Secondary antisera from ThermoFisher Scientific included Alexa Fluor 594 antirabbit IgG , Alexa Fluor 488 anti-mouse IgG and Alexa Fluor 488 anti-guinea pig IgG all used at 1:1000 dilutions. An epifluorescence microscope with a 63× PlanApo objective and ORCA-ER camera was used to capture image z-stacks, through a depth of 2 μm in 0.2 μm z-steps, from the DG outer molecular layer and CA1 stratum radiatum. For slice experiments, 1 z-stack was captured from each of 6 sections per slice. For behavioral/brain studies, 3 z-stacks were captured per section from 3 to 4 spaced sections within a given septo-temporal span of hippocampus . Immunolabeling for the synaptic vesicle protein SYN and for the excitatory synapses postsynaptic scaffold protein PSD-95 served as markers for the presynaptic and postsynaptic compartment, respectively. The incidence and density of immunolabeling for the phosphoprotein co-localized with these compartment markers were then evaluated using wide- field epifluorescence microscopy and fluorescence deconvolution tomography as described elsewhere . Briefly, images within each z-stack were processed for iterative deconvolution and then used to construct a 3-dimensional montage of the sample field. Automated systems were then used to normalize background density, identify immuno labeled elements within the size and eccentricity constraints of synapses, and quantify those double-labeled. Elements were considered double-labeled if there was any overlap in the field labeled with the 2 fluorophores as assessed in 3D.Male Long–Evans rats were handled for 6 sessions, 2 sessions per day, and prior to odor discrimination training. Procedures for animal handling, training, and testing were adapted from Martens et al. as described in detail elsewhere . Sessions of ten 30 s trials on a given odor pair were repeated up to twice daily until rats reached a success rate of 80% correct at which point they were considered to have acquired the odor discrimination task.
On the following day, trained rats were either given 10 training trials on a novel odor pair or transported to but not placed in the test apparatus,cannabis plant growing and killed immediately thereafter for tissue harvest .The CB1R is found on axon terminals throughout the brain including the field of LPP termination in the outer molecular layer of the DG . We confirmed CB1R localization to glutamatergic terminals in the rat LPP field and then compared the effects of cannabinoid receptor agonist WIN 55,212–2 on synaptic physiology in the S–C and LPP projections. In accord with prior work , WIN caused a rapid and pronounced depression of S–C fEPSPs in CA1 that was accompanied by an increase in paired-pulse facilitation and the expected severe impairment of LTP . Very different results were obtained in the LPP: WIN had no effect on baseline fEPSPs or on paired pulse facilitation . Voltage-clamp recordings also detected no effect on EPSCs in the LPP . In contrast to these results for glutamatergic responses in the LPP, WIN produced the canonical depression of IPSCs elicited by single pulse LPP stimulation . We next asked if, despite the lack of effect on baseline responses, WIN influences the machinery that produces the ECBdependent potentiation of the LPP using stimulation that is near threshold for induction. WIN more than doubled the magnitude of lppLTP under these conditions . These results suggest that activation of CB1Rs in the LPP preferentially engages signaling mechanisms leading to potentiated transmission as opposed to the more commonly observed depression of release. CB1R signaling through ERK1/2 effects phosphorylation and degradation of the vesicular protein Munc18-1 leading to reductions in transmitter release . In accord with this, using dual immunofluorescence and FDT , we found that treatment with WIN increased levels of phosphorylated Munc18-1 S241 co-localized with the presynaptic marker SYN in the S–C terminal field: WIN caused both a rightward shift in the pMunc18-1 immuno labeling intensity frequency distribution and increased numbers of terminals with dense pMunc18-1 immuno reactivity . In the same slices, WIN had no effect on presynaptic pMunc18-1 immuno labeling in the LPP terminal field .
