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Suppression of Presynaptic Glutamate Releaseby Postsynaptic Metabotropic NMDA ReceptorSignalling to Pannexin-1
https://doi.org/10.1523/JNEUROSCI.0257-19.2019
Cite as: J. Neurosci 2019; 10.1523/JNEUROSCI.0257-19.2019
Received: 29 January 2019Revised: 26 November 2019Accepted: 3 December 2019
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Suppression of Presynaptic Glutamate Release by Postsynaptic Metabotropic NMDA 1 Receptor Signalling to Pannexin-1 2
3 4 Jennifer Bialecki1, Allison Werner1, Nicholas L. Weilinger1, Catharine M. Tucker1, Haley A. 5
Vecchiarelli1, Jon Egaña2, Juan Mendizabal-Zubiaga2, Pedro Grandes2, Matthew N. Hill1 and 6 Roger J. Thompson1 7
8 9 10
1Hotchkiss Brain Institute 11 Department of Cell Biology and Anatomy 12
University of Calgary 13 Calgary, AB, Canada 14
15 2Department of Neurosciences 16
University of the Basque Country UPV/EHU 17 Leioa, Spain 18
Abbreviated title: Pannexin1 Suppresses Bursting of Glutamatergic Synapses 25 26 Number of figures: 11 27 28 Number of words (Abstract = 185; Introduction = 363; Discussion = 1495) 29
30
Acknowledgments 31
The work was supported by grants from the Canadian Institutes of Health Research to 32
MNH (FDN 143329) and RJT (MOP 136812) and the Natural Sciences and Engineering 33
Research Council of Canada to RJT. Additional support was provided to RJT by the 34
Cumming School of Medicine via the Ronald and Irene Ward Foundation and the 35
Gwendolyn McLean Fund, and from the Hotchkiss Brain Institute. MNH holds a Canada 36
Research Chair Tier 2. This work was also supported by grants from MINECO/FEDER, UE 37
(SAF2015-65034-R) and The Basque Government (BCG IT764-13) to PG. 38
39
2
Abstract 40
The impact of pannexin-1 (Panx1) channels on synaptic transmission is poorly 41
understood. Here, we show that selective block of Panx1 in single postsynaptic 42
hippocampal CA1 neurons from male rat or mouse brain slices causes intermittent, 43
seconds long increases in the frequency of spontaneous excitatory postsynaptic currents 44
(sEPSC) following Schaffer collateral stimulation. The increase in sEPSC frequency 45
occurred without an effect on evoked neurotransmission. Consistent with a pre-synaptic 46
origin of the augmented glutamate release, the increased sEPSC frequency was prevented 47
by bath applied EGTA-AM or tetrodotoxin. Manipulation of a previously described 48
metabotropic NMDAR pathway (i.e. by preventing ligand binding to NMDARs with 49
competitive antagonists or blocking downstream Src kinase) also increased sEPSC 50
frequency similar to that seen when Panx1 was blocked. This facilitated glutamate 51
release was absent in TRPV1 knockout mice and prevented by the TRPV1 antagonist, 52
capsazapine, suggesting it required presynaptic TRPV1. We show presynaptic expression 53
of TRPV1 by immunoelectron microscopy and link TRPV1 to Panx1 because Panx1 block 54
increases tissue levels of the endovanilloid, anandamide. Together, these findings 55
demonstrate an unexpected role for metabotropic NMDARs and postsynaptic Panx1 in 56
suppression of facilitated glutamate neurotransmission. 57
58
Significance Statement 59
The postsynaptic ion and metabolite channel, pannexin-1 is regulated by metabotropic 60
NMDAR signalling through Src kinase. This pathway suppresses facilitated release of 61
presynaptic glutamate during synaptic activity by regulating tissue levels of the TRPV1 62
agonist anandamide. 63
3
Introduction 64
Pannexin-1 (Panx1) is an ion / metabolite channel that is broadly distributed in 65
the brain (Bruzzone et al., 2003; Vogt et al., 2005) and highly expressed in the 66
postsynaptic density (Zoidl et al., 2007). Panx1 opening is implicated in several 67
pathologies, including neuronal death during ischemia (Thompson et al., 2006; Bargiotas 68
et al., 2011; Weilinger et al., 2012), N-methyl-D-aspartate receptor (NMDAR) 69
excitotoxicity (Thompson et al., 2008; Weilinger et al., 2016), inflammasome activation in 70
the gut (Gulbransen et al., 2012), and inflammation in the vasculature (Lohman et al., 71
2015). An important role for Panx1 in ATP release during apoptosis has also been 72
proposed (Chekeni et al., 2010). Despite the clear roles of Panx1 opening in pathology, 73
our understanding of the physiological functions of Panx1 in the brain is limited. 74
We showed recently that Panx1 opening following phosphorylation of its C-75
terminal regulatory domain by sarcoma (Src) kinase contributes to neuronal death during 76
NMDAR excitotoxicity (Weilinger et al., 2016). In other studies it was reported that Src 77
phosphorylation of Panx1 in the channel’s intracellular loop (at Y198) in vascular smooth 78
muscle can regulate ATP release and may contribute to hypertensive pathologies (DeLalio 79
et al., 2019). Thus, Src kinase appears to be a potent regulator of Panx1 activity. Src also 80
phosphorylates NDMARs in neurons and contributes to chronic pain through potentiation 81
of the receptor (Yu et al., 1997; Salter and Kalia, 2004; Yang et al., 2012). Given the impact 82
of Src activity on both NMDARs and Panx1, we tested here if the NMDAR-Src-Panx1 83
signalsome contributes to synaptic activity in the hippocampus. We show that blocking 84
Panx1 in single postsynaptic CA1 neurons generates intermittent facilitation of glutamate 85
release during low frequency Schaffer collateral stimulation. Moreover, blocking Src 86
directly or preventing ligand binding (both glutamate and glycine) to NMDARs caused 87
4
similar stimulation-dependent facilitated glutamate release. We further show that 88
blocking Panx1 increased tissue concentrations of the TRPV1 channel agonist, 89
anandamide (AEA) and that the facilitated release was TRPV1 dependent. Our findings 90
