Maintenance of presynaptic function by AMPA -mediated excitatory postsynaptic activity in adult brain

Sho Kakizawa*†, Taisuke Miyazaki‡, Dai Yanagihara§, Masamitsu Iino†, Masahiko Watanabe‡, and Masanobu Kano*¶

*Department of Cellular Neurophysiology, Graduate School of Medical Science, Kanazawa University, Kanazawa 920-8640, Japan; †Department of Pharmacology, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033, Japan; ‡Department of Anatomy, Hokkaido University School of Medicine, Sapporo 060-8638, Japan; and §Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Tokyo 153-8902, Japan

Edited by Roger A. Nicoll, University of California, San Francisco, CA, and approved November 9, 2005 (received for review May 26, 2005) Activity-dependent synaptic modification occurs in both develop- (AMPA)-type glutamate receptors affects synaptic strength in ing and mature animals. For reliable information transfer and the mature brain. Our results indicate that AMPA receptor- storage, however, once established, synapses must be main- mediated excitatory postsynaptic activity maintains CF’s func- tained stably. We investigated how chronic blockade of neuronal tional release sites and its innervation at PC-shaft dendrites in activity or ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid the mature cerebellum. (AMPA)-type glutamate receptors affects excitatory climbing fiber (CF) to Purkinje cell (PC) synapses in adult mouse cerebellum. Both Results treatments caused reduced glutamate concentration transient at Effects of Chronic Tetrodotoxin (TTX) Treatment. To test whether the synaptic cleft, decreased frequency of quantal excitatory neuronal activity is required for the maintenance of synaptic postsynaptic current, and diminished CF innervation of PC shaft function in the mature brain, we applied TTX locally to the dendrites but no change in CF’s release probability. These results mouse cerebellum by continuous infusion from ethylene-vinyl indicate that, in the mature cerebellum, AMPA receptor-mediated acetate copolymer (Elvax) implants (18). At postnatal day (P)24, excitatory postsynaptic activity maintains CF’s functional gluta- a small piece of Elvax was implanted on the surface of the mate-release sites and its innervation of PC shaft dendrites. cerebellum (lobules 6–8 of the vermis) (15, 16, 19). We esti- mated the duration of the TTX effect from the performance of adult mouse ͉ cerebellar Purkinje cell ͉ climbing fiber ͉ neuronal activity ͉ Elvax-implanted mice on the rotating rod (15). TTX-treated presynaptic function mice, but not vehicle-treated mice, displayed motor discoordi- nation for about 7 days after Elvax implantation, indicating that ynapses undergo activity-dependent changes not only during TTX blocked activity in the cerebellum during this period (see Sdevelopment but also in adulthood (1–6). Various forms of Fig. 5, which is published as supporting information on the PNAS synaptic plasticity, including long-term potentiation and depres- web site). Therefore, in the following experiments, we per- sion, have been found in developing and mature animals and formed electrophysiological analyses 3–7 days after the Elvax have been implicated as cellular bases for learning, memory, and implantation. We prepared parasagittal cerebellar slices from functional neural-circuit formation. Many studies have revealed the vermis and conducted whole-cell recording from visually that neuronal activity is essential for inducing various forms of identified PCs (11, 12, 20, 21). To washout TTX from cerebellar synaptic plasticity, and underlying mechanisms have been stud- tissues, we incubated slices in a reservoir chamber containing the ied intensively (1–6). However, relatively little is known about standard bathing solution for at least 1 h before recording. how the strength of once-established synaptic connection is In the majority of PCs from the TTX- and vehicle-treated maintained stably in the mature brain, crucial for reliable mice, EPSCs were readily elicited in an all-or-none fashion in information transfer and storage. To examine whether neuronal response to CF stimulation (13, 17), indicating that TTX was activity is involved in the maintenance of synaptic strength, it is sufficiently washed out from cerebellar slices at the time of necessary to detect changes in synaptic responses arising from recording. When PCs were sampled in lobules 6–8 (those closest defined inputs after chronic manipulation of neuronal activity. to the Elvax implant), the average amplitude of CF-EPSCs was However, it is difficult to design such experiments, because single significantly smaller in TTX-treated PCs when compared with neurons in the brain generally receive numerous but weak vehicle-treated PCs (Fig. 1 A and B, 77.3 Ϯ 3.7%, n ϭ 16) or synaptic inputs from multiple origins. untreated control PCs (data not shown). However, when PCs The climbing fiber (CF)-to-Purkinje cell (PC) synapse in the were sampled in lobules 1͞2 and 10 (farthest from the implant), cerebellum offers a good model system to assess the possible no significant difference in the EPSC amplitude was noted influence of neuronal activity on synaptic strength. In the adult between the TTX- and vehicle-treated PCs (Fig. 1B). This result cerebellum, single CFs form hundreds of synaptic contacts onto indicates that the effect of TTX was confined to the cerebellar PC’s proximal dendrites and cause strong excitation (7–10). lobules near the implants. In the following analyses, we deal with Although PCs receive the other type of excitatory inputs from numerous parallel fibers (PFs), responses elicited by CFs and those by PFs can be easily distinguished electrophysiologically Conflict of interest statement: No conflicts declared. (11, 12). CF activation elicits large and constant excitatory This paper was submitted directly (Track II) to the PNAS office. postsynaptic currents (EPSCs) with a low coefficient of varia- Abbreviations: AMPA, ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; CF, climb- tion, making precise and reliable electrophysiological examina- ing fiber; Elvax, ethylene-vinyl acetate copolymer; EPSC, excitatory postsynaptic current; tions possible (13, 14). Furthermore, amplitudes and kinetics of CF-EPSC, CF-mediated EPSC; NBQX, 2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzoquinoxa- line-7-sulfonamide; P, postnatal day; PDA, (ϩ)-cic-2,3-piperidine-dicarboxylic acid; PC, CF-mediated EPSC (CF-EPSC) are relatively similar when Purkinje cell; PF, parallel fiber; PPR, paired-pulse ratio; TTX, tetrodotoxin. recorded in cerebellar slices prepared from mice of the same ¶To whom correspondence should be addressed at: Department of Cellular Neurophysiol- strain and age (11, 12, 14–17). We therefore used the CF-to-PC ogy, Graduate School of Medical Science, Kanazawa University, Takara-machi, Kanazawa synapse to examine how chronic blockade of neuronal activity 920-8640, Japan. E-mail: [email protected]. or ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid © 2005 by The National Academy of Sciences of the USA

