One Unknown

The semblance hypothesis has identified multiple lines of indirect evidence supporting the plausibility of the IPL mechanism (see Evidence page). However, no studies have yet investigated inter-neuronal inter-spine interactions with the specific aim to test for the presence of IPLs. In this context, it is worth examining the perisynaptic region for its ionic and electrical properties. 

When two cellular membranes are separated by distances of approximately 1–30 nm, hydration forces rapidly diminish beyond the first few nanometers, and membrane interactions become increasingly influenced by van der Waals forces, screened electrostatic fields, membrane fluctuations, and the composition of the intervening extracellular matrix (ECM) [1, 2]. Since the ECM contains highly negatively charged molecules such as sulfated glycosaminoglycans and proteoglycans, the resulting polyelectrolyte matrix can bind substantial amounts of Ca²⁺ and other cations, creating a specialized ionic microenvironment [3, 4]. Within such a perisynaptic microdomain, activity-dependent opening of Ca²⁺-permeable channels on adjacent neuronal membranes may lead to rapid influx of extracellular Ca²⁺ into the neurons. As local free Ca²⁺ levels decrease, ECM-bound Ca²⁺ may dissociate from negatively charged sites to replenish the extracellular pool, thereby dynamically altering the balance between bound and free calcium and increasing the density of uncompensated negative charges within the matrix [3, 4]. These changes may modify local ionic conditions, extracellular conductivity, charge screening, and the propagation of extracellular electric fields, causing the microdomain to function as a dynamically regulated electrochemical compartment in which neuronal activity, calcium buffering, and ECM charge state are continuously coupled [4, 5]. Although direct transfer of depolarization between neighboring membranes across such extracellular gaps has not been experimentally demonstrated, electrostatic and ephaptic interactions are expected to depend strongly on membrane separation and the ionic properties of the intervening ECM, with the strongest interactions predicted at the smallest separations [2, 5].

The derived solution - the IPL mechanism, consists of inter-dendritic branch inter-spine interactions between spines, mainly belonging to different neurons. Most likely, it involves a spectrum of mechanisms. They can be tested as follows.

1. Simultaneous dual-nanopipette recording from abutted dendritic spines in animals. In awake, behaviourally responsive non-human primates or rodents, two flexible quartz nanopipettes (tip diameters 15–30 nm, spring constant ∼0.08 N/m) coated with quantum dots for two-photon visualization can be independently positioned under visual guidance onto two abutted dendritic spines [6-8]. A gentle touch-and-buzz electroporation protocol is then applied to each pipette to gain intracellular access to each spine head, enabling simultaneous dual recording from both spines [6, 9, 10]. The prediction is that within a 5–20 ms co-activation window, spontaneous or evoked activity in spine A will produce a subthreshold depolarization in spine B with short, fixed latency. Following recording, distinct fluorescent dyes can be injected through each nanopipette into the respective spine heads and allow to diffuse throughout the parent neuron, after which whole-neuron imaging can be performed to verify if the two spines belong to different neurons. Reproducible transfer significantly above control conditions constitute direct evidence for IPLs; falsification requires its absence across a minimum of 50 confirmed inter-neuronal abutted spine pairs. If recordings suggestive of the presence of IPL are present, extracellular spacing can be transiently increased by focal application of hyperosmotic mannitol-based ACSF through a nearby micropipette under two-photon guidance, while neuronal firing rates and membrane excitability are monitored to verify that any change in transfer probability reflects IPL disruption rather than generalized excitability change. The transfer probability and/or amplitude are expected to markedly decrease during extracellular expansion and recovers following washout.

2. Another possible mechanism is localized electrical field that can form between inter-LINKed spines. Given that extracellular potentials can influence nearby neuronal membranes through ephaptic coupling [11, 12], and that abutted spines at IPL-eligible sites are separated by as little as 10–30 nm of extracellular space [13, 14], it is possible to infer that such fields could permit highly reversible, rapid voltage transfer between spine heads without persistent structural change. Importantly, biophysical feasibility of field-mediated, non-synaptic interactions in dense cortical neuropil [15], provides indirect support for exploring how IPL-like structures that could operate even if it doesn't directly address or prove the specific IPL mechanism. Endogenous fields (the brain's natural extracellular voltage gradients, Ve) that spans scales (synapses → single neurons → networks) are not mere epiphenomena but can feedback to modulate membrane potentials, synaptic currents, spiking timing, and network synchronization [15]. 

3. Experimental data suggest that AMPA receptor subunit-containing vesicles with small diameters fuse with spine membrane due to VAMP2 [16]. In addition to delivering AMPA receptor subunit to the postsynaptic membrane [17], this serves another purpose. The vesicles membranes get incorporated with the lateral spine membrane regions of spines to cause regional spine enlargement. The extent of LTP induction correlates with the ability to learn and can be explained in terms of IPL formation [18].

4. Another testable mechanism involves redox-mediated inter-spine bridging. When spines get abutted more closely (e.g., due to spine expansion by released dopamine [19], redox-active amino acids such as C103 of VAMP2 from two abutted spines become exposed to the aqueous extracellular medium. When synaptic activity induces local oxygen depletion [20], this along with the release of metal ions like Zn²⁺ into the extrasynaptic space [21], can facilitate the formation of metal complexes [22] that bridge the spines and facilitate voltage transfer.

5. Postsynaptic SNARE proteins are essential for activity-dependent membrane remodeling at dendritic spine margins [23], affecting membrane tension, curvature, and lipid organization, and bringing abutted spines closer together. SNARE proteins overcome curvature-induced energy barriers, thereby initiating hemifusion [24]. SNAREs also generate force to tightly appose membranes [25], forming characteristic hemifusion intermediates [26]. SNARE-mediated fusion of AMPA receptor-containing vesicles with the spine membrane [27] potentially contributes membrane material to lateral spine regions. Based on the semblance hypothesis, SNARE proteins play a key role in IPL formation during LTP induction by repurposing fusion machinery to facilitate inter-spine interactions [18]. During heightened synaptic activity, this may allow for transient hemifusion or restricted membrane continuity between abutted postsynaptic membranes, that stops short of full fusion. 

6. While conducting the above experiments, it is essential to examine whether a reversible, distance-dependent inter-spine voltage transfer mechanism can be demonstrated that cannot be adequately explained by known dendritic physiology, recurrent collateral activity, or long-range corticothalamic and thalamocortical connectivity.

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