show that metabotropic NMDAR-Panx1 opening in the postsynapse is a homeostatic 91
regulatory mechanism that buffers AEA accumulation and suppresses TRPV1-dependent 92
glutamate release. A portion of these data have posted on the BioRxiv preprint server 93
(Bialecki et al., 2018). 94
95
Materials and Methods 96
Animals: All animal care and use were in accordance with the Canadian Council on Animal 97
Care guidelines and approved by the University of Calgary’s Animal Care and Use 98
Committee. Male Sprague Dawley rats, aged 21-37 days were housed on a 12 hour 99
light/dark cycle with access to Purina Laboratory Chow and water ad libitum. Male Wild 100
type mice (C57BL/6J), conditional pannexin-1 knockout mice ( Panx1fl/fl-wfs1-Cre; 101
Weilinger et al., 2012) and TRPV1 knockout mice (TRPV1-/- Birder et al., 2002) were bred 102
in-house and kept under the same conditions as the rats. Panx1 knockout was achieved 103
by intra-peritoneal tamoxifen injections (100mg/kg) once daily for 5 days (Weilinger et 104
al., 2012). Control conditions for Panx1 knockout were littermates that received 105
injections of the vehicle alone (95% corn oil 5% ethanol) once daily for 5 days. 106
Chemicals and reagents: All salts were from Sigma-Aldrich. Capsazepine (CPZ) at a final in 107
vitro concentration of 10μM) was from Tocris Bioscience. The selective peptide inhibitor 108
of Panx1, 10panx (WRQAAFVDSY) and its scrambled control peptide, sc10panx 109
(FSVYWAQADR) were synthesized by AnaSpec or New England Peptide and used at the 110
final concentration of 100 μM. The C-terminal anti-Panx1 polyclonal antibody (α-panx1; 111
5
0.25ng/μl), which blocks Panx1 when included in the patch pipette (Weilinger et al., 2012, 112
2016), was from Invitrogen (catalog #488100, rabbit polyclonal). Its control (Weilinger et 113
al., 2016) was an anti-connexin-43 polyclonal antibody (α-Cx43; 0.3ng/μl) that was from 114
Abcam (catalog #ab11370). All drugs were dissolved in water, DMSO or ethanol and 115
aliquoted and frozen until use. Drugs were then dissolved into artificial cerebral spinal 116
fluid (aCSF) at their final concentrations. Final concentrations of DMSO or ethanol did not 117
exceed 0.1%. The aCSF was saturated with 95% O2 /5% CO2 and contained of 120 mM 118
NaCl, 26 mM NaHCO3 ,3mM KCl, 1.25 mM NaH2PO4, 1.3 mM MgCl2, 2mM CaCl2, 10 mM 119
glucose and 100 M picrotoxin. 120
Acute hippocampal slice preparation: Rats or mice were anaesthetized by isoflurane 121
inhalation in air and decapitated; the brain was extracted, blocked, mounted on a 122
vibrating slicer (VT1200S; Leica) and submerged in an ice-cold high sucrose solution 123
consisting of the following (in mM): 87 NaCl, 2.5 KCl, 25 NaHCO3, 0.5 CaCl2, 7 MgCl2, 1.25 124
NaH2PO4, 25 glucose, and 75 sucrose, saturated with 95% O2 /5% CO2. Transverse 125
hippocampal slices were cut (370 μm for rats and 300 μm for mice) and placed into a 126
chamber containing aCSF at 33°C for at least 1 h before use. 127
Electrophysiology: Slices were transferred to a recording chamber and constantly 128
perfused with aCSF (33°C-35°C) at a rate of 1-2mL/min. Visualization of hippocampal CA1 129
pyramidal neurons was achieved with differential interference contrast (DIC) microscopy 130
with an Olympus BX51Wi microscope. Electrophysiological data were collected with a 131
MultiClamp 700B amplifier and digitized with a Digidata 1440A analog to digital 132
converter (Molecular Devices) at 10 kHz and low-pass Bessel filtered at 1 kHz. Data were 133
recorded using pCLAMP 10, Clampex 10.3, and Axoscope 10.3 (Molecular Devices) 134
software and stored for future analysis with Clampfit 10.3, GraphPad Prism and Excel 135
6
(Microsoft). Whole cell voltage clamp recordings were performed using borosilicate glass 136
microelectrodes (Sutter Instrument) with a tip resistance of 3-6MΩ that were pulled 137
using a P-1000 Flaming/Brown Micropipette Puller (Sutter Instrument). Microelectrodes 138
were filled with an intracellular solution containing 108 mM potassium gluconate, 2 mM 139
MgCl2, 8mM sodium gluconate, 8 mM KCl, 2.5 mM K2-EGTA, 4 mM K2-ATP, and 0.3 mM 140
Na3-GTP at pH 7.25 with 10 mM HEPES. Access resistance (RA) was monitored over the 141
course of the experiments and was < 8 MΩ (range 7-8 MΩ) after break-in or the cell was 142
discarded. Cells were also discarded from the data set if RA reached 10 MΩ at any point in 143
the recording. The holding potential was -70 mV unless otherwise noted. 144
Panx1 block in single CA1 pyramidal neurons was achieved by inclusion of an anti-145
Panx1 polyclonal antibody ( -Panx1;0.25ng/ L) or its negative control, anti-connexin- 146
43 polyclonal antibody ( -Cx43; 0.3 ng/ml) in the patch pipette, as previously described 147
(Weilinger et al., 2012, 2016). To buffer intracellular Ca2+ we included 10 mM BAPTA in 148
the pipette. In the figures, within cell comparisons are indicated by the straight line 149
joining the data points in the absence (unfilled bars / symbols) and following (filled bars / 150
symbols) Schaffer collateral PPS. 151
In most experiments within-cell comparisons between no stimulation and Schaffer 152
collateral stimulation were used: After formation of the whole-cell configuration we 153
allowed 10 min for equilibration of the intracellular solution, followed by 5 min of 154
recording spontaneous (s)EPSC. A stimulating electrode was then placed in the Schaffer 155
collateral pathway and paired-pulse stimulations (PPS; 1 ms pulse duration and 50 ms 156
interpulse interval) were applied at 0.05 Hz for 5 minutes. Stimulation strength was set at 157
50% of the maximum evoked response for the duration of the recording. Detection and 158
quantification of sEPSC interevent intervals and amplitude was with Clampfit (Molecular 159
