The Journal of Neuroscience, November 1, 1998, 18(21):8863–8874 Silent Synapses in the Developing Rat Visual Cortex: Evidence for Postsynaptic Expression of Synaptic Plasticity Simon Rumpel, Hanns Hatt, and Kurt Gottmann Lehrstuhl fu¨ r Zellphysiologie, Ruhr-Universita¨ t Bochum, D-44870 Bochum, Germany In the developing visual cortex activity-dependent refinement of The selective activation of NMDA receptors at silent syn- synaptic connectivity is thought to involve synaptic plasticity apses could be explained presynaptically by assuming a lower processes analogous to long-term potentiation (LTP). The re- glutamate concentration compared with functional ones. How- cently described conversion of so-called silent synapses to ever, we found no differences in glutamate concentration- functional ones might underlie some forms of LTP.Using whole- dependent properties of NMDA receptor-mediated PSCs, sug- cell recording and minimal stimulation procedures in immature gesting that synaptic glutamate concentration is similar in silent pyramidal neurons, we demonstrate here the existence of func- and functional synapses. Our results thus support a postsyn- tionally silent synapses, i.e., glutamatergic synapses that show aptic mechanism underlying silent synapses, i.e., that they do only NMDA receptor-mediated transmission, in the neonatal rat not contain functional AMPA receptors. Synaptic plasticity at visual cortex. The incidence of silent synapses strongly de- silent synapses might be expressed postsynaptically by mod- creased during early postnatal development. After pairing pre- ification of nonfunctional AMPA receptors or rapid membrane synaptic stimulation with postsynaptic depolarization, silent insertion of AMPA receptors. This conversion of silent synapses synapses were converted to functional ones in an LTP-like to functional ones might play a major role in activity-dependent manner, as indicated by the long-lasting induction of AMPA synaptic refinement during development of the visual cortex. receptor-mediated synaptic transmission. This conversion was Key words: visual cortex; synapse development; synaptic dependent on the activation of NMDA receptors during the plasticity; silent synapses; AMPA receptor; NMDA receptor; pairing protocol. glutamate concentration Activity-dependent refinement of synaptic connectivity during There are two major alternative explanations regarding the the early postnatal development of the visual cortex is thought to molecular mechanisms underlying silent synapses (Kullmann and underlie the emergence of the adult pattern of cortical neuronal Siegelbaum, 1995; Malenka and Nicoll, 1997; Kullmann and Asz- circuits (Goodman and Shatz, 1993; Singer, 1995; Katz and Shatz, tely, 1998). First, silent synapses might be postsynaptically silent; 1996). The cellular and molecular mechanisms involved in the i.e., they might contain either nonfunctional or no AMPA recep- competition of presynaptic axons for their postsynaptic targets tors (Isaac et al., 1995; Liao et al., 1995; Durand et al., 1996). have been proposed to be analogous to synaptic plasticity phe- Second, silent synapses might be presynaptically silent (Kimura et nomena such as long-term potentiation (LTP) and long-term al., 1997); i.e., they might release much less glutamate than depression (Artola and Singer, 1987; Bear et al., 1987, 1992; functional synapses, or they might not release glutamate at all. Tsumoto, 1992; Fox and Daw, 1993; Crair and Malenka, 1995; Because NMDA receptors exhibit a much higher affinity to glu- Kirkwood et al., 1995; Singer 1995; Katz and Shatz, 1996). tamate than do AMPA receptors (Patneau and Mayer, 1990; Recently, a novel mechanism leading to LTP at glutamatergic Hestrin, 1992), NMDA receptors might be activated by low synapses, i.e., the conversion of silent synapses to functional ones, concentrations of glutamate that are not sufficient to elicit a has been described in the hippocampus and in the somatosensory detectable AMPA receptor-mediated PSC. Spillover of glutamate cortex (Isaac et al., 1995, 1997; Liao et al., 1995; Durand et al., from a functional synapse adjacent to a silent synapse releasing 1996). Silent synapses exhibit exclusively NMDA receptor- no glutamate might also provide a low concentration of glutamate mediated synaptic transmission and are functionally silent at the that would activate only NMDA receptors (Kullmann et al., 1996; resting membrane potential because of the strong, voltage- Asztely et al., 1997; Barbour and Ha¨usser, 1997; Kullmann and 21 dependent Mg block of NMDA receptors (McBain and Mayer, Asztely, 1998). 