HIPPOCAMPUS 26:560–576 (2016) LTP Enhances Synaptogenesis in the Developing Hippocampus Deborah J. Watson,1 Linnaea Ostroff,2 Guan Cao,1 Patrick H. Parker,1 Heather Smith,1 and Kristen M. Harris1* ABSTRACT: In adult hippocampus, long-term potentiation (LTP) produces synapse enlargement while preventing the formation of new small dendritic spines. Here, we tested how LTP affects structural synaptic plasticity in hippocampal area CA1 of Long-Evans rats at postnatal day 15 (P15). P15 is an age of robust synaptogenesis when less than 35% of dendritic spines have formed. We hypothesized that LTP might therefore have a different effect on synapse structure than in adults. Theta-burst stimulation (TBS) was used to induce LTP at one site and control stimulation was delivered at an independent site, both within s. radiatum of the same hippocampal slice. Slices were rapidly fixed at 5, 30, and 120 min after TBS, and processed for analysis by three-dimensional reconstruction from serial section electron microscopy (3DEM). All findings were compared to hippocampus that was perfusion-fixed (PF) in vivo at P15. Excitatory and inhibitory synapses on dendritic spines and shafts were distinguished from synaptic precursors, including filopodia and surface specializations. The potentiated response plateaued between 5 and 30 min and remained potentiated prior to fixation. TBS resulted in more small spines relative to PF by 30 min. This TBS-related spine increase lasted 120 min, hence, there were substantially more small spines with LTP than in the control or PF conditions. In contrast, control test pulses resulted in spine loss relative to PF by 120 min, but not earlier. The findings provide accurate new measurements of spine and synapse densities and sizes. The added or lost spines had small synapses, took time to form or disappear, and did not result in elevated potentiation or depression at 120 min. Thus, at P15 the spines formed following TBS, or lost with control stimulation, appear to be functionally silent. With TBS, existing synapses were awakened and then new spines C 2015 The formed as potential substrates for subsequent plasticity. V Authors Hippocampus Published by Wiley Periodicals, Inc. KEY WORDS: synapses; ultrastructure; 3DEM; postnatal day 15; thetaburst stimulation This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. 1 Department of Neuroscience, Center for Learning and Memory, Institute for Neuroscience, University of Texas at Austin, Austin, Texas 78731; 2 Allen Brain Institute, 551 N 34th St, Seattle, Washington 98103 Additional Supporting Information may be found in the online version of this article. Grant sponsor: NIH grants; Grant numbers: NS201184, MH095980, and NS074644 (to K.M.H.) and MH096459 (to D.J.W.); Grant sponsor: Texas Emerging Technologies Fund. *Correspondence to: Kristen M. Harris, Department of Neuroscience, Center for Learning and Memory, Institute for Neuroscience, University of Texas at Austin, Austin TX 78731, USA. E-mail: kmh2249@gmail.com D.J.W. and L.O. contributed equally to this work. Accepted for publication 20 September 2015. DOI 10.1002/hipo.22536 Published online 29 September 2015 in Wiley Online Library (wileyonlinelibrary.com). INTRODUCTION Appropriate synaptogenesis and dendritic spine formation during development are necessary for normal cognitive function. Many neurological disorders are characterized by abnormalities that retain juvenile dendritic spines and filopodia (Fiala et al., 2002b; Lin and Koleske, 2010; Penzes et al., 2011; Kuwajima et al., 2013b). Characterizing processes that underlie normal developmental synaptogenesis is vital to understanding the impact of abnormal structure in neurological diseases. Long-term potentiation (LTP) is a cellular mechanism of learning and memory that is readily induced by theta-burst stimulation (TBS), a pattern that mimics naturally occurring neuronal firing patterns (Staubli and Lynch, 1987; Nguyen and Kandel, 1997; Morgan and Teyler, 2001; Buzsaki, 2002). Synaptic scaling is a homeostatic process that enhances or reduces global synaptic strength after periods of weak or strong activity, respectively (Turrigiano, 2008; Turrigiano, 2012). In adult hippocampal area CA1, the enlargement of some synapses following induction of LTP by TBS is balanced by concurrent suppression of the formation of small dendritic spines (Bourne and Harris, 2011; Bell et al., 2014). These results were the first to show that mature dendrites have a maximal capacity for total synaptic input; they support fewer, larger, and more effective synapses, or more, smaller, and less effective synapses. This balancing of total synaptic input has been named structural synaptic scaling, as it suggests interactions between LTP and homeostatic mechanisms at the structural level. In hippocampal area CA1 of Long-Evans rats, P12 is the onset age for LTP induced by TBS that lasts for more than 3 h (Cao and Harris, 2012). Consistent with the first appearance of dendritic spines in this brain region, we and others have hypothesized that spine structure facilitates changes in signaling processes leading to enduring LTP (Fiala et al., 