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, 112 (3), 691-702

Reduced Expression of Glutamate Receptors and Phosphorylation of CREB Are Responsible for in Vivo Delta9-THC Exposure-Impaired Hippocampal Synaptic Plasticity

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Reduced Expression of Glutamate Receptors and Phosphorylation of CREB Are Responsible for in Vivo Delta9-THC Exposure-Impaired Hippocampal Synaptic Plasticity

Ni Fan et al. J Neurochem.

Abstract

Chronic use of marijuana impairs synaptic plasticity and cognitive function. However, the molecular mechanisms by which marijuana alters long-term synaptic plasticity are largely unknown. Here, we show that repeated in vivo exposures to Delta9-THC for 7 consecutive days significantly impaired hippocampal long-term potentiation (LTP) of excitatory glutamatergic synaptic transmission. The Delta9-THC exposure-induced decrease in LTP was prevented by pharmacological inhibition or deletion of the cannabinoid 1 receptor (CB1R). To determine the molecular mechanisms underlying Delta9-THC-altered LTP, we targeted expression and function of the glutamate receptors (GluR) and phosphorylation status of cAMP-response element-binding protein (CREB). Chronic in vivo exposure to Delta9-THC produced CB1R-dependent decreases in expression of hippocampal GluR1, NR2A, and NR2B, the ratio of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/NMDA receptor-gated currents, and phosphorylation of CREB. Our results suggest that reduced expression and function of the GluR subunits and phosphorylation of CREB may underlie the impaired long-term synaptic plasticity induced by repeated in vivo exposure to Delta9-THC.