1994). A conversion of silent synapses to functional ones can be In the present study, we demonstrate the existence of silent achieved by pairing presynaptic stimulation with postsynaptic synapses in the neonatal rat visual cortex. We further address depolarization, and the conversion is dependent on the activation whether the glutamate concentration is lower at silent synapses of NMDA receptors (Isaac et al., 1995, 1997; Liao et al., 1995; compared with functional ones by comparing the rise times of Durand et al., 1996). NMDA PSCs, which strongly depend on glutamate concentration (Clements and Westbrook, 1991). Furthermore we studied gluta- Received May 4, 1998; revised Aug. 21, 1998; accepted Aug. 21, 1998. mate concentration-dependent block of NMDA PSCs by the This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 509/ rapidly unbinding NMDA receptor antagonist D-aminoadipate C1. We thank Dr. B. Ache for helpful comments on this manuscript and H. Jung and (D-AA) (Clements et al., 1992; Clements, 1996). We found no H. Bartel for excellent technical assistance. Correspondence should be addressed to Dr. Kurt Gottmann at the above address. difference between silent and functional synapses in these gluta- Copyright © 1998 Society for Neuroscience 0270-6474/98/188863-12$05.00/0 mate concentration-dependent properties of NMDA PSCs. Our 8864 J. Neurosci., November 1, 1998, 18(21):8863–8874 Rumpel et al. • Silent Synapses in the Rat Visual Cortex results do not support the hypothesis that the glutamate concen- tration is lower at silent synapses and are thus consistent with the idea that a postsynaptic mechanism underlies synaptic plasticity at silent synapses. MATERIALS AND METHODS Electrophysiology. Coronal slices (300 mm thick) of the visual cortex from 3- to 11-d-old Wistar rats were cut with a vibratome. Slices were incu- bated in artificial CSF (in mM: 125 NaCl, 3 KCl, 1.25 NaH2PO4 ,25 NaHCO3 ,20D-glucose, 2.5 CaCl2 , and 1.5 MgCl2 , saturated with 95% O2 /5% CO2 , pH 7.3) and allowed to recover for at least 1 hr at room temperature. All recordings were made at 28–30°C in a submerged slice chamber perfused with artificial CSF with added picrotoxin (100 mM). Whole-cell patch-clamp recordings were obtained in layers V/VI of the visual cortex using the blind-patch technique (Blanton et al., 1989). Recording electrodes (7–10 MV) were filled with intracellular solution I (in mM: 135 K-gluconate, 20 KCl, 2 MgCl2 , 10 HEPES, 1 EGTA, 4 Na2-ATP, and 0.5 Na2-GTP, and 0.3% Lucifer yellow, adjusted to pH 7.3 with KOH) in current-clamp experiments. In voltage-clamp experiments intracellular solution II (in mM: 135 CsCl, 20 TEA-Cl, 2 MgCl2 ,10 HEPES, 4 Na2-ATP, 0.5 Na2-GTP, and 1 or 10 EGTA, adjusted to pH 7.3 with CsOH) was used. In experiments designed to investigate syn- aptic plasticity, intracellular solution II contained 1 mM EGTA, other- wise10mM. PSCs were evoked at a stimulation frequency of 0.2 Hz with a glass monopolar electrode placed in the visual cortex in different layers within 100–800 mm of the recording electrode. The vast majority of synaptic responses were evoked by stimulation in layers V/VI. The stimulation frequency was not changed during the experiments. The input resistance (1–0.5 GV) was continuously monitored by delivering a voltage step command, and cells were discarded when it changed by .20%. Recordings were made using a HEKA (Lambrecht/Pfalz, Ger- many) EPC-7 patch-clamp amplifier, filtered at 3 kHz, and sampled at 20 kHz (280 mV holding potential) or 2 kHz (140 mV holding potential). Sampling was done with a TL-1 interface using pClamp 5.5.1 Software (Axon Instruments, Foster City, CA), and data were stored on computer Figure 1. Morphology and action potential discharge pattern of imma- disk. ture pyramidal neurons in layers V/VI. A, Reconstruction of a Lucifer Data analysis. Off-line analysis of PSCs was done using AUTESP yellow-filled pyramidal neuron at P3. Orientation to the pial surface is software (H. Zucker, Max-Planck-Institut fu¨r Psychatrie, Martinsried, indicated by the arrow. At P3 pyramidal neurons typically respond with a Germany). Briefly, response amplitudes at 280 mV holding potential single action potential to current injection. Amplitude of injected current were measured as the difference between the amplitude during a 0.8 msec is indicated at the corresponding membrane voltage record. B, Recon- window including the peak of the PSC and the amplitude during a struction of a Lucifer yellow-filled pyramidal neuron at P10. These more window 2 msec before the stimulus. To avoid contamination of measure- differentiated pyramidal neurons showed a train of action potentials after ments by the stimulus artifact, averaged traces of visually identified current injection. failures were subtracted before analysis. Noise amplitudes were mea- sured as the difference between the amplitude during the window 2 msec similar to that in nonbursting cells (Kasper et al., 1994a). Pyra- before the stimulus and a third window 6 msec before the stimulus. PSC midal neurons in slices obtained from younger animals (P3–P4) amplitudes at 140 mV holding potential were measured analogously, but subtraction of failures was omitted, and the size of the windows used was exhibited a pattern of action potential firing that was even more 8 msec. To estimate the amplitude of the NMDA receptor-mediated immature, typically showing only one action potential after cur- component of the PSC, the window was positioned 30 msec after the rent injection (Fig.