19180–19185 ͉ PNAS ͉ December 27, 2005 ͉ vol. 102 ͉ no. 52 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0504359103 Downloaded by guest on September 29, 2021 Fig. 1. Reduced glutamate release from CFs caused by chronic TTX application to the cerebellum. (A and B) Reduced CF-EPSC amplitude after chronic TTX treatment. Specimen traces (A) were obtained from PCs in cerebellar lobules 6–8. Summary bar graphs (B) show that TTX was effective in lobules 6–8 (closest to the implants; n ϭ 14 for vehicle, n ϭ 16 for TTX) but not in lobules 1͞2 and 10 (farthest from the implants; n ϭ 5 for vehicle, n ϭ 5 for TTX). (C and D) Reduced glutamate-concentration transients at the CF synaptic cleft after chronic TTX treatment. Specimen traces (C) and summary graphs (D)(n ϭ 6–7 for vehicle, n ϭ 4–7 for TTX) with increasing concentrations of PDA, a rapidly unbinding competitive antagonist of AMPA receptors. (E–G) No change in the amplitude (E and F) but significant reduction in the frequency (E and G) of quantal CF-EPSCs after chronic TTX treatment. Specimen records (E) and cumulative histograms (F and G) were obtained from the same PCs. **, P Ͻ 0.01; ***, P Ͻ 0.001; comparison between the TTX- and vehicle-treated samples by Student t test.

