Fatty-acid-binding protein 5 controls retrograde endocannabinoid signaling at central glutamate synapses

Abstract

Endocannabinoids (eCBs) are lipid-signaling molecules involved in the regulation of numerous behaviors and physiological functions. Released by postsynaptic neurons, eCBs mediate retrograde modulation of synaptic transmission and plasticity by activating presynaptic cannabinoid receptors. While the cellular mechanisms by which eCBs control synaptic function have been well characterized, the mechanisms controlling their retrograde synaptic transport remain unknown. Here, we demonstrate that fatty-acid-binding protein 5 (FABP5), a canonical intracellular carrier of eCBs, is indispensable for retrograde eCB transport in the dorsal raphe nucleus (DRn). Thus, pharmacological inhibition or genetic deletion of FABP5 abolishes both phasic and tonic eCB-mediated control of excitatory synaptic transmission in the DRn. The blockade of retrograde eCB signaling induced by FABP5 inhibition is not mediated by impaired cannabinoid receptor function or reduced eCB synthesis. These findings indicate that FABP5 is essential for retrograde eCB signaling and may serve as a synaptic carrier of eCBs at central synapses.

Keywords: 2-AG; AMPA; dorsal raphe; endocannabinoid; synapse.

Fig. 1.

Inhibition of FABP impairs phasic 2-AG signaling in the DRn. (A) Inhibition of FABPs with SBFI26 reduces the DSE. (Lower) The DSE obtained in control (●, n = 10) and in the presence of SBFI26 (10 μM, O, n = 10). (Upper) The AMPAR-EPSC traces taken at the time indicated by numbers. (B) FABP inhibition blocks the α₁-AR-LTD induced by phenylephrine (PE, 100 μM). (Lower) The α₁-AR-LTD obtained in control (●, n = 10) and in slices treated with SBFI26 (10 μM, O, n = 10). (Upper) AMPAR-EPSCs traces taken at the corresponding time points. (C) Summary of the effect of SBFI-26 (10 μM) on AMPAR-EPSC amplitude. (Upper) Sample AMPAR-EPSC traces collected at the corresponding time points. (D) FABP inhibition does not alter the function of CB1R. (Lower) Summary of the depression of AMPAR-EPSCs induced by WIN55, 212–2 (10 μM) in control (●, n = 9) and in slices treated with SBFI26 (10 μM, O, n = 9). (Upper) Sample AMPAR-EPSC traces taken at the corresponding time points. (E) Effect of FABP inhibition on 2-AG (Left) and AEA levels (Right) in the DRn. (F) Inhibition of FABP does not alter Pr of glutamate release. (Left) The average PPR of AMPAR-EPSCs obtained in control (O, n = 7) and SBFI26-treated slices (O, n = 7, P > 0.05). (Right) The corresponding AMPAR-EPSC traces. (Scale bars: 50 pA, 10 ms.)

Fig. 2.

Inhibition of FABP blocks tonic 2-AG signaling in the DRn. (A) Tonic eCB controls baseline glutamatergic transmission in the DRn. (Left) The potentiation of AMPAR-EPSC amplitude induced by AM251 (3 μM, n = 8). (B) Summary of the effect of AM 251 on the PPR of AMPAR-EPSC. (C) Tonic eCB signaling is mediated by 2-AG. (Lower) The AM251-induced potentiation of AMPAR-EPSCs obtained in control (●, n = 10) and in slices treated with the DAGLα inhibitor THL (10 µM, O, n = 10). (Upper) The corresponding AMPAR-EPSC traces. (D) Inhibition of FABPs abolishes tonic 2-AG–mediated control of AMPAR-EPSCs. (Lower) The AM251-induced potentiation of AMPR-EPSCs obtained in control (●, n = 10) and in SBFI26-treated slices (10 µM, O, n = 10). Treatment with SBFI26 prevented the AM251-induced potentiation of AMPAR-EPSCs. (Upper) AMPAR-EPSC traces taken at the indicated time points. (Scale bars: 100 pA, 10 ms.)

Fig. 3.

FABP5 is expressed and localized in DRn synapses. (A) Immunohistochemical distribution of FABP5 in the DRn of WT (Left) and FABP5 KO mice (Right). Note the expression of FABP5 (red) in all DRn subdivisions of WT but not KO mice. Nuclei (blue) are labeled with DAPI. (B) EM images of the cellular distribution of FABP5 in the DRn of WT (Left panels) and FABP5KO (Right panels). Arrows indicate a synapse location. (C) Specific RLI were calculated from immunogold distribution in the DRn of wild-type and FABP5 KO mice.

Fig. 4.

Genetic deletion of FABP5 abolishes phasic and tonic 2-AG signaling in the DRn. (A) FABP5 deletion blocks the DSE. (Left) DSE in WT (●) and FABP5 KO mice (O, n = 8). (Right) The corresponding AMPAR-EPSC traces collected at the indicated time points. (B) FABP5 deletion abolishes the AM251-induced potentiation of AMPAR-EPSCs. (Lower) A summary of AM251-induced potentiation of AMPAR-EPSCs in WT (●, n = 12) and FABP5 KO mice (O, n = 12). (Upper) AMPAR-EPSC traces collected at the corresponding time points. (C) Genetic deletion of FABP5 blocks the JZL184-induced depression of AMPAR-EPSCs. (Lower) The depression of AMPAR-EPSCs induced by JZL184 (1 μM) in WT (●, n = 9) and FABP5 KO mice (O, n = 9). (Upper) AMPAR-EPSCs collected before and during JZL184. (Scale bars: 100 pA, 20 ms.)