins that activate phospholipase C (PLC) (Maejima et al., 2001
; Varma et al., 2001
; Ohno-Shosaku et al., 2002
; Brown et al., 2003
; Brenowitz and Regehr, 2005
). Cartwheel cells express multiple Gq/11
-coupled metabotropic receptors including mGluR1 (Petralia et al., 1996
; Wright et al., 1996
; Molitor and Manis, 1997
; Fujino and Oertel, 2003
) and M1/M3 mAchRs (Chen et al., 1995
). To test whether PLC-coupled metabotropic receptors contribute to spike-evoked endocannabinoid release, we performed experiments using the PLC inhibitors U73122 (5 μM) and edelfosine (10 μM). Slices were incubated in either U73122 or edelfosine for at least 30 minutes, and physiological recordings were performed in the continued presence of the drug. Suppression of PF EPSPs during 15-second spike trains was not affected by pharmacological block of PLC with either inhibitor ().
Figure 5 Suppression of parallel fiber synapses following sustained firing of cartwheel cells is independent of PLC pathway activation. A. Left, EPSPs recorded in the presence of the PLC inhibitor U73122, before and after a 15-second spike train. Right, Time course (more ...)
Recent studies have shown that the endocannabinoid 2-arachidonylglycerol (2-AG) is responsible for DSE, DSI and PLC-dependent endocannabinoid release in several brain regions (Zhao et al., 2009
; Gao et al., 2010
; Tanimura et al., 2010
). However, the identity of the endocannabinoid released during sustained firing of CWCs is not known. Therefore, to further explore the mechanism of endocannabinoid release from CWCs during spike trains we examined the effects of tetrahydrolipostatin (THL), an inhibitor of diacylglycerol lipase, the major biosynthetic enzyme for 2-AG. We found that application of THL (10 μM) strongly reduced suppression of EPSPs evoked by 15-second tonic firing of CWCs (, 17±.4% suppression in THL vs. 86±4% suppression under control conditions, n=4). Consistent with a recent study (Zhao et al., 2009
), we also observed that THL greatly reduces DSE at PF-CWC synapses (, 14±2% suppression in THL vs. 69±5% under control conditions, n=3). Together, our results indicate that Gq/11
-coupled metabotropic receptors do not contribute to spike-evoked endocannabinoid release, and suggest that elevation of dendritic calcium evoked by backpropagating action potentials or direct depolarization causes synthesis and release of 2-AG from CWCs that results in PF suppression.
Calcium dependence of spiking-evoked PF suppression
Based on our findings that dendritic calcium levels are dependent on CWC firing rates (), we next examined the calcium dependence of spiking-evoked PF suppression in CWCs by combining physiological recordings of EPSPs with calcium measurements in CWC dendrites. In these experiments, we applied current injections (200–500 pA) during 15-second spike trains to vary the CWC firing rate between 5–50 Hz. As in the case of DSE experiments, we first compared calcium transients in proximal and distal regions of the CWC dendrite to determine the range of postsynaptic calcium levels at parallel fiber synapses throughout the CWC. Despite the differences in peak calcium levels in response to a single action potential (), calcium levels were not different in proximal and distal dendrites during 15 second spike trains (). Moreover, calcium levels in spines and the parent dendrite were not different in either proximal or distal regions (). Therefore in the following experiments to determine the calcium dependence of spiking-evoked PF suppression by CWCs, we focused on measuring calcium transients in dendritic shafts.
Figure 6 Submicromolar elevation of dendritic calcium in CWCs during tonic spiking evokes endocannabinoid release and suppression of parallel fiber synapses. A. Calcium transients in proximal (top) and distal (bottom) dendrite of a representative CWC during a (more ...)
