Presynaptic NMDA receptors facilitate short-term plasticity and BDNF release at hippocampal mossy fiber synapses
- Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, United States
- Instituto de Investigación en Discapacidades Neurológicas (IDINE), Facultad de Medicina, Universidad Castilla-La Mancha, Spain
- Department of Psychiatry and Behavioral Sciences, Albert Einstein College of Medicine, United States
Figures
Figure 1 with 3 supplements
Anatomical and functional evidence for preNMDARs at mossy fiber synapses.
(A) Image of a mossy fiber (mf) giant bouton and postsynaptic spines (s). (B, C) Higher magnification of mf synapses. Arrows indicate postsynaptic GluN1, whereas arrowheads indicate presynaptic …
Figure 1—figure supplement 1
Immunogold-EM reveals negligible presynaptic AMPAR particle distribution.
(A, B) Images of mossy fiber (mf) and associational–commissural (ac) synapses, postsynaptic spines (s). (C) AMPAR immuno-particle distribution (30 nm bins), mf: 102 synapses, eight presynaptic …
Figure 1—figure supplement 2
Stable low-frequency facilitation of mf-CA3 synaptic transmission in naïve slices.
(A) Stable low-frequency facilitation (LFF) of AMPAR-EPSCs. In naïve slices (interleaved experiments), LFF remained unchanged throughout the recording session (baseline 335 ± 62%, naïve 363 ± 63%, n …
Figure 1—figure supplement 3
Intracellular MK-801 effectively blocked postsynaptic NMDARs.
Representative NMDAR-EPSCs (Vh = +40 mV) from CA3 pyramidal neurons patch-loaded with 2 mM MK-801 (left) or naïve internal solution (right). Mf inputs were stimulated with a bipolar electrode …
Figure 2
GluN1 deletion from GCs reduces mf-CA3 facilitation.
(A) Representative images showing GCs patch-loaded with Alexa 594 (35 µM) (left), and GFP expression in GCs (right). (B) Representative EPSCs recorded from control (GFP+) and Grin1-cKO (Cre-GFP+) …
Figure 3 with 1 supplement
Reduced facilitation by NMDAR antagonism is independent of the GC somatodendritic compartment.
(A) KAR-EPSCs were recorded at Vh = −70 mV in the presence of 15 µM LY303070 and 100 µM picrotoxin. In addition, NMDAR-mediated transmission was blocked intracellularly by loading MK-801 (2 mM) in …
Figure 3—figure supplement 1
Targeting preNMDARs located in mf axons, but not granule cells.
(A) Field view of a representative hippocampal slice showing a surgical cut between DG and CA3. (B) Local D-APV puff application (vertical arrow, two puffs at 0.1 Hz) blocks NMDAR currents recorded …
Figure 4 with 3 supplements
PreNMDARs contribute significantly to burst-induced facilitation and spike transfer.
(A) Representative images showing expression of GFP-Cre (left) and ChIEF-tdTomato (right) in the DG of control and Grin1-cKO animals. (B) Representative AMPAR-EPSCs from control (left) and Grin1-cKO …
Figure 4—figure supplement 1
PreNMDARs contribute to burst-induced facilitation in more physiological conditions: 1.2 mM Mg+2, 1.2 mM Ca+2 and 35°C.
KAR-EPSCs were recorded from CA3 pyramidal cells loaded with 2 mM MK-801 in the presence of 15 µM LY303070 and 100 µM picrotoxin. (A) Bath application of MK-801 (50 µM) significantly reduced …
Figure 4—figure supplement 2
PreNMDARs contribute to action potential firing elicited by AMPAR-mediated transmission.
(A) Bath application of MK-801 (50 µM) reduced action potentials induced by 5 pulses at 25 Hz burst stimulation (baseline 2.47 ± 0.27, MK-801 1.9 ± 0.24, n = 6 cells, five animals; p=0.036, Wilcoxon …
Figure 4—figure supplement 3
Intracellular MK-801 effectively blocked postsynaptic NMDARs elicited by burst stimulation (5 pulses at 25 Hz).