The above results indicate that WIN-initiated CB1R signaling at LPP terminals is biased “away from” the ERK1/2-to-Munc18-1 cascade through which ECBs suppress neurotransmitter release and toward a route that promotes plasticity. They also raise the question of whether signaling to Munc18-1 and release suppression in CA1 is engaged by normally occurring patterns of physiological activity. Blocking the CB1R with the inverse agonist AM251 had no effect on S–C fEPSPs elicited by single pulses . Thus, we tested for an effect using short trains of low-frequency gamma stimulation. This pattern occurs routinely in hippocampal and entorhinal fields in behaving animals and is thought to be associated with processing of complex information . Within-slice comparisons were made between responses collected before and after 40 min perfusion of vehicle or 5 μM AM251. In CA1, S–C responses to low gamma stimulation showed the rapid, within-train frequency facilitation described in prior work . AM251 did not affect baseline responses but clearly enhanced S–C response facilitation during the gamma train . Effects of AM251 were greatest in later portions of the train, as anticipated for contributions of “on-demand” ECB production. Very different results were obtained for the LPP. Within-train facilitation was less pronounced in the LPP than in the S–C system, and this effect was not altered by AM251 . These findings are consistent with the hypothesis that CB1R signaling leading to a depression of transmitter release is more readily engaged in the S–C projections than in the LPP.The above results were unexpected because prior studies showed that physostigmine causes a suppression of excitatory transmission in the LPP and other hippocampal pathways that is blocked by AM251 .Therefore, we tested if physostigmine increases hippocampal 2-AG levels, as anticipated, and then used the FDT technique employed above to determine if it also triggers Munc18-1 S241 phosphorylation in the LPP. Infusion of physostigmine elicited a marked increase in slice levels of 2-AG but not other lipids ; it also produced a reliable increase in SYN+ terminals with dense concentrations of pMunc18-1 in both LPP and S–C fields.
As predicted,vertical grow system physostigmine effects on both projections were dramatically reduced in slices prepared from Munc18-1+/− mice relative to those from wild types although the mutation had no effect on the input/output curve or paired-pulse facilitation in the LPP . A recent study showed that the locally synthesized neurosteroid pregnenolone reduces both CB1R signaling through ERK1/2 and neurotransmitter release suppression normally mediated by the ECB receptor . We tested for this effect in hippocampus beginning with the pronounced fEPSP depression produced by CB1R activation in the S–C system: treatment with 10 μM pregnenolone prevented the synaptic response depression elicited by WIN . Pregnenolone was similarly effective in the LPP where it blocked actions of physostigmine on presynaptic pMunc18-1 immuno reactivity and synaptic transmission . These findings point to the conclusion that the pregnenolone/ CB1R/Munc18-1 system, as found in the S–C projections, is present in the LPP although it is not engaged by either the CB1R agonist WIN or repetitive afferent activity. There remains the possibility, however, that it is activated by the short high-frequency gamma trains used to induce lppLTP and participates in subsequent stabilization of the potentiated state of LPP terminals. We conducted multiple tests of this argument. Pregnenolone at the concentration that eliminates physostigmine effects on transmission and pMunc18-1 immuno reactivity in CA1 had no detectable effect on lppLTP induced by near threshold stimulation . Conventional stimulation trains failed to influence Munc18-1 phosphorylation in LPP terminals and induction of lppLTP was fully intact in Munc18-1+/− mice . Considered together with evidence that lppLTP is both 2-AG and CB1R-dependent , the present results suggest that potentiation in the LPP involves a second CB1R signaling pathway that has not been evaluated in work using physiological activation of hippocampal synapses. Finally, the results obtained with pregnenolone afforded a means for testing if increases in 2-AG content produced by physostigmine promote lppLTP in the absence of the response suppression associated with Munc18-1 phosphorylation. We tested this intriguing point and found that physostigmine more than doubled the magnitude of lppLTP induced by threshold level stimulation . This result is consistent with our earlier observation that reducing 2-AG breakdown, and thereby increasing hippocampal slice 2-AG levels, with the monoacylglycerol lipase inhibitor JZL184 similarly augments lppLTP .Prior results showed that lppLTP is blocked by presynaptic actions of latrunculin A , a toxin that selectively blocks the assembly of actin filaments. This raises the possibility that the CB1R promotes lppLTP via actions on actin regulatory signaling, an idea in alignment with evidence that CB1R initiates actin reorganization in dissociated cells and rapidly activates both FAK and the small GTPase RhoA in N18TG2 neuroblastomacells . FAK is a non-receptor tyrosine kinase that mediates integrin effects on the actin cytoskeleton throughout the body .
