Development of Schaffer collateral and mossy fiber synapses in slice culture

Synapse development is incomplete at birth. Indeed, structural and functional changes occur in animals such as rodents throughout the life time of the animal. In the present study, I investigated the ultrastructure of mossy fiber and Schaffer collateral synapses at two developmental time points (DIV14 and DIV28) in the mouse hippocampus. My data demonstrate a developmental increase in the spatial density of docked synaptic vesicles at both Schaffer collateral and mossy fiber active zones between DIV14 and DIV28.

Mossy fiber and Schaffer collateral synapses undergo structural and functional maturation between the ages of P3 and P21 (Amaral and Dent, 1981; Battistin and Cherubini, 1994; Buchs et al., 1993; Helassa et al., 2018; Ho et al., 2007; Hussain and Carpenter, 2001; LaVail and Wolf, 1973; Marchal and Mulle, 2004; Mori-Kawakami et al., 2003; Münster-Wandowski et al., 2013; Rose et al., 2013; Schmitz et al., 2003; De Simoni et al., 2003; Wilke et al., 2013).

Structural maturation of mossy fiber boutons in cultured slices closely resembles the time course described in vivo (Amaral and Dent, 1981; Galimberti et al., 2006; Wilke et al., 2013).

Since previous studies have concluded that presynaptic structural maturation of mossy fiber boutons peaks at P14, with only minor changes beyond that, including a pruning of filopodial extensions and an increase in bouton volume (Amaral and Dent, 1981; Galimberti et al., 2006;

Wilke et al., 2013), my observations yield novel insight into mossy fiber synapse development at the level of fine structural organization at the active zone. Synaptogenesis and spine maturation in CA1 pyramidal neurons has been described (Fiala et al., 1998; Harris and Weinberg, 2012; Harris et al., 1992). Schaffer collateral spines undergo structural maturation from P1 to P40 in both the proportion of spine morphologies and presence of specialized organelles such as spine apparatuses (Fiala et al., 1998; Harris and Weinberg, 2012; Harris et al., 1992; De Simoni et al., 2003). By P12 in rats, most excitatory synapses in the stratum lucidum of the CA1 are located at either the dendritic shaft, or the tip of postsynaptic spines (Fiala et al., 1998). Further, from P15 to adult (P70) in the rat hippocampus, spine morphologies change in their relative proportion and density (Harris et al., 1992). Harris and colleagues found a higher proportion of thin spines and, inversely, a lower proportion of stubby spines in the CA1 at P70 compared to P15 in rats (Harris et al., 1992). The

126 heterogeneity of spine morphologies in the CA1 has been postulated to influence structural plasticity during high-activity states, however this remains an open question (Harris and Weinberg, 2012).

The developmental increase in the spatial density of docked synaptic vesicles in Schaffer collateral and mossy fiber synapses was not paralleled by corresponding changes in the efficacy of neurotransmitter release as measured by patch clamp electrophysiology. Neither mean evoked EPSC amplitudes nor paired-pulse ratios changed significantly during this developmental window. This observation was not entirely unexpected, since unlike structural development, the functional changes in both Schaffer collateral and mossy fiber synapses observed in past studies occurs mostly before P14 and after P28 in vivo (Battistin and Cherubini, 1994; Ho et al., 2007; Hussain and Carpenter, 2001; Marchal and Mulle, 2004;

Mori-Kawakami et al., 2003; Rose et al., 2013; Schiess et al., 2010; Schmitz et al., 2003). In acute rat slices, mossy fiber synapses exhibit similar paired-pulse facilitation and post-tetanic potentiation between P10 and P20 (Hussain and Carpenter, 2001). However, between P14 and P21 mossy fiber synapses in acute mouse slices exhibit increased frequency facilitation that is not dependent on postsynaptic receptors (Marchal and Mulle, 2004). Developmental changes in Schaffer collateral synaptic function in acute slices also occur before P14 and after P28 in rodents (Hussain and Carpenter, 2001; Schiess et al., 2010).

Understanding why the observed increase in the spatial density of docked synaptic vesicles does not translate to detectable changes in postsynaptic responses requires the consideration of the complex interplay between multiple factors. These factors include presynaptic calcium channels and buffers, as well as developmental structural and molecular changes in the postsynaptic compartment, and in the abundance and properties of postsynaptic transmitter receptors.