data obtained from PCs in lobules 6–8. Reversal potential, rise reduced glutamate transients in the TTX-treated PCs results time and decay time constant for CF-EPSC, and PC’s membrane from decreased glutamate release from CFs. capacitance were unchanged by the TTX treatment (see Table 1, which is published as supporting information on the PNAS Effects of Chronic 2,3-Dioxo-6-Nitro-1,2,3,4-Tetrahydrobenzoquinoxa- web site). These results demonstrate that transmission efficacy at line-7-Sulfonamide (NBQX) Treatment. To examine whether excita- CF–PC synapse is weakened by a few days’ blockade of neuronal tory postsynaptic activity is required for the maintenance of CF’s activity. presynaptic function, we applied the AMPA- We tested whether the effect of TTX on CF-EPSCs is NBQX locally to the cerebellum by means of Elvax. We esti- reversible. In seven mice, we implanted TTX-containing Elvax mated that the duration of the NBQX effect was Ϸ7 days after and confirmed that TTX was effective by examining the per- Elvax implantation by the performance of Elvax-implanted mice formance of the mice on the rotating rod. Then, we removed on the rotating rod (15) (Fig. 5). By performing in vivo extra- Elvax 4 or 7 days after the implantation and performed elec- cellular recording in the cerebellum of anesthetized mice, we trophysiological examination 10–14 days after the removal of confirmed that NBQX effectively blocked CF-mediated excita- Elvax. Amplitudes and other parameters of CF-EPSCs were tion of putative PCs (see Supporting Text and Fig. 6, which are normal in these mice (data not shown), indicating that the effect published as supporting information on the PNAS web site). We of TTX on CF-EPSCs is reversible. also examined whether NBQX influences glutamate release Reduction of transmission efficacy can result from either from CFs in acute slices. CF-EPSCs were elicited by paired CF stimuli at an interval of 100 ms, and paired-pulse ratio (PPR), decreased transmitter release from presynaptic terminals or which is known to be a good measure of release probability (26), from reduced receptor sensitivity at the postsynaptic mem- was monitored. As detailed in Supporting Text and Fig. 7, which brane. To assess a possibility of presynaptic change, we asked is published as supporting information on the PNAS web site, whether chronic TTX treatment alters glutamate concentra- NBQX did not affect PPR, indicating that NBQX has no effect tion at CF synaptic clefts. We estimated sizes of glutamate on glutamate release from CFs. transients by analyzing nonequilibrium inhibition of postsyn- ϩ We then examined the effects of chronic NBQX treatment and aptic AMPA receptors by ( )-cic-2,3-piperidine-dicarboxylic found that it had essentially the same effects on CF-PC synapses acid (PDA) (22), a rapidly unbinding competitive antagonist of as those exerted by the TTX treatment (Fig. 1). For PCs sampled AMPA receptors. Because PDA is easily displaced by released in cerebellar lobules 6–8 but not in lobules 1͞2 and 10, the glutamate (23), its inhibitory effect depends on the amplitude average CF-EPSC amplitude was significantly smaller in the and time course of glutamate in the synaptic cleft. We found NBQX-treated than in the vehicle-treated mice (Fig. 2 A and B, that PDA antagonized CF-EPSCs of the TTX-treated PCs 52.1 Ϯ 4.3%, n ϭ 13). Basic electrophysiological parameters of more effectively than those of the vehicle-treated PCs (Fig. 1

