Michael A. Chirillo

Dynamics of nascent and active zone ultrastructure as synapses enlarge during long-term potentiation in mature hippocampus.

Nascent zones and active zones are adjacent synaptic regions that share a postsynaptic density, but nascent zones lack the presynaptic vesicles found at active zones. Here dendritic spine synapses were reconstructed through serial section electron microscopy (3DEM) and EM tomography to investigate nascent zone dynamics during long‐term potentiation (LTP) in mature rat hippocampus. LTP was induced with theta‐burst stimulation, and comparisons were made with control stimulation in the same hippocampal slices at 5 minutes, 30 minutes, and 2 hours post‐induction and to perfusion‐fixed hippocampus in vivo. Nascent zones were present at the edges of ∼35% of synapses in perfusion‐fixed hippocampus and as many as ∼50% of synapses in some hippocampal slice conditions. By 5 minutes, small dense‐core vesicles known to transport active zone proteins moved into more presynaptic boutons. By 30 minutes, nascent zone area decreased, without significant change in synapse area, suggesting that presynaptic vesicles were recruited to preexisting nascent zones. By 2 hours, both nascent and active zones were enlarged. Immunogold labeling revealed glutamate receptors in nascent zones; however, average distances from nascent zones to docked presynaptic vesicles ranged from 170 ± 5 nm in perfusion‐fixed hippocampus to 251 ± 4 nm at enlarged synapses by 2 hours during LTP. Prior stochastic modeling suggests that decrease in glutamate concentration reduces the probability of glutamate receptor activation from 0.4 at the center of release to 0.1 just 200 nm away. Thus, conversion of nascent zones to functional active zones likely requires the recruitment of presynaptic vesicles during LTP. J. Comp. Neurol. 522:3861–3884, 2014.

Presynaptic ultrastructural plasticity along CA3→CA1 axons during long-term potentiation in mature hippocampus.

In area CA1 of the mature hippocampus, synaptogenesis occurs within 30 minutes after the induction of long‐term potentiation (LTP); however, by 2 hours many small dendritic spines are lost, and those remaining have larger synapses. Little is known, however, about associated changes in presynaptic vesicles and axonal boutons. Axons in CA1 stratum radiatum were evaluated with 3D reconstructions from serial section electron microscopy at 30 minutes and 2 hours after induction of LTP by theta‐burst stimulation (TBS). The frequency of axonal boutons with a single postsynaptic partner was decreased by 33% at 2 hours, corresponding perfectly to the 33% loss specifically of small dendritic spines (head diameters <0.45 μm). Docked vesicles were reduced at 30 minutes and then returned to control levels by 2 hours following induction of LTP. By 2 hours there were fewer small synaptic vesicles overall in the presynaptic vesicle pool. Clathrin‐mediated endocytosis was used as a marker of local activity, and axonal boutons containing clathrin‐coated pits showed a more pronounced decrease in presynaptic vesicles at both 30 minutes and 2 hours after induction of LTP relative to control values. Putative transport packets, identified as a cluster of less than 10 axonal vesicles occurring between synaptic boutons, were stable at 30 minutes but markedly reduced by 2 hours after the induction of LTP. APV blocked these effects, suggesting that the loss of axonal boutons and presynaptic vesicles was dependent on N‐methyl‐D‐aspartic acid (NMDA) receptor activation during LTP. These findings show that specific presynaptic ultrastructural changes complement postsynaptic ultrastructural plasticity during LTP. J. Comp. Neurol. 521:3898–3912, 2013.

Hippocampal Spine Head Sizes are Highly Precise

Hippocampal synaptic activity is probabilistic and because synaptic plasticity depends on its history, the amount of information that can be stored at a synapse is limited. The strong correlation between the size and efficacy of a synapse allowed us to estimate the precision of synaptic plasticity. In an electron microscopic reconstruction of hippocampal neuropil we found single axons making two or more synaptic contacts onto the same dendrites which would have shared histories of presynaptic and postsynaptic activity. The postsynaptic spine heads, but not the spine necks, of these pairs were nearly identical in size. The precision is much greater than previous estimates and requires postsynaptic averaging over a time window many seconds to minutes in duration depending on the rate of input spikes and probability of release. One Sentence Summary Spine heads on the same dendrite that receive input from the same axon are the same size.

Mitochondrial support of persistent presynaptic vesicle mobilization with age-dependent synaptic growth after LTP.