7
Devices Inc.) using a template-based protocol. The sEPSC template was the average of all 160
the events during the 5 min baseline recording. During analysis, the user manually 161
approved (or not) each sEPSC event that was initially identified by Clampfit. User-162
rejected events were very rare when RA remained < 10 MΩ. sEPSC peak amplitude and 163
interevent interval for the 5 min baseline were compared to the subsequent 5 min period 164
were Schaffer collaterals were stimulated. 165
Amplitudes were determined by single Gaussian fits to the distribution of peak 166
sEPSC amplitudes. We compared the cumulative amplitude distributions of the last 200 167
sEPSC events from the baseline (i.e. no Schaffer collateral stimulation) to the 168
experimental period (with Schaffer collateral stimulation) using Kolmogorov-Smirnov 169
statistics (K-S; Graphpad Prism 7). Significance was set as p<0.001 for comparison of 170
distributions because the datasets are large (Kim and Alger, 2010). 171
The frequency of sEPSCs was determined as the inverse of the average interevent 172
interval for the 5 min recording segments. Cumulative interevent interval distributions 173
were compared using K-S tests (with p<0.001). Under each experimental condition, all of 174
the baseline (i.e. prestimulation) events for each neuron were compared to all of the 175
sEPSC events during the 5 min stimulation period. Paired-pulse ratios were calculated as 176
the ratio of the peak amplitudes of the (evoked) eEPSC peak 2 divided by the amplitude of 177
evoked peak 1; here a between cells design was necessary (i.e. control conditions were 178
not the same cells as Panx1 blocked). Data were compared using nonparametric statistics 179
with either the Wilcoxon matched-pairs signed rank test (for paired data from within cell 180
experimental design), the Kruskal-Wallis test (with Dunn’s multiple comparisons) for 181
more than 2 conditions that shared the same baseline, or Mann-Whitney U test for 182
unpaired populations of neurons. Significance was set at p≤ 0.05. All results are 183
8
presented as means ±SEM with n equal to the number of neurons. Only one neuron was 184
evaluated per hippocampal slice and at least 3 animals were used for each condition. 185
RNA isolation and reverse transcription: Hippocampi and dorsal root ganglia tissue from 186
both male and female mice was collected from C57BL/6J or TRPV1-/- strains. Total RNA 187
was extracted from tissue using the RNeasy® Plus Micro Kit (Qiagen). Total RNA (1.0 μg) 188
was reverse-transcribed into cDNA by using Superscript™ IV VILO™ Master Mix with 189
ezDNase (Invitrogen). The resulting cDNA was used as a template for PCR amplification 190
and stored at -20°C if not used immediately. 191
Polymerase Chain Reaction (PCR): PCR amplification was carried out using 1.0 μL of cDNA 192
and KAPA HiFi HotStart ReadyMix (Kapa Biosystems) with TRPV1 primers (forward 5’ - 193
CATGCTCATTGCTCTCATGG and reverse 5’ - GCCTTCCTCATGCACTTCAG) in an Eppendorf 194
Mastercycler Gradient GSX1 Thermal Cycler. The PCR program used consisted of a 5-min 195
incubation at 95°C followed by 35 cycles of 98°C for 20 s, 60°C for 15 s, 72°C for 15 s, and 196
a final incubation of 72°C for 5 min. PCR amplification products were separated on a 1.0% 197
agarose gel and stained with Safe-Red™ to verify their size using a 100 base pair (bp) DNA 198
ladder. 199
Quantitative PCR (qPCR): qPCR was performed using 1.0 μL of cDNA and PowerUp SYBR® 200
Green Master Mix (Applied Biosystems), following the Fast PCR protocol, in a QuantStudio 201
3 Real-Time PCR System. Mouse β-actin QuantiTect Primer Assay (Qiagen) was purchased 202
as a control gene and TRPV1 primers used were as mentioned above for PCR. TRPV1 203
primer quality was validated through a qPCR standard curve. Assays were run on 204
MicroAmp® Fast Optical 96-Well Reaction Plates (applied biosystems) in triplicates. Fold 205
expression of TRPV1 mRNA levels in hippocampi compared to dorsal root ganglia were 206
9
determined using the 2-∆∆Ct method after normalization to internal control β-actin RNA 207
levels. 208
Electron Microscopy: 5 wildtype TRPV1 and 5 TRPV1-/- adult mice of either sex were used 209
in this study. TRPV1-/- mice (C57BL/6J background; (Birder et al., 2002)) were originally 210
from The Jackson Laboratory (Strain Name: B6.129X1-Trpv1tm1Jul/J, Bar Harbor, ME). 211
Experimental animals were genotyped by polymerase chain reaction (PCR) under 212
standard buffer conditions using the primer pair 5'-CCT GCT CAA CAT GCT CAT TG-3' and 213
5'-TCC TCA TGC ACT TCA GGA AA -3' for the wild-type locus. The primer pair 5'- CAC GAG 214
ACT AGT GAG ACG TG -3'and 5'-TCC TCA TGC ACT TCA GGA AA -3' was used to detect a 215
fragment in the Neo cassette, specific for the mutant TRPV1 locus. All four primers were 216
used together in the reaction mix (94ºC/3min; 35x[94ºC/30 sec, 64ºC/1 min, 72ºC/1 217
min]; 1x72ºC/2 min; 1x10ºC hold). 218
Homozygous TRPV1-/- and wild-type littermates (TRPV1+/+) from heterozygous breedings 219
were used for experiments. They were deeply anesthetized by intraperitoneal injection of 220
ketamine/xylazine (80/10 mg/kg body weight) and then transcardially perfused at room 221
temperature (RT) with phosphate buffered saline (PBS 0.1M, pH 7.4) for 20 seconds, 222
followed by the fixative solution made up of 4% formaldehyde (freshly depolymerized 223
from paraformaldehyde), 0.2% picric acid and 0.1% glutaraldehyde in phosphate buffer 224
(PB 0.1M, pH 7.4) for 10-15 minutes. Brains were then removed from the skull and 225
postfixed in the fixative solution for approximately one week at 4°C. Afterwards, brains 226
were stored at 4°C in 1:10 diluted fixative solution until used. 227