Representative NMDAR-EPSCs (Vh = +40 mV) from CA3 pyramidal neurons patch-loaded with 2 mM MK-801 (left) or naïve internal solution (right). Mf inputs were stimulated with a bipolar electrode …
Figure 5 with 1 supplement
preNMDARs contribute to presynaptic Ca2+ rise.
(A) Representative images showing GCs patch-loaded with Alexa 488 (35 µM) to confirm expression of mCherry (bottom). Representative AMPAR-EPSCs recorded from control (top) or Grin1-cKO (middle) GCs. …
Figure 5—figure supplement 1
NMDAR antagonism reveals a reduction in presynaptic Ca+2 rise in the presence of 1.3 mM Mg+2 and 2.5 mM Ca+2.
(A, B) Granule cells were patch-loaded with Fluo-5F (200 µM) and Alexa 594 (35 µM). Line scan analysis of mf giant bouton calcium transients (CaTs) in response to action potential (AP) stimulation …
Figure 6 with 3 supplements
Uncaging glutamate induces Ca2+ rise in mossy fiber boutons.
(A) Representative images showing dendritic spines in GCs (left) and mf boutons (right), and the associated line scan image of calcium transients (CaTs) elicited by uncaging of MNI-glutamate (see …
Figure 6—figure supplement 1
Grin1-cKO exhibit reduced CaTs at varying uncaging laser power intensities.
(A) Representative images of CaTs from control (top) and Grin1-cKO animals (bottom) after MNI-glutamate uncaging (2 mM, 3 pulses at 25 Hz) on GC dendritic spines. Dotted line (yellow) indicates line …
Figure 6—figure supplement 2
Bouton CaTs can be detected after repetitive uncaging of MNI-glutamate.
(A) Representative images of CaTs from single-trial: 1 pulse (top) and 5 pulses, 25 Hz (bottom) of MNI-glutamate uncaging (2 mM). Dotted line (yellow) indicates line scan, and blue dots indicate 2PU …
Figure 6—figure supplement 3
NMDAR antagonism with D-APV blocks CaTs elicited by glutamate 2PU.
(A) Representative image of baseline glutamate uncaging driven CaTs in mf boutons (top). D-APV application (100 µM) blocks CaTs (bottom). (B) Quantified ΔG/R signals before and after D-APV …
Figure 7 with 1 supplement
preNMDARs contribute significantly to BDNF release following repetitive activity.
(A) Representative images showing expression of BDNF-pHluorin in the DG and CA3 area (arrows indicate mf axon, arrowheads indicate mf boutons). Control images (top), Grin1-cKO images (bottom). (B) …
Figure 7—figure supplement 1
preNMDARs contribute significantly to BDNF release following a more physiological pattern of burst stimulation.
(A) Representative images of BDNF-pHluorin signal intensity at baseline and after burst stimulation of mfs (5 pulses, 100 Hz, ×50, every 0.5 s). Control images (left), Grin1-cKO images (right), …
Figure 8 with 1 supplement
preNMDARs contribute to synaptic facilitation of mossy fiber inputs onto mossy cells, but not onto CA3 inhibitory interneurons.
(A) Representative images showing a CA3 IN and a hilar MC patch-loaded with Alexa 594 (35 µM) for morphological identification in acute rat hippocampal slices. (B) AMPAR-EPSCs were recorded from CA3 …
Figure 8—figure supplement 1
Stability experiments for mf-Interneuron and mf-mossy cell short-term plasticity.
