THE JOURNAL OF COMPARATIVE NEUROLOGY 484:183–190 (2005) Synaptogenesis on Mature Hippocampal Dendrites Occurs via Filopodia and Immature Spines during Blocked Synaptic Transmission LARA J. PETRAK,1 KRISTEN M. HARRIS,2,4 AND SERGEI A. KIROV3,4* 1Department of Biology, Boston University, Boston, Massachusetts 02215 2Department of Neurology, Medical College of Georgia, Augusta, Georgia 30912 3Department of Neurosurgery, Human Brain Laboratory, Medical College of Georgia, Augusta, Georgia 30912 4Synapses and Cognitive Neuroscience Center, Medical College of Georgia, Augusta, Georgia 30912 ABSTRACT During development, dendritic spines emerge as stubby protrusions from single synapses on dendritic shafts or from retracting filopodia, many of which have more than one synapse. These structures are rarely encountered in the mature brain. Recently, confocal and two- photon microscopy have revealed a proliferation of new filopodia-like protrusions in mature hippocampal slices, especially when synaptic transmission was blocked. It was not known whether these protrusions have synapses nor whether they are accompanied by the other immature spine forms. Here, reconstruction from serial section electron microscopy (ssEM) was used to answer these questions. Acute hippocampal slices from mature male rats, ages 56 and 63 days, were maintained in vitro in control medium or in a nominally calcium-free medium with high magnesium, glutamate receptor antagonists, and sodium and calcium channel blockers. At the end of each 8-hour experiment, all slices were fixed, coded, and processed for ssEM. In agreement with light microscopy, there were more filopodia along dendrites in slices with blocked synaptic transmission. These filopodia were identified by their pointy tips and either the absence of synapses or presence of multiple synapses along them. There was also a proliferation of stubby spines. Filopodia along mature dendrites were typically shorter than developmental filopodia, with outgrowth likely being constrained by reduced extracellular space and compact neuropil, providing numerous candidate presynap- tic partners in the vicinity of the mature dendrites. These findings suggest that synaptogen- esis and spine formation are readily initiated under conditions of reduced activity in the mature brain. J. Comp. Neurol. 484:183–190, 2005. © 2005 Wiley-Liss, Inc. Indexing terms: spinogenesis; structural synaptic plasticity; mature CA1 neurons; dendritic spines; serial section electron microscopy Most excitatory synapses in the adult brain occur on den- with estrogen (Woolley et al., 1990; Gould et al., 1990), dritic spines (Gray, 1959; Sorra and Harris, 2000). Changes after exposure to enriched environments (Greenough and in dendritic spines alter synapse function and connectivity, thereby impacting information storage in the brain (Harris and Kater, 1994; Stepanyants et al., 2002). Despite more than a century of studying dendritic spines (Ramo´n y Cajal, Grant sponsor: National Institutes of Health; Grant number: KO1MH02000 (to S.A.K.); Grant number: NS21184; Grant number: 1891), the pivotal question of whether new spines form to NS33574 (to K.M.H.); Grant sponsor: Packard Foundation. support experience-dependent synaptic plasticity, especially Lara J. Petrak’s present address is Department of Cell Biology, Harvard in the mature brain, remains one of the most challenging Medical School, Boston, MA 02115. questions in neuroscience and is crucial for our understand- *Correspondence to: Sergei A. Kirov, Department of Neurosurgery, Med- ical College of Georgia, 1120 15th Street, CB-2607, Augusta, GA 30912. ing of memory (Chang and Greenough, 1982; Desmond and E-mail: [email protected] Levy, 1990; Bailey and Kandel, 1993; Geinisman, 2000; Mul- Received 6 October 2004; Revised 23 November 2004; Accepted 30 No- ler et al., 2002; Harris et al., 2003). vember 2004 Recent findings show that new spines can indeed form DOI 10.1002/cne.20468 on mature neurons. Spines increase on mature neurons Published online in Wiley InterScience (www.interscience.wiley.com). © 2005 WILEY-LISS, INC. 184 L.J. PETRAK ET AL. Bailey, 1988; Bailey and Kandel, 1993), with spatial learn- mM KCl, 26 mM NaHCO3, 1 mM NaH2PO4, 2.5 mM ing (Moser et al., 1994), or with prolonged sensory stimu- CaCl2, 1.3 mM MgSO4, and 10 mM glucose, pH 7.4. Trans- lation (Knott et al., 2002). New spines form on neurons of verse slices were cut from the middle third of the hip- mature mice in the barrel cortex in vivo over a period of pocampus. The block medium had this same solution ex- weeks (Trachtenberg at al., 2002), but are more stable in cept that it had 0 mM Ca2ϩ and8mMMg2ϩ and the primary visual cortex (Grutzendler et al., 2002). New sodium channel blocker tetrodotoxin (TTX, 1 ␮M), the spines emerge from the soma of dentate granule cells in ionotropic glutamate receptors antagonists 6-cyano-7- mature hippocampal slices (Wenzel et al., 1994). More nitroquinoxaline-2,3-dione (CNQX, 20 ␮M), D,L-2-amino- spines also occur along the dendrites of mature CA1 neu- 5-phosphonovaleric acid (APV, 50 ␮M), L-type calcium rons in hippocampal slices during