Preembedding immunogold method for TRPV1 electron microscopy (EM): Coronal 50μm-228
thick hippocampal vibrosections were collected in 0.1M PB at RT. Then, they were 229
preincubated in a blocking solution of 10% bovine serum albumin (BSA), 0.1% sodium 230
10
azide and 0.02% saponine prepared in Tris-HCl buffered saline (TBS 1X, pH 7.4) for 30 231
minutes at RT. Sections were incubated with the primary goat TRPV1 antibody (1:100, 232
VR1 (P-19), sc-1249, Santa Cruz Biotechnology) prepared in the blocking solution but 233
with 0.004% saponin, for 2 days at 4ºC. After several washes, tissue sections were 234
incubated with 1.4nm gold-labeled rabbit antibody to goat IgG (Fab fragment, 1:100, 235
Nanoprobes Inc., Yaphank, NY, USA) prepared in the same solution as the primary 236
antibody for 3 hours at RT. Tissue was washed overnight at 4ºC and postfixed in 1% 237
glutaraldehyde for 10 minutes. After several washes with 1% BSA in TBS, gold particles 238
were silver-intensified with a HQ Silver Kit (Nanoprobes Yaphank, NY, USA) for 12 239
minutes in the dark. Then, sections were osmicated, dehydrated and embedded in Epon 240
resin 812. Finally, ultrathin sections were collected on mesh nickel grids, stained with 241
lead citrate and examined in a PHILIPS EM208S electron microscope. Tissue preparations 242
were photographed by using a digital camera coupled to the electron microscope. 243
Specificity of the immunostaining was assessed by incubation of the TRPV1 antiserum in 244
TRPV1-/- hippocampal tissue in the same conditions as above. 245
Statistical analysis of TRPV1 in the CA1 hippocampus: 50μm-thick CA1 hippocampal 246
sections from TRPV1+/+ and TRPV1-/- mice (n=5 each) showing good and reproducible 247
silver-intensified gold particles were cut at 80nm. Electron micrographs (18,000–248
28,000X) were taken from grids (2 mm x 1 mm slot) with ultrathin sections showing 249
similar labeling intensity indicating that selected areas were at the same depth. 250
Furthermore, to avoid false negatives, only ultrathin sections in the first 1.5 μm from the 251
surface of the tissue block were examined. Positive labeling was considered if at least one 252
immunoparticle was within approximately 30nm from the plasmalemma. 253
11
TRPV1 metal particles on axon terminals were visualized and counted in randomly taken 254
electron micrographs from both animal types. The number of positive terminals was 255
normalized to the total number of terminals in the images to identify the proportion of 256
TRPV1-positive profiles in TRPV1+/+ versus TRPV1-/-. Results were expressed as means of 257
independent data points ± S.E.M. Statistical analyses (Unpaired Student’s t-test) were 258
performed using GraphPad software 5.0 (GraphPad Software Inc, San Diego, USA) and 259
significance was set a p<0.05. 260
Mass Spetrometrical Quantification of Anandamide Levels: Hippocampal slices were 261
prepared as described above and underwent whole-cell patch clamp recording with 262
Schaffer collateral stimulation in aCSF or aCSF with 100 μM 10panx or 10 μM CPZ prior to 263
lipid extraction as previously described (Qi et al., 2015). In brief, tissue samples were 264
weighed and placed in borosilicate glass culture tubes containing 2 ml of acetonitrile with 265
5 pmol of [2H8] AEA for extraction. These samples were homogenized with a glass rod, 266
sonicated for 30 min, incubated overnight at -20°C to precipitate proteins, then 267
centrifuged at 1500 g for 5 min to remove particulates. Supernatants were removed to a 268
new glass culture tube and evaporated to dryness under N2 gas, re-suspended in 300 μl of 269
acetonitrile to recapture any lipids adhering to the tube and re-dried again under N2 gas. 270
The final lipid extracts were suspended in 200 μl of acetonitrile and stored at -80°C until 271
analysis. AEA contents within lipid extracts were determined using isotope-dilution, 272
liquid chromatography-tandem mass spectrometry (LC-MS/MS) as previously described 273
(Qi et al., 2015). 274
275
276
277
12
Results 278 279 Block of postsynaptic Panx1 facilitates glutamate release following Schaffer 280
collateral stimulation 281
To evaluate putative roles of Panx1 in spontaneous and evoked neurotransmission, 282
we selectively inhibited the channels in single postsynaptic CA1 neurons by inclusion of a 283
blocking antibody, α-panx1 (0.25 ng/μl) in the patch pipette (Weilinger et al., 2012, 284
2016). Properties of spontaneous excitatory postsynaptic currents (sEPSC) over a 5 min 285
baseline were compared to those in the subsequent 5 mins when paired pulse 286
stimulations were applied to the Schaffer collaterals (PPS; 0.05 Hz, 1 ms pulse duration, 287
50ms interstim interval), which also allowed for evaluation of evoked responses. Note 288
that all bathing solutions contained 100 M picrotoxin to block GABAA receptors. 289
Figures 1A-F illustrate responses of a CA1 neuron to PPS with the control pipette 290
solution, and Figures G-L are from a different CA1 neuron with α-panx1 in the pipette. Fig 291
1A shows 20 s long recordings before (left) and after (right) PPS of the Schaffer collaterals 292
and Figs 1B and 1C show the instantaneous frequency of sEPSCs for the entire 5 min 293
recoding periods of this neuron. As expected, PPS did not alter the cumulative interevent 294
interval (Fig. 1D; n=28 neurons from 28 slices, K-S test; p>0.99; K-S D=0.0019) nor the 295
amplitude distribution of sEPSCs (Fig. 1D; n=28; K-S test; p>0.99; K-S D=0.0003) under 296
control conditions. In contrast, inclusion of α-panx1 in the pipette solution (Fig 1G) 297
caused a significant leftward shift in the cumulative interevent interval distribution 298
during PPS, indicating higher frequency (Figs. 1H-J; n=31; K-S test; p<0.0001; K-S 299