Our experimental approach for measuring the relationship between dendritic calcium and suppression of PF EPSPs is illustrated in for a representative neuron. Dendritic calcium was measured during 15-second spike trains at 5–50 Hz. In this neuron the range of action potential firing rates raised dendritic calcium to levels that did not exceed 1 μM () yet produced strong suppression of PF EPSPs (). Calcium levels were dependent on CWC firing rates () and there was a clear trend towards greater amounts of PF suppression with higher firing rates (). The results indicate that PF EPSPs are suppressed by >50% with submicromolar levels of calcium. This analysis was repeated in a group of 9 neurons, and the data was fitted to the Hill equation (). The parameters of the fit indicated that half-maximal suppression of EPSPs occurred at mean dendritic calcium levels of 0.40±0.07 μM. Compared to DSE, which was half-maximal at 3.9 μM calcium (), this reflects roughly a 10-fold increase in calcium sensitivity. Thus, submicromolar elevation of dendritic calcium that occurs under conditions where CWC firing rates exceed ~25 Hz for 15 seconds causes strong suppression of parallel fiber synapses mediated by retrograde endocannabinoid signaling.
If the same calcium-dependent signaling pathway is responsible for both DSE and spike-evoked PF suppression in CWCs, it is possible that the long duration of the low-level calcium signal during spike trains compensates for the lower peak requirement compared to DSE. To examine this possibility, we calculated the integral of the calcium transient for experiments in which DSE (from ) and spike-evoked PF suppression (from ) were measured (). The integrated calcium transient is proportional to the total calcium influx evoked by either voltage steps or spike trains and can be directly compared because the same concentration of calcium indicator was used for both voltage clamp and current clamp experiments (Neher and Augustine, 1992
; Schneggenburger et al., 1993
). This analysis indicates that although calcium levels driving endocannabinoid release during CWC spike trains are ~10-fold lower than during DSE trials, a larger total calcium influx is required to obtain a given amount of PF suppression during a spike train compared to a DSE trial.
Endocannabinoids released from cartwheel cells during sustained tonic firing do not suppress inhibitory synapses to cartwheel cells
In addition to glutamatergic PF synaptic inputs, cartwheel cells receive glycinergic and GABAergic inputs from other cartwheel cells and superficial stellate cells (Zhang and Oertel, 1993
; Davis and Young, 2000
; Roberts et al., 2008
). Therefore, we tested the possibility that endocannabinoids released by CWC dendrites during spike trains also suppress these inhibitory synapses. Spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded from voltage-clamped CWCs at a holding potential of −70 mV in extracellular solution supplemented with DNQX (10 μM) to block AMPA receptors. The intracellular concentrations of chloride ions was adjusted so that ECl
= ~ −40 mV. Synaptic currents recorded under these conditions were confirmed to be glycinergic/GABAergic as all events could be completely blocked by strychnine (1 μM) and picrotoxin (40 μM) (). sIPSCs were also confirmed to arise from presynaptic spiking because they were eliminated by application of tetrodotoxin (0.5 μM) (). To test for suppression of sIPSCs by CWC spiking, sIPSCs were recorded in voltage clamp for 20 seconds before the amplifier was switched to current-clamp mode to deliver a 15 second current injection. After termination of CWC firing, the amplifier mode was switched back to voltage clamp and sIPSCs were recorded for 40 seconds. A representative example of this protocol is shown in in which the CWC fired in tonic mode for 15 seconds at a rate of 39.2 Hz. When sIPSCs were collected and averaged over a 5-second period immediately before and after the spike train, no change was observed in the sIPSC amplitude (). For the 5 neurons tested (), the average firing rates were 36±3 Hz, a range sufficient to evoke endocannabinoid release and significant suppression of PF synapses (–). Individual events were collected, binned in 1 s intervals, and sIPSC amplitudes were averaged and plotted for each 1 second interval. shows that sustained firing of CWCs had no effect on sIPSC amplitudes.
Figure 7 Endocannabinoids released from cartwheel cells during sustained firing do not suppress inhibitory cartwheel or stellate cell inputs. A. sIPSCs were recorded in voltage-clamp at −70 mV (ECl = ~−40 mV) in continuous presence of DNQX (10 (more ...)