Other experiments found that CB1R acting through FAK initiates actin remodeling in pancreatic cells resulting in enhanced insulin release . Accordingly, we used FDT to test if WIN activates FAK, via Y397 phosphorylation, in LPP terminals . WIN produced a pronounced rightward skew in the immunofluorescence intensity-frequency distribution for pFAK Y397 co-localized with SYN . RhoA and its downstream effector, ROCK2, represent a primary route whereby FAK signals to actin . In the LPP terminal field, WIN increased levels of pROCK S1366 co-localized with SYN but not with the postsynaptic density marker PSD-95 . In the same hippocampal slices, WIN increased presynaptic pROCK levels in CA1 but this effect was substantially smaller than that in the LPP. We quantified the regional difference by converting the data into cumulative probability curves and then subtracting the WIN treatment values at each density bin for each slice from the mean curve for the vehicle group. The rightward shift in pROCK immuno labeling produced by WIN was over 2-fold greater in the LPP than in CA1 . In all, the CB1R agonist WIN had a much greater effect on Munc18-1phosphorylation in CA1 than in the LPP and a much greater effect on markers of actin signaling in the LPP than in CA1. We conclude from this that the CB1R response to WIN is biased toward different signaling streams in the 2 projections. We further tested if CB1R signaling to ROCK is more prominent in the LPP than in CA1 using physostigmine to elevate 2-AG levels and signaling. Physostigmine produced a reliable increase in presynaptic pROCK in the LPP that was blocked by CB1R antagonism but had no reliable effect on presynaptic pROCK levels in CA1 of the same hippocampal slices . A similar pattern of results was obtained in an analysis of the most densely pROCK immunoreactive terminal boutons . Collectively, these results describe a CB1R-FAK-ROCK route by which 2-AG generated and released during high-frequency stimulation could facilitate presynaptic cytoskeletal changes required for production of stable lppLTP. In accord with this proposal, high-frequency bursts of LPP stimulation caused a rightward shift in the density frequency distribution of presynaptic pFAK in slices harvested 2 min after stimulation.Together, these results describe a second CB1R signaling pathway in LPP terminals that, unlike the ERK1/2-Munc18-1 route, is directly involved in the production of lppLTP.An important question raised by the above results is why activation of FAK and ROCK by pharmacological CB1R stimulation, augmented lppLTP but did not by itself induce potentiation. One possibility is that electophysiological stimulation of the LPP engages elements that are not downstream from CB1R activation but nonetheless are required for shifting LPP terminals into the enhanced release state. We tested if integrin class adhesion proteins, which co-operate with CB1R in actin regulatory signaling in cultured cells , fill this critical role. Integrins are dimeric transmembrane adhesion receptors for extracellular matrix and cell surface proteins that are expressed throughout the brain by neurons and glia . In hippocampus the majority of integrins contain the β1 subunit and β1 integrins have been localized to both pre- and postsynaptic compartments . We previously demonstrated that, in hippocampal slices, infusion of β1 neutralizing antisera disrupts activity-induced actin polymerization and LTP in field CA1 . Here, we tested if similar treatments influence potentiation in the LPP. Treatment with anti-β1 had no effect on baseline LPP responses but caused a near complete suppression of lppLTP . In contrast, neutralizing anti-αV integrin left potentiation intact .