Cortical Presynaptic Boutons Progressively Engulf Spinules As They Mature

Abstract

Despite decades of discussion in the neuroanatomical literature, the role of the synaptic spinule in synaptic development and function remains elusive. Canonically, spinules are finger-like projections that emerge from postsynaptic spines and can become enveloped by presynaptic boutons. When a presynaptic bouton encapsulates a spinule in this manner, the membrane apposition between the spinule and surrounding bouton can be significantly larger than the membrane interface at the synaptic active zone. Hence, spinules may represent a mechanism for extrasynaptic neuronal communication, and/or may function as structural anchors that increase the stability of cortical synapses. Yet despite their potential to impact synaptic function, we have little information on the percentages of developing and adult cortical bouton populations that contain spinules, the percentages of these cortical spinule-bearing boutons (SBBs) that contain spinules from distinct neuronal/glial origins, or whether the onset of activity or cortical plasticity are correlated with increased prevalence of cortical SBBs. Here, we employed 2D and 3D electron microscopy to determine the prevalence of spinules in excitatory presynaptic boutons at key developmental time points in the primary visual cortex (V1) of female and male ferrets. We find that the prevalence of SBBs in V1 increases across postnatal development, such that ~25% of excitatory boutons in late adolescent ferret V1 contain spinules. In addition, we find that a majority of spinules within SBBs at later developmental time points emerge from postsynaptic spines and adjacent boutons/axons, suggesting that synaptic spinules may enhance synaptic stability and allow for axo-axonal communication in mature sensory cortex.

Invaginating Structures in Synapses – Perspective

“…Invaginating structures originating from presynaptic terminals in many animals vary from small spinules (Figure 1H) to larger structures and are often filled with presynaptic vesicles (Figures 1D,E). In the mammalian forebrain, some spinules that invaginate into presynaptic terminals originate from adjacent axons or presynaptic terminals, from ∼12% in the CA1 region of the rat hippocampus (Spacek and Harris, 2004) to ∼35% in the visual cortex of the ferret (Campbell et al, 2020). Invaginating structures from adjacent presynaptic terminals that are filled with synaptic vesicles often enter each other; these "pseudopodial indentations" or "PSIs" are described in some vertebrate synaptic terminals and can sometimes form complex intertwinings (Boyne and Mcleod, 1979;Boyne and Tarrant, 1982; see invertebrate examples in Figures 1B, 2).…”

Synaptic spinules are reliable indicators of excitatory presynaptic bouton size and strength and are ubiquitous components of excitatory synapses in CA1 hippocampus

“…In our previous 3D analysis of SBBs within a FIBSEM image volume of late adolescent ferret primary visual cortex, we found that the primary sources of spinules within SBBs were from AdjAx (35.2%), PSs (37.0%), AdjD (14.8%), and AdjS (7.4%) (Campbell et al, 2020). Thus, while SBBs in each brain area may preferentially envelop small percentages of spinules from specific sources based on distinct microcircuit functions, AdjAx and PSs spinules seem to be primary sources for spinules within SBBs across brain areas.…”

“…Perforated PSDs are associated with increases in cortical activity (Calverley and Jones, 1990;Geinisman et al, 1993;Toni et al, 1999), and recent findings have suggested that spinule-bearing spines have higher rates of perforated PSDs (Campbell et al, 2020;Zaccard et al, 2020). Thus, we next sought to examine whether PSs and AdjS spinule-bearing spines (SB spines, n = 92) differed from spines without spinules (non-SB spines, n = 150).…”

“…Our detailed FIB-SEM analysis protocols have previously been published (Campbell et al, 2020;McCoy and Nahmani, 2021). Briefly, we used ImageJ (i.e., FIJI) to scroll through the image volume at 5 nm z-resolution in order to locate every excitatory synapse and spinule within excitatory boutons in the volume (Schindelin et al, 2012).…”

“…These enveloped protrusions are evolutionarily conserved features across animal phyla, from invertebrate cnidarians (Westfall, 1970;Case et al, 1972) to humans (Zhang and Houser, 1999;Dall'Oglio et al, 2015). Indeed, recent data suggests they are ubiquitous components of mammalian excitatory cortical synapses (Campbell et al, 2020). Yet, synaptic spinule function(s) remain obscure due to a lack of investigations into their prevalence, morphological characteristics, and impact on synaptic function.…”