D=0.524). The distribution of amplitudes was not changed (Fig. 1KL; p>0.999; K-S test, K-300
S D=0.0005). 301
13
Mean±SEM frequency for the 5 min baseline (i.e. without stim and without α-302
panx1) was 3.1±0.2 Hz, which was significantly increased to 6.2±0.5 Hz with PPS and 303
pipette α-panx1 (Fig. 2A; Kruskal-Wallis test; p<0.0001, K-W statistic = 33.17; all other 304
conditions vs control p>0.99). The mean±SEM amplitudes of sEPSCs are shown in Fig. 2B 305
and they did not significantly change from the control level of 16.5±0.4 pA under any 306
condition (Kruskal-Wallis test; p=0.09, K-W statistic = 6.51). As an important control for 307
intracellular α-panx1 we evaluated a polyclonal antibody against connexin-43 (α-Cx43 at 308
0.3ng/μl) in the pipette (Weilinger et al., 2016) and found no change in the cumulative 309
sEPSC frequency distribution (n=8; K-S D=0.0011, p>0.99; data not shown) or the mean 310
sEPSC frequency when Shaffer collateral PPS was applied (Fig 2A; n=8; p=0.46; Wilcoxon 311
matched pairs W=1.0). 312
We tested the peptidergic blocker of Panx1, 10panx (100 μM) and its scrambled 313
control (sc10panx; 100 μM) (Thompson et al., 2008) because these are bath applied and 314
block the channels through a mechanism distinct from α-panx1. 10panx decreased 315
cumulative sEPSC interevent intervals during PPS (n=6; p=0.0006; K-S D=0.5357) with a 316
significant increase in sEPSC frequency from 3.1±0.4 Hz to 6.7±0.9 Hz (Fig. 2A; n=6; 317
p=0.01, Wilcoxon matched pairs, W=21). The scrambled control, sc10panx, with PPS did 318
not alter the mean sEPSC frequency (Fig. 2A; n=7, p=0.38, Wilcoxon matched pairs, W=-319
12). Taken together, these data show that 2 distinct Panx1 blockers, one acting 320
intracellularly in single patched cells and the other bath applied, result in facilitated 321
glutamate release during low frequency Schaffer collateral stimulation. 322
Interestingly, the increased sEPSC frequency was not persistent throughout the 323
full 5 min recording, rather it appeared as intermittent bursts that lasted on average for 324
13±0.5s (Fig 1I, n=28; range 1.4-19.8 s). Bursts were defined to start when the 325
14
instantaneous frequency exceeded the baseline frequency by a factor of 2.5 and to 326
terminate when it returned to baseline. On average, a CA1 neuron would burst 3.2±0.4 327
times (range 0-9; median=3.5) in the 5 min stimulation period. sEPSCs bursting events 328
reached a maximum frequency of 28.9±1.3 Hz (n=31), which was significantly larger 329
compared to the maximum frequency of 14.6±2.3 Hz (n=28) without α-panx1 in the 330
pipette (Mann-Whitney U test, p<0.0001, U=30.5). The appearance of the high frequency 331
sEPSC bursts were not clearly time-locked with synaptic stimulation, but they only 332
occurred during PPS when Panx1 was blocked (Fig. 2A). We evaluated a range of 333
intervals between PPS stimulations from 10 to 60 s. Significant increases in mean sEPSC 334
frequency were observed with PPS intervals of 10 s (Fig 2C; Wilcoxon test, p=0.0234; n=8; 335
W=32), 20 s (Fig 1 and 2A; stats reported above) and 30 s (Fig 2C; Wilcoxon test, p=0.031; 336
n=6; W=21) Note that bursts were seen for the 40 s PPS interval, but when averaged over 337
the 5 min recordings did not reach a statistically significant difference. 338
To rule out the possibility of presynaptic Panx1 block by extracellular 339
accumulation of α-panx1 leaking from the pipette due to positive pressure in the 340
recording electrode, we locally puffed α-panx1 while recording from CA1 neurons with 341
the control pipette solution. Puffing α-panx1 within 100 μm from the soma of patched 342
neurons had no effect on sEPSC frequency (data not shown; 2.3±0.3 Hz without PPS and 343
glutamate release with the TRPV1 blocker, capsazapine and did not detect increased 631
sEPSC frequency in TRPV1-/- mice. There are numerous reports of functional TRPV1 in 632
the hippocampus (Gibson et al., 2008; Chávez et al., 2010, 2014; Jensen and Edwards, 633
2012) and a unique splice variant in the hypothalamus (Sharif Naeini et al., 2006). 634
Indeed, in the original systematic mapping of brain TRPV1 it was reported that the 635
27
vanilloid activated channels are expressed in hippocampus, but only in Cajal-Retzius (CR) 636
cells (Cavanaugh et al., 2011). CR cells are reelin expressing glutamatergic neurons that 637
make synaptic contacts with interneurons in the molecular layer and pyramidal neurons 638
in the CA1 region (Anstötz et al., 2016). Recently it was shown that TRPV1 is functional in 639
CR neurons and capsaicin-activation of the channels augmented sEPSC frequency 640
(Anstötz et al., 2018). Thus, it will be of interest to determine if Panx1 block is leading to 641
activation of TRPV1 in CR to CA1 synapses (Fig. 11) because this would be consistent with 642
a presynaptic unsilencing mechanism. It may also reveal if there are currently 643
unidentified physiological roles for Panx1’s suppression of facilitated glutamate release 644
because CR cells are a component of an intra-hippocampal microcircuit (Anstötz et al., 645
2018). 646
Capsaicin perfusion onto hippocampal slices led to a persistent augmentation of 647
glutamate release from CR neurons onto interneurons (Anstötz et al., 2018). In contrast, 648
in afferents of the NTS, or microglia in the anterior cingulate cortex, TRPV1 activation 649
induces transient (10s of ms) asynchronous glutamate release (Peters et al., 2010; 650
Marrone et al., 2017). So why is the facilitated glutamate release that we observe when 651
Panx1 is blocked intermittent and seconds long instead of very brief (ms) or persistent? 652
It may reflect a sustained increase in TRPV1 ligand (i.e. AEA) concentration when Panx1 is 653