(A) Stable CA3 IN burst-induced facilitation of mf-CA3 transmission (baseline 273 ± 30%, naïve 294 ± 33%, n = 10 cells, six animals; p=0.298, paired t-test, baseline vs naïve). (B) Stable …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information | |
|---|---|---|---|---|---|
| Strain, strain background (Rattus norvegicus male and female) | Rat: Sprague-Dawley | Charles River | Strain code: 400 | ||
| Strain, strain background (Mus musculus, male and female) | Mouse: Grin1fl/fl(B6.129S4-Grin1tm2Stl/J) | Dr. Michael Higley/The Jackson Laboratory | RRID:IMSR_JAX005246 | ||
| Strain, strain background (Mus musculus male and female) | Mouse: C57Bl6/J | Charles River | Strain code: 027 | ||
| Antibody | (Include host species and clonality) Mouse, Monoclonal, anti-NMDAR1 | Millipore | Cat# MAB363 | 10 μg/mL | |
| Antibody | Rabbit, Polyclonal, anti-GluA1-4, (pan-AMPA) | Dr. Elek Molnar/Bristol University | Generated by Dr. Elek Molnar | 10 μg/mL | |
| Antibody | Goat anti-rabbit IgG conjugated gold particles | Nanoprobes Inc | #2003–0.5 ML | (1:100) | |
| Recombinant DNA reagent | AAV5-CaMKII-GFP-Cre | Penn Vector Core | AV-5-PV2521 | Available on Addgene | |
| Recombinant DNA reagent | AAV5-CaMKII-eGFP | Penn Vector Core | AV-5-PV1917 | Available on Addgene | |
| Recombinant DNA reagent | AAV5-CaMKII-mcherry-Cre | UNC Vector Core | See website | https://wwwmed.unc.edu/genetherapy/vectorcore/in-stock-aav-vectors/ | |
| Recombinant DNA reagent | AAV5-CaMKII-mcherry | UNC Vector Core-Dr. Karl Deisseroth Control Fluorophores | See website | https://wwwmed.unc.edu/genetherapy/vectorcore/in-stock-aav-vectors/ | |
| Recombinant DNA reagent | AAV-DJ-flex-OChIEF-tdTomato | Dr. Pascal Kaeser PMID:29398114 | Generated at UNC Vector Core | Custom Order | |
| Recombinant DNA reagent | AAV-DJ-DIO-BDNF-phluorin | Dr. Hyungju Park PMID:25467984 | Generated at UNC Vector Core | Custom Order | |
| Chemical compound, drug | Ketamine | Merial | Cat# 03661103001904 | ||
| Chemical compound, drug | Xylazine | Calier | Cat# 20100–003 | ||
| Chemical compound, drug | Paraformaldehyde | Scharlau | PA0095 | ||
| Chemical compound, drug | Glutaraldehyde | Electron Microscopy Sciences | Cat# 16210 | ||
| Chemical compound, drug | Picric Acid | Panreac | Cat# 141048.1609 | ||
| Chemical compound, drug | Phosphate Buffer | Scharlau | SO03321000 | ||
| Chemical compound, drug | Human serum albumin | SigmaMillipore | A-1653 | ||
| Chemical compound, drug | TBS | TRIZMA BASE | SigmaMillipore | T1503 | |
| Trizma HCl | T3253 | ||||
| Chemical compound, drug | Triton X-100 | SigmaMillipore | T8787 | ||
| Chemical compound, drug | Polyethylene glycol | SigmaMillipore | 25322-68-3 | ||
| Chemical compound, drug | Uranyl acetate | Electron Microscopy Sciences | Cat# 22400 | ||
| Chemical compound, drug | Reynold’s lead citrate | Electron Microscopy Sciences | #17800 | ||
| Chemical compound, drug | Picrotoxin | SigmaMillipore | Cat# P1675 | ||
| Chemical compound, drug | LY303070 | ABX Chemical Co. | N/A | Custom Order | |
| Chemical compound, drug | MK-801 | Tocris Bioscience | Cat# 0924 | ||
| Chemical compound, drug | DCG-IV | Tocris Bioscience | Cat# 0975 | ||
| Chemical compound, drug | D-APV | Tocris Bioscience | Cat# 0106 | ||
| chemical compound, drug | D-APV | NIMH Chemical Synthesis Program | N/A | ||