Mitochondria support synaptic transmission through production of ATP, sequestration of calcium, synthesis of glutamate, and other vital functions. Surprisingly, less than 50% of hippocampal CA1 presynaptic boutons contain mitochondria, raising the question of whether synapses without mitochondria can sustain changes in efficacy. To address this question, we analyzed synapses from postnatal day 15 (P15) and adult rat hippocampus that had undergone theta-burst stimulation to produce long-term potentiation (TBS-LTP) and compared them to control or no stimulation. At 30 and 120 min after TBS-LTP, vesicles were decreased only in presynaptic boutons that contained mitochondria at P15, and vesicle decrement was greatest in adult boutons containing mitochondria. Presynaptic mitochondrial cristae were widened, suggesting a sustained energy demand. Thus, mitochondrial proximity reflected enhanced vesicle mobilization well after potentiation reached asymptote, in parallel with the apparently silent addition of new dendritic spines at P15 or the silent enlargement of synapses in adults. DOI: http://dx.doi.org/10.7554/eLife.15275.001

Presynaptic ultrastructural plasticity along CA3→CA1 axons during LTP in Mature Hippocampus

In area CA1 of the mature hippocampus, synaptogenesis occurs within 30 minutes after the induction of long‐term potentiation (LTP); however, by 2 hours many small dendritic spines are lost, and those remaining have larger synapses. Little is known, however, about associated changes in presynaptic vesicles and axonal boutons. Axons in CA1 stratum radiatum were evaluated with 3D reconstructions from serial section electron microscopy at 30 minutes and 2 hours after induction of LTP by theta‐burst stimulation (TBS). The frequency of axonal boutons with a single postsynaptic partner was decreased by 33% at 2 hours, corresponding perfectly to the 33% loss specifically of small dendritic spines (head diameters <0.45 μm). Docked vesicles were reduced at 30 minutes and then returned to control levels by 2 hours following induction of LTP. By 2 hours there were fewer small synaptic vesicles overall in the presynaptic vesicle pool. Clathrin‐mediated endocytosis was used as a marker of local activity, and axonal boutons containing clathrin‐coated pits showed a more pronounced decrease in presynaptic vesicles at both 30 minutes and 2 hours after induction of LTP relative to control values. Putative transport packets, identified as a cluster of less than 10 axonal vesicles occurring between synaptic boutons, were stable at 30 minutes but markedly reduced by 2 hours after the induction of LTP. APV blocked these effects, suggesting that the loss of axonal boutons and presynaptic vesicles was dependent on N‐methyl‐D‐aspartic acid (NMDA) receptor activation during LTP. These findings show that specific presynaptic ultrastructural changes complement postsynaptic ultrastructural plasticity during LTP. J. Comp. Neurol. 521:3898–3912, 2013.

Complexity of dendritic SER increases at enlarging synapses during LTP

Smooth endoplasmic reticulum (SER) forms a membranous network that extends throughout neurons. SER regulates intracellular calcium and the posttranslational modification and trafficking of membrane and proteins. As the structure of dendritic SER shifts from a tubular to a more complex, branched form, the movement of membrane cargo slows and delivery to nearby spines increases. Here we discovered changes in the structural complexity of SER that have important functional implications during long-term potentiation (LTP) in adult rat hippocampus. By 2 hours after the induction of LTP with theta-burst stimulation, synapse enlargement was greatest on spines that contained SER. More spines had an elaborate spine apparatus than a simple tubule of SER. The SER in dendritic shafts became more complex beneath spines with both polyribosomes and SER, and less complex along aspiny dendritic regions. The findings suggest that local changes in dendritic SER support enhanced growth of specific synapses during LTP.

DYNAMICS OF NASCENT AND ACTIVE ZONE ULTRASTRUCTURE AS SYNAPSES ENLARGE DURING LTP IN MATURE HIPPOCAMPUS

Nascent zones and active zones are adjacent synaptic regions that share a postsynaptic density, but nascent zones lack the presynaptic vesicles found at active zones. Here dendritic spine synapses were reconstructed through serial section electron microscopy (3DEM) and EM tomography to investigate nascent zone dynamics during long‐term potentiation (LTP) in mature rat hippocampus. LTP was induced with theta‐burst stimulation, and comparisons were made with control stimulation in the same hippocampal slices at 5 minutes, 30 minutes, and 2 hours post‐induction and to perfusion‐fixed hippocampus in vivo. Nascent zones were present at the edges of ∼35% of synapses in perfusion‐fixed hippocampus and as many as ∼50% of synapses in some hippocampal slice conditions. By 5 minutes, small dense‐core vesicles known to transport active zone proteins moved into more presynaptic boutons. By 30 minutes, nascent zone area decreased, without significant change in synapse area, suggesting that presynaptic vesicles were recruited to preexisting nascent zones. By 2 hours, both nascent and active zones were enlarged. Immunogold labeling revealed glutamate receptors in nascent zones; however, average distances from nascent zones to docked presynaptic vesicles ranged from 170 ± 5 nm in perfusion‐fixed hippocampus to 251 ± 4 nm at enlarged synapses by 2 hours during LTP. Prior stochastic modeling suggests that decrease in glutamate concentration reduces the probability of glutamate receptor activation from 0.4 at the center of release to 0.1 just 200 nm away. Thus, conversion of nascent zones to functional active zones likely requires the recruitment of presynaptic vesicles during LTP. J. Comp. Neurol. 522:3861–3884, 2014.

