CA3 also gives rise to the very large Schaffer–Commissural projection to pyramidal neurons in field CA1, where further integration likely occurs. CA1 directly and through intermediaries innervates the deep layers of entorhinal cortex . It is widely held that through this circuitry the hippocampus converts a sequence of inputs from cortex into representations that include semantic, spatial, and temporal components . Multiple studies suggest that transmission of this code back to cortex is essential for the construction and retrieval of episodic memory, a fundamental ingredient of orderly thought . Consistent with this role, each of the above hippo campal connections undergoes stable potentiation of synaptic trans mission, long-term potentiation , following brief periods of afferent stimulation including activity patterns exhibited during learning. Much has been learned about the synaptic events that induce, express, and rapidly consolidate the potentiated state, particularly for the CA3–CA1, S–C connection: enduring changes occur post synaptically and involve reorganization of the spine cytoskeleton, enlargement of the postsynaptic density, and an increase in membrane neurotransmitter receptors . Appreciation of how hippocampus executes its memory encoding operations will depend on the extent to which other connections in the circuit use this or other forms of plasticity. Recent work suggests that pronounced pathway-specific differences are in fact present. Specifically, the lateral perforant path afferents from lateral entorhinal cortex to the DG exhibit a form of potentiation that depends on postsynaptic induction but is expressed presynaptically as an increase in evoked transmitter release . The retrograde messenger required by this arrangement proved to be the endocannabinoid 2-arachidonoylglycerol which is synthesized in dendritic spines and diffuses to CB1 receptors on axon terminals. The 2-AG system is present at many types of synapses where its direct retrograde signaling has been shown to transiently depress release ; nevertheless,4 x 8 grow tray it serves the very different purpose of promoting potentiation of transmission in the LPP.
Recent studies demonstrated that presynaptic signaling through ERK1/2 to the vesicular protein Munc18-1 is critical for CB1R-mediated depression of release . Here, we show that CB1R agonists readily activate this signaling cascade at S–C synapses but not in the LPP. Instead, at LPP terminals the CB1R/2-AG system is biased toward a second cascade involving β1 integrins and presynaptic actin regulatory signaling. The finding that a specialized form of synaptic potentiation is used to encode semantic information processed by hippo campus calls for substantial revisions to current hypotheses about how the structure contributes to the formation of memory. It also provides a new perspective for understanding the abnormal encoding of episodes produced by cannabinoid drugs.Animals used for extracellular field recordings were 5- to 8- week-old male rats and mice. The mice included Munc18-1 heterozygote knockouts and background strain matched wild types for comparison, and conditional β1-integrin KOs created by crossing mice with floxed β1-integrin exon 3 with mice expressing Cre recombinase under control of the CaMKIIα promoter ; in the progeny the expression of Cre by excitatory hippocampal and cortical neurons leads to excision of β1 exon 3 and disruption of β1 protein expression beginning at 3 weeks of age. The present studies used β1 KOs at 8 weeks of age. The preparation of hippocampal slices and their maintenance in an interface recording chamber has been described in detail elsewhere . Animals were killed by decapitation under deep isofluorane anesthesia and the brain was quickly submerged into oxygenated, ice-cold, high-magnesium artificial cerebral spinal fluid containing : 124 NaCl, 3 KCl, 1.25 KH2PO4, 5 MgSO4, 26 NaHCO3, and 10 dextrose. For rats, slices from the middle third of the hippo campal septo temporal axis were sectioned at a thickness of 330–400 μm using a McIlwain tissue chopper; for mice 375 μm thick sections were prepared on the horizontal plane using a Leica Vibrating Slicer . In both cases, slices were collected into oxygenated, high-magnesium ACSF and then transferred onto an interface recording chamber and continuously perfused with preheated oxygenated ACSF containing : 124 NaCl, 3 KCl, 1.25 KH2PO4, 1.5 MgSO4, 26 NaHCO3, 2.5 CaCl2, and 10 dextrose at a rate of 60–70 ml/h. Experiments were initiated about 1.5 h after slices were placed on the recording chamber.