blocked, which could arise from high local (i.e. synaptic) levels or diffusion out of the 654
synapse. While we have focussed on AEA in this paper it is possible that the effective 655
ligand is another endovanilloid. It has been reported that N-aracidonyl-dopamine 656
(NADA) is a potent ligand for TRPV1 (De Petrocellis et al., 2004) and interestingly, brief 657
application of NADA can induce prolonged TRPV1 opening and glutamate release lasting 658
10s of seconds (Medvedeva et al., 2008), similar to that reported here. 659
28
Several possible mechanisms could account for termination of the augmented 660
sEPSC frequency that we report here. Depletion of the vesicular pool is likely explanation 661
considering the very high instantaneous frequency rates that are reached. Vesicle 662
refilling and availability of AEA may determine when subsequent bursts occur, accounting 663
for the intermittent nature of the response. Other possibilities (that are not mutually 664
exclusive) are TRPV1 endocytosis (Sanz-Salvador et al., 2012) or desensitization 665
(Numazaki et al., 2003). 666
Panx1 involvement in AEA clearance 667
The mechanisms that regulate synaptic levels of AEA and other endovanilloids (i.e. 668
transport and clearance) are not well understood (Hillard and Jarrahian, 2005). Our 669
findings suggest that AEA removal from the synapse is facilitated by Panx1 because 670
blocking the channel increased total AEA concentration, presumably by preventing access 671
of AEA to the intracellular enzyme, FAAH that degrades it (Blankman and Cravatt, 2013). 672
Inhibition of FAAH had a similar effect to blocking Panx1 on sEPSC frequency, indicating 673
that blocking AEA degradation is sufficient to cause Shaffer collateral stimulation induced 674
bursting. It is notable that during the revision of this manuscript a paper from the Fowler 675
group investigated the putative role of Panx1 in AEA transport in human-derived T84 676
colon cancer cell lines (Alhouayek et al., 2019). While they report no obvious role for 677
Panx1, their experiments were conducted under very different timescales compared to 678
ours and the T84 cells lack a synaptic structure to mimic that seen in brain slices. Our 679
data published on the BioRxiv preprint server (Bialecki et al., 2018) show that Panx1 680
expression is directly linked to augmented AEA clearance in hippocampal slices and 681
uptake when ectopically expressed in HEK293T cells. We are now determining if Panx1 682
facilitates AEA clearance from brain slices with focus on several possibilities, such as 683
29
direct transport of AEA by Panx1 or Panx1 regulation of other putative transporters 684
(Hillard et al., 1997; Hillard and Jarrahian, 2005; Fowler, 2013). 685
In conclusion, we have shown here that NMDARs signal to Panx1 through Src 686
kinase to facilitate uptake of AEA from brain tissue and that when we block Panx1, AEA 687
levels increase to a level sufficient to activate presynaptic TRPV1 and augment glutamate 688
release. We propose that these TRPV1 channels are expressed in Cajal-Retzius cells, the 689
only confirmed site of functional TRPV1 expression (i.e. through measurement of ionic 690
currents) in the hippocampus. While our work has not yet identified a physiological role 691
of Panx1’s regulation of AEA and TRPV1 activity, it is possible that it plays a role in 692
regulation of CR-CA1 excitatory synapses. 693
694
695
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869
Figure Legends 870
871
Fig 1. Block of Panx1 in single postsynaptic neurons enhances facilitated glutamate 872
release during Schaffer collateral stimulation. A) Sample 20s traces taken from 5 min 873
recordings (as indicated) from the same CA1 pyramidal neuron without Schaffer 874
collateral stimulation (black) and with paired pulse stimulation (PPS; grey traces) using 875
control pipette solution. B) Time (5 min total) versus instantaneous frequency under 876
control conditions for the cell in A. C) Time (5 min total) versus instantaneous frequency 877
during PPS of the Schaffer collaterals (vertical dashed lines) for the cell in A. D) 878
Cumulative distribution of the interevent interval of spontaneous excitatory postsynaptic 879
currents (sEPSC) for the population of neurons. Comparisons were made by 880
Kolomogorov-Smirnov tests (at p<0.001). No change in the distribution of interevent 881
intervals were detected after Schaffer collateral stimulation under control conditions. E) 882
Families of sEPSCs from the cell in A and B. Mean sEPSCs are shown as the thick dark 883
lines. F) Comparison of the distribution of sEPSC amplitudes before and after PPS deliver 884
to the Shaffer collaterals. Amplitude was determined by fit of single Gaussian to the 885
distribution. The population of distributions were compared by K-S tests and were not 886
different for the 29 neurons evaluated. G) Sample 20s recordings under the condition 887
where 0.25ng/μl α-panx1 (anti-pannexin-1 blocking antibody) is included in the patch 888
pipette to selectively block postsynaptic Panx1. The traces are without (left) and with 889
(right) PPS of the Schaffer collaterals taken at the indicated time from the total 5 min 890
experiment. Notice that there is an increase in the frequency of sEPSC after PPS of the 891
35
Schaffer collaterals. H and I) Instantaneous sEPSC frequency vs. time plots for the cell in 892
G showing that PPS with α-panx1 in the pipette (right panel) evoked increases in sEPSC 893
frequency compared to the non-stimulated control (right). J) Comparison of the 894
cumulative sEPSC frequency distribution for the population of neurons. A significant 895
(P<0.001) was confirmed by K-S test. K) Families of sEPSCs with α-panx1 in the pipette. 896