1998; Kirov et al., 2004; Bourne and Harris, 2008; Colgan and Yasuda, 2014). By P15, spine density along oblique dendritic branches has reached only 33% of adult levels, whereas by P21 spine density is 80% of adult levels (Harris et al., 1992; Fiala et al., 2003; Kirov et al., 2004). Thus, P15 is an age of robust synaptogenesis and an important developmental stage to determine whether the structural effects of LTP differ C 2015 THE AUTHORS HIPPOCAMPUS PUBLISHED BY WILEY PERIODICALS, INC. V LTP ENHANCES SYNAPTOGENESIS from adults (Bourne and Harris, 2011). For example, LTP induced in the developing hippocampus might facilitate spine formation (unlike adults) or trigger active elimination of redundant synapses (Oh et al., 2015). Reconstruction in three dimensions through serial section electron microscopy (3DEM) was used to distinguish nonsynaptic filopodia from synaptic dendritic spines, and to measure accurately the spine and synapse densities and dimensions. The results provide new 3DEM data at P15 in vivo, and in acute slices following induction of LTP or control test pulse stimulation. The findings show profound contrasts between P15 and mature hippocampus suggesting a basis for new understanding about how the same treatments could result in opposing effects, depending on age. MATERIALS AND METHODS All animal use procedures were approved by the Institutional Animal Care and Use Committee at the University of Texas at Austin and complied with the NIH requirements for the humane use of laboratory rats. At the time animals were taken for experimentation, they were all of comparable age (P15-16) and had similar features (weights, open eyes, behaviorally responsive, full bellies, and clean bodies, indicating good maternal care). Hippocampal Slice Preparation, Stimulation, and Recording Hippocampal slices (400 lm) were rapidly prepared from P15-16 male Long-Evans rats (n > 100, including the initial test experiments), and 2–3 of the very best experiments at each time point were subsequently chosen for 3DEM analyses (n 5 7, see statistics below). Animals were decapitated, the brain was removed, and the left hippocampus was dissected. Slices were taken from the middle third of the left hippocampus at an angle 708 transverse to the long axis on a tissue chopper (Stoelting, Wood Dale, IL) at room temperature (258C) in oxygenated aCSF containing (in mM) 117 NaCl, 5.3 KCl, 26 NaHCO3, 1 NaH2PO4, 2.5 CaCl2, 1.3 MgSO4, and 10 D-glucose at pH 7.4. Within 5 min, the slices were transferred to a static–pool interface chamber and were recovered for at least 1 h at 33–348C on a supporting net at the interface of humidified 95% O2 and 5% CO2 atmosphere until recordings began. Next, a single glass extracellular recording pipette (filled with 120 mM NaCl) was placed in the middle of s. radiatum in area CA1 between two concentric bipolar stimulating electrodes (100 mm outer diameters, Fredrick Haer, Brunswick, ME). Prior work showed that a total separation of 300–400 mm between the two stimulating electrodes was sufficient to ensure independent activation of subpopulations of synapses (Sorra and Harris, 1998; Ostroff et al., 2002; Bourne and Harris, 2011). Thus, the separation between the two stimulating electrodes was roughly doubled (Fig. 1a). 561 Stable baseline recordings were obtained from both sites for at least 40 min following the initial recovery period. Extracellular field excitatory postsynaptic potentials (fEPSPs) were estimated by linear regression over 400 ms along the maximal initial slope (mV/ms) of the test pulses consisting of 100 ls of constant, biphasic current. The stimulus intensity was set to evoke a 1/2 maximal fEPSP slope, based on a stimulus/response curve for each experiment, and was then held constant for the entire experiment. The stimulation data acquisition protocols were administered using custom designed Igor software (WaveMetrics, Lake Oswego, OR). Then, TBS (8 trains of 10 bursts at 5 Hz of 4 pulses at 100 Hz delivered 30 s apart over a total of 3.5 min, Fig. 1b) was administered to one of the two stimulating electrodes. Following TBS, test pulse stimulations resumed, alternating between the control and LTP sites once every 2 min at a 30 s interval between electrodes. Physiological responses were monitored for 5 min (Fig. 1c), 30 min (Fig. 1d), or 120 min (Fig. 1e) after the first train of TBS and then rapidly fixed as described below. The stimulating electrode assigned to deliver TBS was counter-balanced for side of the recording electrode (towards CA3 or subiculum) within times and across experiments. Tissue Preparation and Processing for 3DEM Within a few seconds of the end of the experiment, electrodes were removed and each slice was transferred still on its net to 5 ml of mixed aldehydes (6% glutaraldehyde, 2% paraformaldehyde in 100 mM cacodylate buffer with 2 mM CaCl2, and 4 mM MgSO4) and microwaved at full power (700W microwave oven) for 10 s to enhance penetration of the fixative through the depth of the slice with a final temperature of 0.20 lm2 (F(2, 53) 5 4.19, P 5 0.020), (e) at 30 min, for PSD areas
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