Field excitatory postsynaptic potentials were recorded by positioning a glass recording electrode and bipolar stimulating electrode in 2 hippocampal pathways . For studies of the LPP innervation of the DG, recording and stimulating electrodes were both positioned in the outer molecular layer , adjacent to the hippocampal fissure. All evoked responses were initially tested with paired pulse stimuli to confirm specificity of potentials and thus electrode placement: LPP responses show paired-pulse facilitation whereas the adjacent medial perforant path shows paired-pulse depression . For studies of S–C innervation of field CA1 stratum radiatum, recording and stimulating electrodes were positioned in CA1b and CA1c, respectively, at comparable distance from the pyramidal cell layer. Test pulses were delivered at 0.05 Hz and baseline stimulation intensity was adjusted to 50–60% of the maximum spike-free fEPSP. Stable baseline recordings were collected for at least 20 min prior to pharmaco logical manipulation or induction of LTP. For the LPP, LPT was induced using 1 of 2 paradigms: two 100 Hz trains, lasting 1 s and separated by 1 min with stimulus duration and intensity increased to 100% and 50% above baseline levels, respectively, and one 100 Hz train, lasting 1 s, no current changes . Stimulus strength was returned to baseline levels after induction. LTP in field CA1 was induced using one 100 Hz trains, lasting 1 s. The level of potentiation was assessed using 0.05 Hz pulses delivered for ≥1 h after inducing stimulation. In all instances, initial slopes and amplitudes were measured from digitized fEPSPs and normalized to mean responses over the last 20 min of the baseline period. Assessments of the level of potentiation were made for the period from 50 to 60 min after delivery of the inducing 100 Hz stimulation. For analysis of pharmacological treatment effects on synaptic responses, statistical tests considered slice mean responses over the last 5 min of the recording period unless otherwise specified.The present studies provide a first detailed description of the mechanisms underlying a novel form of LTP in a primary cortical input to hippocampus. Given that this projection conveys semantic information to hippocampus , the findings are of basic importance to the development of neurobiologically based theories of episodic memory. The substrates for lppLTP include a repurposing of elements utilized in conventional,vertical racking postsynaptic potentiation, as described for the intensively studied S–C projection to CA1, supplemented with novel features.
Both forms of potentiation are induced post synaptically via NMDARs and increased calcium levels , and both require activation of β1 integrins and their downstream effector ROCK . Integrins are a primary membrane regulator of the actincytoskeleton and their prominent role in both types of plasticity suggests that activity-induced structural changes constitute a shared endpoint. Potentiation of S–C synapses in field CA1 entails integrin-driven assembly of stable actin networks in dendritic spines and an associated shift in spine/synapse morphology . Morphometric studies have yet to be performed for lppLTP but results obtained with the toxin latrunculin A, which selectively blocks actin filament assembly, confirm that in this system the cytoskeletal reorganization is located presynaptically . Thus, integrin regulation of the cytoskeleton, via signaling common to cell adhe sion junctions throughout the body, underlies both forms of potentiation but in the case of lppLTP these processes are active on the presynaptic side. This specialized feature of the LPP results in a new form of LTP. Earlier work showed that LTP induction in the LPP requires stimulation of mGluR5 receptors, something that was not the case for field CA1 or the medial perforant path . The mGluR5 receptor forms a postsynaptic signalosome with the scaffolding protein Homer and the 2-AG synthesizing enzyme diacylglycerol lipase-α . Activation of this unit results in de novo 2-AG production and retrograde signaling to presynaptic CB1Rs, 2 events shown to be essential for lppLTP . The present results establish that this dependency is not due to canonical CB1R signaling, which produces depression of transmitter release at sites throughout the brain. Instead, the critical contribution of CB1Rs to lppLTP involves activation of the integrin-associated kinase FAK and its downstream effector ROCK with high-frequency afferent activity. We found that the CB1R inverse agonist AM251 prevented such activation, whereas the CB1R agonist WIN mimicked it and also lowered