L) Comparison of the distribution of sEPSC amplitudes before and after PPS delivery to 897
the Shaffer collaterals with α-panx1 in the pipette. Amplitude was determined by fit of 898
single Gaussian to the distribution and distributions were compared by K-S tests and 899
were not different. 900
901
Fig. 2. Summary of the frequency and amplitude of sEPSCs during Panx1 block and 902
Schaffer collateral stimulation. A) All Shaffer collateral PPS was delivered at 0.05 Hz 903
(20 s inter-PPS interval). Block of Panx1 with either α-panx1 in the pipette or 10panx 904
(100 μM) in the bath augmented sEPSC frequency following afferent stimulation. Note 905
the lack of effect of the respective controls, α-Cx43 and sc10panx. * denotes statistically 906
significant (as indicated) at p<0.05 using the non-parametric Wilcoxon matched-pairs 907
signed rank test. B) Summary of the sEPSC amplitudes under control (n=12) and with α-908
panx1 (n=31) in the pipette in the presence or absence of PPS as indicated. Note that a 909
within cell design was used as for the frequency analysis and no significant difference was 910
detected. C) Comparison of different inter-PPS intervals as indicated above each bar. 911
Frequency was determined for the entire 5 min recordings and was significantly 912
increased at 10 and 30 s inter-PPS intervals. 913
914
36
Fig. 3. Conditional knockout of Panx1 increases sEPSC frequency following Schaffer 915
collateral stimulation. A) Traces from a CA1 neuron in hippocampal slice from wildtype 916
(C57Bl/6J) mouse with α-panx1 in the pipette without (left) or with (right) paired-pulse 917
(PPS) Schaffer collateral stimulation. B) Traces are from a CA1 neurons in slice from a 918
tamoxifen treated Panx1fl/fl-wfs1-Cre mice to knockout Panx1. Knockout of Panx1 when 919
paired with PPS of the Shaffer collaterals increased the frequency of sEPSCs without α-920
panx1 in the pipette. C) Cumulative interevent intervals for the cells shown in A (left) and 921
B (right). D) Summary of the sEPSC frequency from wildtype mice or those carrying 922
floxed Panx1 with and without α-panx1 in the pipette and with and without PPS of the 923
Shaffer collaterals as indicated. Treatment with tamoxifen alone or with intracellular α-924
panx1 (as indicated) resulted in an increase in sEPSC frequency upon PPS of the Shaffer 925
collaterals. These data indicate that Panx1 is responsible suppressing augmented sEPSC 926
frequency under control conditions and that α-panx1 is selective. * significance at p<0.05 927
using the Wilcoxon matched-pairs signed rank test. 928
929
Fig. 4. Preventing ligand binding to NMDARs and Src kinase activity induced 930
facilitated glutamate release during Schaffer collateral stimulation. A) Traces from a 931
CA1 neuron in hippocampal slice (rat) with the competitive blockers for both ligand 932
binding sites of the NMDAR. D-APV (50 μM) and CGP (1 μM) were in the bath and α-panx1 933
in the pipette. Note that PPS of the Schaffer collaterals caused an increase sEPSC 934
frequency. B) Cumulative sEPSC frequency distributions with either D-APV (top) or CGP 935
(bottom) in the bath. Neither competitive antagonist alone altered the distribution of 936
interevent intervals as determined by K-S analysis. C) In contrast to either competitive 937
antagoinist alone, the combined appliciaton of D-APV+CGP caused a significant (P<0.001) 938
37
shift in the distribution of interevent intervals and in the mean ± SEM sEPSC frequencies 939
as depicted in (D). E) Traces from a neuron with Src activation of Panx1 blocked by 940
inclusion of TAT-Panx308 (10 μM) in the pipette before and after Schaffer collateral 941
stimulation. F) cumulative interevent distributions for the population of neurons with 942
TAT-Panx308 in the pipette. G) cumulative interevent distributions for the population of 943
neurons with the Src blocker, PP2 in the bath. H) Comparison of the mean sEPSC 944
frequency under conditions of Src block. Note that α-panx1 was not in the patch pipette 945
under any conditions depicted in this figure. 946
947
Fig 5. Chelating postsynaptic Ca2+ does not prevent Panx1-block induced increases 948
in sEPSC frequency. A) Sample traces from the same neuron showing the presence of 10 949
mM BAPTA and α-panx1 in the pipette did not alter the increase in frequency of sEPSCs 950
when Schaffer collaterals receive PPS (lower trace). B) Plots of instantaneous frequency 951
reveal that synaptic stimulation (lower panel; vertical lines) causes bursts of sEPSC 952
release when BAPTA and α-panx1 are in the pipette. C) Summary of the paired changes in 953
frequency upon synaptic stimulation. * denotes p<0.05 (Wilcoxon matched-pairs signed 954
rank test). 955
956
Fig. 6. Blockers of CB1 receptors and nitric oxide synthase do not prevent the Panx1 957
block induced facilitated glutamate release. A and B are exemplar recordings from 2 958
different CA1 neurons testing the role of the retrograde transmitter receptor, CB1R, and 959
nNOS in synaptic bursting. Both were ruled out because application of their specific 960
antagonists, AM251 and L-NAME, respectively, did not alter the appearance of increased 961
38
sEPSC frequency. C) Summary of the mean sEPSC frequency. *Denotes significance at 962
p<0.05 via Wilcoxon matched-pairs signed rank test. 963
964
Fig 7. The block of postsynaptic Panx1-induced increase in sEPSCs does not alter 965
evoked EPSCs, AMPA/NMDA receptor ratios but requires increased Ca2+. A) Paired-966
pulse ratio (PPR) is not altered by blocking postsynaptic Panx1. Average (n=15 pairs) 967
PPRs (at 0.05 Hz, 1 ms duration, 50ms interstim interval) are shown under control 968
(black) and with α-panx1 in the pipette (red). The population response of the PPR is 969