Learning (by) osmosis: an approach to teaching osmolarity and tonicity

Understanding osmolarity and tonicity is one of the more challenging endeavors undertaken by students of the natural sciences. We asked students who completed a course in animal physiology to submit an essay explaining what they found most perplexing about this subject, and what in-class activities proved most useful to them. Students had difficulty distinguishing osmolarity from tonicity and determining tonicity based on the solutions composition. The most useful activities were questions requiring simultaneous consideration of both osmolarity and tonicity. Problems that require calculating osmotic concentration and the volumes of body fluid compartments after administration or loss of various solutions emphasize the significance of osmolarity and tonicity in the context of systemic homeostasis and clinical medicine. We hope that our approach to teaching osmolarity and tonicity will prove useful to physiology lecturers who are looking for new ways of introducing this complicated topic to their health professions students.

Dynamics of nascent and active zone ultrastructure as synapses enlarge during long-term potentiation in mature hippocampus: Nascent and Active Zone Structural Dynamics

Nascent zones and active zones are adjacent synaptic regions that share a postsynaptic density, but nascent zones lack the presynaptic vesicles found at active zones. Here dendritic spine synapses were reconstructed through serial section electron microscopy (3DEM) and EM tomography to investigate nascent zone dynamics during long‐term potentiation (LTP) in mature rat hippocampus. LTP was induced with theta‐burst stimulation, and comparisons were made with control stimulation in the same hippocampal slices at 5 minutes, 30 minutes, and 2 hours post‐induction and to perfusion‐fixed hippocampus in vivo. Nascent zones were present at the edges of ∼35% of synapses in perfusion‐fixed hippocampus and as many as ∼50% of synapses in some hippocampal slice conditions. By 5 minutes, small dense‐core vesicles known to transport active zone proteins moved into more presynaptic boutons. By 30 minutes, nascent zone area decreased, without significant change in synapse area, suggesting that presynaptic vesicles were recruited to preexisting nascent zones. By 2 hours, both nascent and active zones were enlarged. Immunogold labeling revealed glutamate receptors in nascent zones; however, average distances from nascent zones to docked presynaptic vesicles ranged from 170 ± 5 nm in perfusion‐fixed hippocampus to 251 ± 4 nm at enlarged synapses by 2 hours during LTP. Prior stochastic modeling suggests that decrease in glutamate concentration reduces the probability of glutamate receptor activation from 0.4 at the center of release to 0.1 just 200 nm away. Thus, conversion of nascent zones to functional active zones likely requires the recruitment of presynaptic vesicles during LTP. J. Comp. Neurol. 522:3861–3884, 2014.

“Presynaptic ultrastructural plasticity along CA3→CA1 axons during LTP in mature hippocampus”: Presynaptic Ultrastructural Plasticity During LTP

In area CA1 of the mature hippocampus, synaptogenesis occurs within 30 minutes after the induction of long‐term potentiation (LTP); however, by 2 hours many small dendritic spines are lost, and those remaining have larger synapses. Little is known, however, about associated changes in presynaptic vesicles and axonal boutons. Axons in CA1 stratum radiatum were evaluated with 3D reconstructions from serial section electron microscopy at 30 minutes and 2 hours after induction of LTP by theta‐burst stimulation (TBS). The frequency of axonal boutons with a single postsynaptic partner was decreased by 33% at 2 hours, corresponding perfectly to the 33% loss specifically of small dendritic spines (head diameters <0.45 μm). Docked vesicles were reduced at 30 minutes and then returned to control levels by 2 hours following induction of LTP. By 2 hours there were fewer small synaptic vesicles overall in the presynaptic vesicle pool. Clathrin‐mediated endocytosis was used as a marker of local activity, and axonal boutons containing clathrin‐coated pits showed a more pronounced decrease in presynaptic vesicles at both 30 minutes and 2 hours after induction of LTP relative to control values. Putative transport packets, identified as a cluster of less than 10 axonal vesicles occurring between synaptic boutons, were stable at 30 minutes but markedly reduced by 2 hours after the induction of LTP. APV blocked these effects, suggesting that the loss of axonal boutons and presynaptic vesicles was dependent on N‐methyl‐D‐aspartic acid (NMDA) receptor activation during LTP. These findings show that specific presynaptic ultrastructural changes complement postsynaptic ultrastructural plasticity during LTP. J. Comp. Neurol. 521:3898–3912, 2013.