incubation in vitro (Ki- channels blocker nimodipine (5 ␮m), and the metabotropic rov et al., 1999; Johnson and Ouimet, 2004). The new glutamate receptor antagonist (S)-␣-methyl-4-carboxy- spines form rapidly, with more than half of them appear- phenylglycine (MCPG, 500 ␮M). ing during the first 30 minutes in vitro (Kirov et al., Immediately after preparation, slices were transferred 2004a). Blocking synaptic transmission further elevates into the recording chamber (Stoelting, Wood Dale, IL) and this spine number (Kirov and Harris, 1999; Kirov et al., incubated in the control or block medium for a total of 8 2004b). Nevertheless, little is known about the process of hours at an interface of humid atmosphere (95% O2,5% new spine formation and synaptogenesis in the mature CO2) at 32°C. Previously, we have demonstrated a signif- brain. icant increase in the protrusion number on mature den- Dendritic filopodia are the prominent structures during drites during 8 hours of blocking synaptic transmission developmental synaptogenesis in the hippocampus (Dai- (Kirov and Harris, 1999). Here the same time point was ley and Smith, 1996; Fiala et al., 1998; Boyer et al., 1998; chosen to obtain measurements using the more labor- Dunaevsky et al., 1999). During the first few postnatal intensive ssEM approach. After 8 hours the slices were days most of the synapses are located on dendritic shafts immersed in mixed aldehydes (6% glutaraldehyde, 2% or filopodia, although there are also many filopodia with- paraformaldehyde, 1 mM CaCl2, and 2 mM MgCl2, in 0.1 out synapses (Fiala et al., 1998; Boyer et al., 1998). By M cacodylate buffer at pH 7.4) and exposed for 8 seconds postnatal day 15, short stubby spines predominate. These to microwave irradiation for rapid fixation. observations suggest that filopodia may be direct precur- To confirm slice viability field excitatory postsynaptic sors of dendritic spines (Ziv and Smith, 1996; Marrs et al., potentials (fEPSPs) were recorded in the control slices 2001) and also serve to recruit nascent synapses to the within an hour of fixation using the Axopatch 200 ampli- dendritic shaft from which stubby spines emerge (Fiala et fier (Axon Instruments, Foster City, CA). Signals were al., 1998). During the next week, thin and mushroom filtered at 2 kHz, digitized at 10 kHz with Digidata 1200 dendritic spines become prominent and are ultimately the D/A interface board (Axon Instruments), and analyzed dominant forms in adults (Harris et al., 1992; Fiala et al., with pClamp 8 software (Axon Instruments). The slope 1998). function (mV per ms) of the fEPSP was measured from the Confocal microscopy revealed the highest proliferation steepest 400 ␮s segment of the negative field potential of filopodia-like protrusions when synaptic transmission over a series of stimulus intensities, and the half-maximal was blocked on mature CA1 pyramidal cells (Kirov and responses were used to monitor the stable fEPSPs. Slices Harris, 1999). Here, reconstruction from serial section maintained with activity antagonists were also tested to electron microscopy (ssEM) revealed a selective elevation ensure that no synaptic response could be elicited, even at in dendritic protrusions with developmental features, sug- high stimulus intensities. Only experiments in which the gesting a recapitulation of developmental synaptogenesis control slices had a healthy sigmoidal input/output re- during periods of reduced synaptic transmission in the sponse function and a stable response at half maximal mature brain. stimulation were used. Blocking synaptic transmission with activity antago- nists alone also causes an increase in the number of den- MATERIALS AND METHODS dritic protrusions detected by confocal microscopy, al- though to a lesser extent than when extracellular calcium Acute hippocampal slices is controlled (Kirov and Harris, 1999). Due to the labor- Hippocampal slices (400 ␮m) were prepared from ma- intensive nature of analysis by ssEM, only the experimen- ture male rats of the Long-Evans strain, ages 56 and 63 tal treatment containing activity antagonists, high extra- days according to standard protocols (Kirov and Harris, cellular magnesium, and calcium-free medium was 1999). All procedures followed the National Institutes of evaluated because it produced the greatest increase in Health Guidelines for the Care and Use of Laboratory spine density revealed by confocal microscopy (Kirov and Animals and all efforts were made to minimize animal Harris, 1999; Kirov et al., 2004b). suffering and to reduce the number of animals used. An- TTX was acquired from Calbiochem (La Jolla, CA), imals were deeply anesthetized with metofane (methoxy- CNQX and nimodipine from Research Biochemicals flurane) and decapitated. The right hippocampus was (Natick, MA) and MCPG from Tocris Cookson (Bristol, mounted on an agar block in the slicing chamber of a UK). All other drugs and chemicals were from Sigma vibrating-blade microtome (VT1000 S, Leica Instruments, Chemical (St. Louis, MO). MCPG was solubilized at 100ϫ Nussloch, Germany) containing partially frozen oxygen- final concentration in 1.1 eq.