shown in the right panel. In the presence of α-panx1 in the pipette (n=31) the PPR over 970
the 5 min recordings was unchanged compared to the control (n=28). When the 971
increased sEPSC frequency overlapped with the evoked responses (blue bar; n=12) the 972
PPR was still unchanged. B) AMPAR / NMDAR ratio is not altered by block of postsynaptic 973
Panx1. Families from both control (normal internal solution; n=7) and with α-panx1 974
(n=9) in the pipette (red) are shown. Downward traces are the AMPAR component 975
collected at Vm=-70 mV in 1.2 mM Mg2+ and upward traces are the NMDAR component 976
collected at Vm=+40 mV in 0 Mg2+ and 10 μM DNQX. Calculated ratios are shown in the 977
right hand plot. C) Incubation of hippocampal slices with EGTA-AM prevented the 978
increased sEPSC frequency arising from Schaffer collateral stimulation when α-panx1 was 979
in the pipette. Example 10 s traces are shown. The arrow depicts paired pulse 980
stimulation. The plot (right) illustrates a failure of the mean frequency of sEPSCs to 981
increase, suggesting that bursting is Ca2+ dependent. 982
983
Fig. 8. Expression of TRPV1 in the CA1 region is at a portion of presynaptic 984
terminals. A) Demonstration of TRPV1 mRNA by PCR in hippocampus and dorsal root 985
39
ganglia (DRG). Note that a PCR product is not observed in TRPV1-/- mice. B) Comparison 986
of TRPV1 expression relative to DRG. qPCR levels were first normalized to β-actin levels 987
and then expressed as a fraction of the level in DRG. Note that the hippocampus has a very 988
small, but detectable, amount of TRPV1 mRNA relative to DRG and that TRPV1 mRNA was 989
not detectable in knockout mice. C) Sample immuno EM images of TRPV1 labelling in 990
wild type (wt; top) and knockout (TRPV1-/-; bottom) mice. Arrows indicate TRPV1 991
immunogold particles in terminals. Presynaptic terminals are colored red and CA1 spines 992
blue. Scale bars represent 1 μm. D) Summary of the percentage of labelled terminals in 993
the CA1. * in B and D denotes p<0.05 determined by unpaired student’s t-test. 994
995
Fig. 9. TRPV1 activity is required for Panx1-block mediated synaptic bursting. 996
A) Sample 20 s traces from the same cell that have α-panx1 in the pipette are shown 997
during baseline (top), PPS stimulation of the Schaffer collaterals (middle) and during PPS 998
stimulation with the TRPV1 antagonist, capsazapine (CPZ) in the bath (bottom). Note that 999
CPZ blocks the PPS-induced increase in sEPSC frequency. B) Summary of the changes in 1000
sEPSC frequency in rats with α-panx1 in the pipette. Each neuron was treated with all 4 1001
conditions and within cell comparisons are indicated by the lines joining individual 1002
points. Note the block of the increase in sEPSC frequency with bath application of 10 μM 1003
CPZ. C) The Ih blocker, ZD7288 (1 μM) did not induce increased sEPSC frequency when 1004
PPS was applied, nor did it prevent the increase when α-panx1 was present. This is an 1005
important control for off target effects of CPZ. D) Knockout of TRPV1 (TRPV1-/-) prevents 1006
Schaffer collateral stimulation induced increases in sEPSC frequency. Throughout the 1007
figure, * denotes p<0.05 using the Wilcoxon matched-pairs rank test and n.s. (not 1008
significant; p>0.05). 1009
40
1010
Fig. 10. Panx1 regulates tissue levels of the TRPV1 agonist, anandamide. 1011
A) Quantification of tissue (brain slice) concentrations of anandamide (AEA) by mass 1012
spectrometry. Note that the bath applied Panx1 blocker, 10panx (100 μM) increased AEA 1013
concentration but the TRPV1 blocker, CPZ (10μM) did not. B) The FAAH antagonist URB 1014
597 (1 μM), which blocks AEA metabolism caused an increase in sEPSC frequency upon 1015
PPS delivery to Shaffer collaterals. Note that αPanx1 was not included in the pipette. 1016
These data suggest that Panx1 regulates tissue AEA levels, which is a key step in 1017
activating presynaptic TRPV1 and causing increased sEPSC frequency. 1018
1019
Fig. 11. A model of the proposed pathway for suppression of glutamate release by 1020
postsynaptic metabotropic NMDAR signalling to Panx1. The left side of the cartoon. 1021
“functional Panx1”, shows the scenario when Panx1 channels are operating normally. 1022
This physiological state is indicated as steps 1-3 (boxed numbers) where the binding of 1023
glutamate and glycine / D-serine to NMDARs (step 1) initiates Src-mediated 1024
phosphorylation of Panx1 (step 2), which facilitates clearance of anandamide (AEA) (step 1025
3) for degradation by FAAH. Our evidence for this sequence is that blocking ligand 1026
binding to NMDRs (with D-APV+CGP; at step 1) but not postsynaptic increases in Ca2+ 1027
(with BAPTA), or blocking Src activation of Panx1 (with TAT-Panx308 or PP2; at step 2), or 1028
inhibiting Panx1 (with -panx1, 10panx, or Panx1-/-; at step 3) all augmented sEPSC 1029
frequency. The right side of the figure, “blocked Panx1”, depicts the sequence of events 1030
occurring when Panx1 or metabotropic NMDARs have been blocked (steps 4-6; dashed 1031
arrows). In the current study, direct block of Panx1 increases tissue AEA levels (step 4), 1032
and we propose that AEA acts on presynaptic TRPV1 to increase Ca2+ (step 5) and 1033
41
facilitate activity-dependent glutamate release (step 6). Our evidence for this sequence is 1034
that facilitated glutamate release was prevented by TRPV1 block (with CPZ or in TRPV1-/-; 1035
at step 4), or by bath applied (EGTA-AM; at step 5). We also quantified TRPV1 expression 1036
in the hippocampus (PCR, qPCR) and found it at presynaptic sites (by immuno EM) and 1037
propose that these are Cajal-Retzius (CR) cells synapsing onto CA1 neurons, because CR 1038
cells are the only confirmed site of TRPV1 expression. 1039