Modifications to presynaptic calcium channels such as the relative proportion of VGCC subtype can shape presynaptic function. Spontaneous and evoked synaptic currents increase during development in dissociated hippocampal neurons (Basarsky et al., 1994). Dissociated cultured neurons had a developmental increase in calcium influx that coincided with an increase of the proportion of P/Q calcium channel expression as the neurons matured (Basarsky et al., 1994). At mossy fiber synapses (at P20-23), the number of P/Q-type calcium

127 channels estimated per active zone is approximately 23 (Vyleta and Jonas, 2014). It is, however, not known how this number is developmentally regulated.

Developmental changes in the spatial distribution of presynaptic calcium channels have been described in the rat cerebellum (Miki et al., 2017). Between P14 and P28, calcium channels decrease in number and form organized clusters that preferentially aggregate at the periphery of the active zone (Miki et al., 2017). Consequently, Miki and colleagues found that the number of docked vesicles correlated with the number of calcium channel clusters indicating fewer vesicles were docked at active zones at P28 (Miki et al., 2017).

Bornschein and colleagues found that there was a developmental decrease in the calcium channel-sensor coupling distance in somatosensory layer-V pyramidal neurons from wild-type mice between the ages of P10 and P24 (Bornschein et al., 2019). Despite tighter coupling, there was no change in the RRP and release probability between the two ages (Bornschein et al., 2019), indicating that developmental changes can occur without affecting the functional output of a given synapse. In mossy fiber synapses, calcium channel-synaptic vesicle coupling distance is around 70 nm, much larger than other synapses with a higher release probability (~20 nm in the Calyx of Held) (Chen et al., 2015). An unanswered question is whether mossy fiber or Schaffer collateral synapses have a developmental change in calcium channel-sensor coupling distance.

The unchanged functional output of both mossy fiber and Schaffer collateral synapses could also be due to an increase in endogenous calcium buffers (Blatow et al., 2003; Luiten et al., 1994). For example, calbindin is a fast-acting endogenous calcium buffer that influences synaptic functional properties in mossy fiber synapses (Blatow et al., 2003; Dumas et al., 2004). In the developing rat hippocampus, calbindin expression steadily increases in granule cells and mossy fiber synapses from P5 to P20 (Luiten et al., 1994).

Presynaptic mitochondria can rapidly sequester presynaptic calcium in synapses to maintain a low release probability (Kwon et al., 2016). Presynaptic mitochondria are more abundant in mossy fiber boutons compared to Schaffer collateral synapses (Amaral and Dent, 1981;

Shepherd and Harris, 1998; Smith et al., 2016). Indeed, as assessed by 2D transmission EM, in the untreated slice cultures at DIV28, nearly every mossy fiber bouton contained at least one mitochondrion whereas approximately one third of Schaffer collateral synapses harbored a

128 presynaptic mitochondrion (see Figure 34 in the appendix). However, developmental changes in the presence of presynaptic mitochondria are poorly understood.

Postsynaptic development includes changes in glutamate receptor subtype expression, and in the localization of receptors in relation to synaptic release sites (Ho et al., 2007; Marchal and Mulle, 2004; Sans et al., 1996). For example, CA3 pyramidal neurons undergo a developmental increase in kanaite receptor expression at the mossy fiber-CA3 postsynaptic compartment that marks the onset of frequency facilitation and potentiation observed at these synapses (Marchal and Mulle, 2004). In another study, expression of calcium-permeable AMPA receptors underlies early post-natal long-term depression exhibited at mossy fiber-CA3 synapses (Ho et al., 2007). In Schaffer collateral synapses, Sans and colleagues found an increase in the expression of proteins associated with the postsynaptic density involved in anchoring NMDA receptors at the synaptic junction (Sans et al., 1996).

They postulated that during development, the accumulation of NMDA-anchoring molecules helps localize NMDA receptors closer to release sites (Sans et al., 1996).

The factors outlined above, perhaps in combination, may occlude the detection of functional changes associated with my observed developmental changes in vesicle docking. As described in other synapses, the structural alteration in presynaptic vesicle organization may be silent, exerting no influence on release probability or on short-term facilitation (Bornschein et al., 2019).

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