the threshold for induction of lppLTP. Conversely, the release depression function of the CB1R was poorly developed in the LPP and not involved in the potentiation effect. ECB-mediated release suppression is reportedly sensitive to locally synthe sized pregnenolone and involves phosphory lation of the vesicular protein Munc18-1 . These effects were evident in S–C projections to CA1 but not in the LPP. Specifically, treatment with CB1R agonist WIN increased phosphorylation of Munc18-1 and caused a Munc18-1 dependent, pregnenolone-sensitive depression of synaptic responses in field CA1. In contrast, neither pregnenolone nor reductions in Munc18- 1 expression affected lppLTP. We propose that a shift in the bias of CB1R signaling away from the Munc18-1 pathway and toward facilitation of the ROCK/FAK, integrin signaling cascade constitutes a projection system-specific specialization that enables the thus far singular form of LTP found in the LPP . Ligands for CB1R, including the ECBs, synthetic cannabinoids such as WIN and phytocannabinoids such as Δ9 -tetrahydrocannabinol, all bind different residues on the receptor. This has been suggested to give rise to ligand-specific conformational changes in CB1R leading to activation of different downstream signaling pathways . Moreover, the functional selectivity of a given ligand can be cell-type-specific . In line with these observations, we found cell-type-specific differences in CB1R-mediated responses to a given ligand and differences in CB1R response to different ligands for a given cell-type . The evidence for projection-specific differences in CB1R signaling gives rise to the prediction that modulation of synaptic transmission by ECBs during behaviorally relevant patterns of synaptic activity, a topic that has received surprisingly little attention, will differ between the S–C and LPP systems. Tests confirmed that stimulation in the low-frequency gamma range, selected to simulate the activity in fields CA3 and CA1 during exploration , engages CB1Rs to depress synaptic responses generated by the S–C, but not LPP, projections. Thus, the projection-specific bias in CB1R function is likely to differentially influence throughput across the nodes of the primary hippocampal network. An interesting issue for future research concerns the extent to which the contribution of ECBs to synaptic function is frequency-tuned and differs across hippocampal rhythms associated with various behaviors. Prior work showed that blocking or enhancing the production of 2-AG produces corresponding effects on both the magnitude of lppLTP and the encoding of olfactory cues carried to hippocampus by the LPP system . The present experiments demonstrate that olfactory discrimination learning elicits evidence for potentiation in the form of increased presynaptic pROCK within the LPP. Questions thus arise about the functional significance of using a specialized form of plasticity to encode the semantic information carried by the LPP. One possibility is that the specialization helps to maintain cue identity through downstream hippocampal processing. The novel lppLTP effect changes the frequency facilitation char acteristics of the LPP, as evidenced by paired-pulse measurements, something that would be expected to alter the spiking response of granule cells to patterned input. This would serve to differentiate the DG outputs produced by learned versus unlearned cues and help maintain cue identity through down stream processing. It would also further distinguish the response of granule cells to input arriving over the LPP from those elicited by the subjacent medial perforant path . Related to this, insertion of a 2-AG step in the mechanisms for encoding opens the way for modulation of lppLTP by afferents arising from sites other than entorhinal cor tex. Cholinergic inputs from the medial septum and diagonal bands are of particular interest in this regard because enhancing constitutive transmission in this projection elevates 2-AG levels and related CB1R signaling in terminals. We used this effect to confirm that the Munc18-1 release suppression system is present in the LPP although not engaged by WIN or afferent stimulation. The studies also demonstrated that increasing cholinergic transmission has a strong positive effect on the production lppLTP, particularly when release suppression machinery is blocked with pregnenolone. It is, therefore, possible that particular patterns of firing by septal afferents or levels of pregnenolone synthesis promote the presynaptic LTP in the LPP while depressing the postsynaptic variant found in the medial perforant path.