Figures

Figure 1
Figure 1
Repeated in vivo exposures to Δ9-THC induce a CB1 receptor-dependent decrease in hippocampal LTP. A1. Representative traces of fEPSPs recorded before (1) and after (2) TBS in hippocampal slices from CB1R wild-type (WT) animals that received vehicle or Δ9-THC (10 mg/kg) once a day for 7 consecutive days. LTP was measured 24 hrs after cessation of last injection. A2. Time course of the Δ9-THC-induced changes in fEPSP slopes. A3. Mean values of the potentiation of fEPSPs averaged from 36 to 40 min following TBS (Veh: 11 recordings/6 animals, THC: 11 recordings/6 animals). **P<0.01 compared with the vehicle control. B1. Representative traces of fEPSPs recorded before (1) and after (2) TBS in hippocampal slices from CB1R WT animals that received repeated injections with SR141716 (SR, 5 mg/kg) or SR plus Δ9-THC (10 mg/kg) for 7 consecutive days. B2. Time course of the Δ9-THC-induced changes in fEPSP slopes. B3. Mean values of the potentiation of fEPSPs averaged from 36 to 40 min following TBS (SR: 11 recordings/5 animals, SR+THC: 12 recordings/6 animals). C1. Representative traces of fEPSPs recorded before (1) and after (2) TBS in hippocampal slices from CB1R knockout (KO) mice that received vehicle or Δ9-THC (10 mg/kg) once a day for 7 consecutive days. LTP was measured 24 h after cessation of injections. C2. Time course of the Δ9-THC-induced changes in fEPSP slopes. C3. Mean values of the potentiation of fEPSPs averaged from 36 to 40 min following TBS (Veh: 8 recordings/6 animals, THC: 11 recordings/6 animals). Scale bar: 0.3 mV/10 msec.
Figure 2
Figure 2
Chronic in vivo exposure to Δ9-THC reduces expression of AMPA and NMDA receptor subunits. A1. Immunoblot analysis of NMDA subunits NR1, NR2A, NR2B, and AMPA subunits GluR1 and GluR2 in hippocampal tissues from CB1 wild type mice that received repeated exposures to Δ9-THC (10 mg/kg) for 7 days. Proteins were detected 24 hrs after cessation of last injection. A2. Quantification of the total subunits in vehicle- and Δ9-THC-treated animals (6 animals per group). B1. Surface expression of NR1, NR2A, NR2B, GluR1, and GluR2 subunits is reduced in animals that were chronically exposed to Δ9-THC for 7 days. Surface proteins were biotinylated with EZ-link sulfo-NHS-LC biotin. B2. Quantification of the surface NR1, NR2A, NR2B, GluR1, and GluR2 subunits (3 animals per group). C1. Intracellular NR2A, NR2B and GluR1 subunits in hippocampal tissues from vehicle- or Δ9-THC-treated animals. Extracellular proteins were cross-linked using BS3 (Bis [Sulfosuccinimidyl] suberate). C2. Quantification of the intracellular NR2A, NR2B and GluR1 subunits (3 animals per group). *P<0.05; **P<0.01 compared with vehicle controls.
Figure 3
Figure 3
Real-time PCR analysis of NR1, NR2A, NR2B, GluR1, and GluR2 expressions in hippocampal tissue from mice treated with vehicle or Δ9-THC (10 mg/kg) for 7 days. Results are from six independent observations (6 animals per group) with duplicate wells. *P<0.05, **P<0.01 compared with vehicle controls.
Figure 4
Figure 4
The CB1 receptor mediates Δ9-THC-reduced expression of AMPA and NMDA receptor subunits. A1–A2. Δ9-THC-reduced expression of NR2A, NR2B and GluR1 subunits is prevented by pharmacological inhibition of the CB1 receptor. CB1R wild-type (WT) animals were administrated with vehicle or Δ9-THC (10 mg/kg) plus SR (5 mg/kg) for 7 days. Subunit proteins were analyzed 24 hrs after cessation of last injection (3 animals per group). B1–B2. Δ9-THC-reduced expression of NR2A, NR2B and GluR1 subunits is prevented by genetic deletion of the CB1 receptor. CB1R knockout (KO) mice were injected with vehicle or Δ9-THC (10 mg/kg) for 7 days (3 animals per group).
Figure 5
Figure 5
Exposure to Δ9-THC induces changes in the ratio of AMPA/NMDA receptor-gated currents. A1. Representative traces of the AMPA- and NMDA-gated currents at hippocampal perforant path synapses in CB1R WT animals that received repeated injections with vehicle or Δ9-THC for 7 days. Membrane potential was held at +40 mV, and the AMPA-mediated current was isolated by AP5 (50 µM) and bicuculline (10 µM). The NMDA-mediated current was obtained by subtraction of the AMPA component from the total currents. Scale bar: 50 pA/100 msec. A2. Chronic in vivo exposure to Δ9-THC reduces the ratio of AMPA/NMDA-mediated currents (Veh: 12 recordings/5 animals, THC: 15 recordings/5 animals). *P<0.05 compared with vehicle controls. B1. Representative traces of the AMPA- and NMDA-gated currents at hippocampal perforant path synapses in CB1R KO animals that received repeated injections with vehicle or Δ9-THC for 7 days. B2. Repeated in vivo exposures to Δ9-THC fail to change the ratio of AMPA/NMDA-mediated currents in CB1KO mice (Veh: 13 recordings/5 animals, THC: 12 recordings/5 animals).
Figure 6
Figure 6
Chronic in vivo Δ9-THC exposure enhances basal synaptic transmission. A1. Representative fEPSP waveforms recorded at hippocampal perforant path synapses in the dentate gyrus of animals injected with vehicle and Δ9-THC (10 mg/kg) for 7 days. A2. Input-output function curves (Veh: 10 recordings/5 animals, THC: 9 recordings/5 animals). Stimulus intensity was normalized to the maximum intensity. Scale bar: 0.3 m V/10 msec. B1. Representative EPSC traces recorded in granule neurons in response to perforant path stimuli from animals injected with vehicle and Δ9-THC (10 mg/kg) for 7 days. EPSCs were induced by a paired-pulse protocol with varying interpulse intervals. B2. Paired-pulse ratios (EPSC2/EPSC1) in vehicle- and Δ9-THC-treated animals (Veh: 27 recordings/6 animals, THC: 25 recordings/6 animals). Scale bar: 300 pA/100 msec
Figure 7
Figure 7
Repeated in vivo exposures to Δ9-THC enhance probability of synaptic glutamate release. A1–A2. Representative sweeps of spontaneous excitatory postsynaptic currents (sEPSCs) recorded in granule neurons in hippocampal slices from animals injected with vehicle or Δ9-THC (10 mg/kg) for 7 days. The membrane potential was held at −70 mV. Bicuculline (10 µM) was included in the external solution. The synaptic events were analyzed using the MiniAnalysis program. B1–B2. Cumulative probability of sEPSC frequency and amplitude recorded in neurons from animals treated with vehicle or Δ9-THC. C1–C2. Mean percentage changes in the frequency and amplitude of sEPSCs (Veh: 28 recrodings/5 animals, THC: 29 recordings/5 animals). *P<0.05 compared with the vehicle control (The Kolmogorov-Smirnov test). Scale bar: 20 pA/1 sec.
Figure 8
Figure 8
Repeated in vivo exposures to Δ9-THC produce a CB1 receptor-dependent reduction of CREB phosphorylation. A1–A2. Western blot analysis of hippocampal CREB and its phosphorylation in CB1WT animals that received repeated exposures to Δ9-THC for 7 days (6 animals per group). B1–B2. Repeated Δ9-THC exposure-induced decreases in hippocampal CREB and its phosphorylation are blocked by inhibition of the CB1 receptor in CB1R WT mice (Veh: 3 animals, THC+SR: 3 animals). C1–C2. Chronic in vivo exposure to Δ9-THC fails to induce decreases in hippocampal CREB and its phosphorylation in CB1R KO animals (3 animals per group).

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