Parallel fiber excitation elevates CWCs firing rates and evokes endocannabinoid release
We next tested whether the ongoing spontaneous activity of CWCs produces tonic release of endocannabinoids by comparing PF EPSPs before and after a 15 second period in which the CWC fired spontaneously with a bias current of 0 pA (). During the spontaneous firing period, CWCs fired almost exclusively simple spikes at 21.3±0.8 Hz (n=6). PF EPSPs were not suppressed by this spontaneous firing (EPSP amplitudes were 100±9.2% relative to pre-firing amplitudes, n=6), indicating that spontaneous CWC firing rates are not sufficiently high to produce tonic release of endocannabinoids.
Figure 8 Pathway-specific endocannabinoid release evoked by combined PF stimulation and CWC spontaneous firing. Two stimulus electrodes were used to activate independent populations of PF synapses (paths 1 and 2) onto a postsynaptic CWC. Data from the same representative (more ...)
One mechanism that could increase CWC firing rates and evoke endocannabinoid release is excitatory input from parallel fibers. Therefore we performed a series of experiments to examine the role of PF excitation in elevating CWC firing rates and causing suppression of PF synapses through release of endocannabinoids from CWCs. Stimulation of PF inputs at 10 Hz for 15 seconds while maintaining the CWC at a hyperpolarized membrane potential resulted in strong post-tetanic potentiation of PF EPSPs () that decayed over 20–30 seconds. We next combined 10 Hz PF stimulation with a current injection that maintained the CWC at a holding current of 0 pA (). Despite increasing CWC firing rates to 28.6±2.0 Hz (n=6), PF EPSPs remained strongly potentiated (209±23% relative to pre-firing amplitudes, n=6). When this protocol was repeated in the presence of AM251, the amplitudes of PF EPSPs showed only a slight enhancement (, 214±23% relative to preconditioning amplitudes, p>0.05, n=4). These results indicate that any potential effect of endocannabinoid release from the CWC on PF EPSP amplitudes was obscured by the strong potentiation of EPSPs following sustained 10 Hz stimulation.
Therefore, to test for global endocannabinoid release during elevated rates of CWC firing, we performed experiments in which a second stimulus electrode placed in the molecular layer of the DCN was used to activate an independent set of parallel fiber inputs to the postsynaptic CWC (). The independence of the two parallel fiber pathways was tested by demonstrating the absence of paired-pulse facilitation when alternate pathways were stimulated at 20–50 msec intervals (Brown et al., 2003
; Tzounopoulos et al., 2007
). In these experiments we monitored the amplitudes of EPSPs evoked by stimulation of parallel fiber pathway 1 before and after a 15-second conditioning stimulus during which the second pathway was activated at 10 Hz during the 15-second period of spontaneous CWC firing. Stimulation of PF pathway 2 increased the firing rates of CWCs from 21.3±0.8 Hz to 29.7±2.1 Hz (, n=6). Following this stimulus protocol, PF EPSPs from pathway 1 were suppressed to 57.3±4.0% (, n=6 neurons, 12 pathways) of their pre-conditioning amplitudes. In each neuron, the ability of each parallel fiber pathway to suppress the other was reciprocally tested. This suppression of PF EPSPs was abolished in the presence of AM251 (, 101.7±10.1% relative to preconditioning amplitudes, n=4). These results demonstrate that parallel fiber stimulation can elevate the CWC firing rate and cause global release of endocannabinoids. This form of endocannabinoid-mediated synaptic plasticity is heterosynaptic in nature because it is restricted to the population of parallel fiber inputs that are not repetitively activated during the conditioning stimulus. Under these conditions, highly active synapses that increase CWC firing rates became potentiated, whereas synaptic inputs that were inactive exhibit short-term suppression that recovered over 10–15 seconds as a result of endocannabinoid release from the CWC.