| Chemical compound, drug | R-CPP | Tocris Bioscience | Cat# 0247 | ||
| Chemical compound, drug | NBQX | Cayman Chemical Co. | Cat# 14914 | ||
| Chemical compound, drug | Fluo5-F pentapotassium salt cell impermeant | Invitrogen Molecular Probes | Cat# F14221 | ||
| Chemical compound, drug | Alexa Fluor 594 Hydrazide | Invitrogen Molecular Probes | Cat# A10438 | ||
| Chemical compound, drug | Alexa Fluor 488 Hydrazide | Invitrogen Molecular Probes | Cat# A10436 | ||
| Chemical compound, drug | D-Serine | Tocris Bioscience | Cat# 0226 | ||
| Chemical compound, drug | MNI-caged-L-glutamate | Tocris Bioscience | Cat# 1490 | ||
| Chemical compound, drug | Sucrose | SigmaMillipore | Cat# S9378 | ||
| Chemical compound, drug | KCl | SigmaMillipore | Cat# P3911 | ||
| Chemical compound, drug | NaH2PO4 | SigmaMillipore | Cat# S9638 | ||
| Chemical compound, drug | CaCl2 | SigmaMillipore | Cat# C8106 | ||
| Chemical compound, drug | MgCl2 | SigmaMillipore | Cat# M2670 | ||
| Chemical compound, drug | MgSO4 | SigmaMillipore | Cat# M1880 | ||
| Chemical compound, drug | Glucose | SigmaMillipore | Cat# G8270 | ||
| Chemical compound, drug | NaCl | SigmaMillipore | Cat# S7653 | ||
| Chemical compound, drug | NaHCO3 | SigmaMillipore | Cat# S6014 | ||
| Chemical compound, drug | Cesium hydroxide | SigmaMillipore | Cat# 23204 | ||
| Chemical compound, drug | D-gluconic acid | SigmaMillipore | Cat# G1951 | ||
| Chemical compound, drug | EGTA | SigmaMillipore | Cat# E4378 | ||
| Chemical compound, drug | HEPES | SigmaMillipore | Cat# H3375 | ||
| Chemical compound, drug | Potassium gluconate | SigmaMillipore | Cat# G4500 | ||
| Chemical compound, drug | MgATP | SigmaMillipore | Cat# A9187 | ||
| Chemical compound, drug | Na3GTP | SigmaMillipore | Cat# G0635 | ||
| Chemical compound, drug | NMDG | SigmaMillipore | Cat# M2004 | ||
| Chemical compound, drug | Sodium ascorbate | SigmaMillipore | Cat# A4034 | ||
| Chemical compound, drug | Thiourea | SigmaMillipore | Cat# T8656 | ||
| Chemical compound, drug | Sodium pyruvate | SigmaMillipore | Cat# P2256 | ||
| Chemical compound, drug | KMeSO4 | SigmaMillipore | Cat# 83000 | ||
| Chemical compound, drug | Na2ATP | SigmaMillipore | Cat# A2383 | ||
| Chemical compound, drug | NaGTP | SigmaMillipore | Cat# 51120 | ||
| Chemical compound, drug | Sodium phosphocreatine | SigmaMillipore | Cat# P7936 | ||
| Chemical compound, drug | NH4Cl | SigmaMillipore | Cat# A9434 | ||
| Chemical compound, drug | KOH | EMD Millipore | Cat# 109108 | ||
| Chemical compound, drug | HCl | Fisher Chemical | Cat# SA49 | ||
| Software, algorithm | IgorPro7 | Wavemetrics | https://www.wavemetrics.com/ | ||
| Software, algorithm | Origin Pro 9 | Origin Lab | https://www.originlab.com/ | ||
| Software, algorithm | ImageJ | ImageJ | http://imagej.net/Welcome | ||
| Software, algorithm | Multiclamp 700B | Molecular Devices | https://www.moleculardevices.com/ | ||
| Software, algorithm | Prairie View 5.4 | Bruker Corp. | https://www.pvupdate.blogspot.com/ | ||
Additional files
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Source data 1
Source datasets for all figures.
- https://cdn.elifesciences.org/articles/66612/elife-66612-data1-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/66612/elife-66612-transrepform-v2.pdf