PCs were not altered by the NBQX treatment (Table 1). PDA NEUROSCIENCE C and D). To determine the locus of this difference, we antagonized CF-EPSCs of the NBQX-treated PCs more effec- ϩ recorded quantal CF-EPSCs in the presence of Sr2 , which tively than those of the vehicle-treated PCs (Fig. 2 C and D). The causes asynchronous transmitter release (24, 25) (Fig. 1E). The mean amplitude of quantal CF-EPSCs and their distribution mean amplitude of quantal CF-EPSCs and their distribution were unchanged (P ϭ 0.4962, Kolmogorov–Smirnov test; Fig. 2 were unchanged by the TTX treatment (P ϭ 0.2111, Kolmog- E and F), whereas the frequency was significantly (P ϭ 0.0011) orov–Smirnov test; Fig. 1 E and F), indicating no change in the lower in the NBQX-treated PCs (Fig. 2 E and G). These results quantum size. In contrast, the frequency of quantal CF-EPSCs clearly indicate that a few days’ blockade of postsynaptic AMPA was significantly (P ϭ 0.0267) lower in the TTX-treated PCs receptors significantly reduces glutamate release from CFs, with (Fig. 1 E and G). These results strongly suggest that the no perceptible changes in postsynaptic glutamate sensitivity.

Kakizawa et al. PNAS ͉ December 27, 2005 ͉ vol. 102 ͉ no. 52 ͉ 19181 Downloaded by guest on September 29, 2021 Fig. 2. Reduced glutamate release from CFs caused by chronic NBQX application to the cerebellum. (A and B) Reduced CF-EPSC amplitude after chronic NBQX treatment. Specimen traces from PCs recorded near the Elvax implants (A) and summary bar graphs for CF-EPSC amplitudes (B) in lobules 6–8 (n ϭ 14 for vehicle, n ϭ 13 for NBQX) and lobules 1͞2 and 10 (n ϭ 5 for vehicle, n ϭ 5 for NBQX). (C and D) Glutamate-concentration transients at synaptic cleft. Sample traces (C) and summary graphs (D)(n ϭ 6–7 for vehicle, n ϭ 7 for NBQX) with increasing concentrations of PDA. (E–G) No change in the amplitude (E and F) but significant reduction in the frequency (E and G) of quantal CF-EPSCs after chronic NBQX treatment. Specimen records (E) and cumulative histograms (F and G) were obtained from the same PCs. **, P Ͻ 0.01; ***, P Ͻ 0.001; comparison between the NBQX- and vehicle-treated samples by Student t test.

To check whether TTX and NBQX have additive effects on indicating that glutamate transients resulting from single synap- CF synapses, we tested Elvax containing both TTX and NBQX. tic vesicles are the same. Furthermore, blocking effects of PDA We found that the effect of TTX plus NBQX (54.0 Ϯ 5.0% of in normal (2 mM) and low (0.5 mM) external Ca2ϩ were almost control, n ϭ 10) was similar to that of NBQX alone (52.1 Ϯ 4.3% the same for the TTX- (Fig. 3C) and the NBQX-treated (Fig. of control; n ϭ 13) (see Fig. 8, which is published as supporting 3D) PCs. Therefore, the difference in glutamate transients in information on the PNAS web site), indicating that NBQX normal external Ca2ϩ is most likely to result from the lower occludes the effect of TTX. This result suggests that TTX and probability of multivesicular release in the TTX- or NBQX- NBQX exert their actions through common mechanisms. treated PCs. Multivesicular release depends critically on both release Chronic TTX or NBQX Treatment Induces Reduction of CF’s Functional probability and the number of functional release sites per Release Sites. It has recently been clarified that multivesicular synapse. To estimate release probability, we examined paired- release occurs at CF–PC synapses at normal extracellular Ca2ϩ pulse plasticity (26). In low extracellular Ca2ϩ (0.25 mM and (14, 27, 28). Decreasing release probability by lowering extra- 0.5 mM), in which CF-EPSCs were not saturated (28), PPR was cellular Ca2ϩ to 0.5 mM or lower results in one-site-one-vesicle similar for the three conditions of PCs (Fig. 3 E and F). This release (27). To elucidate the causes of smaller glutamate result indicates that there is no significant difference in CF’s transients in the TTX- or NBQX-treated PCs, we estimated release probability in the three conditions of PCs. In normal glutamate transients resulting from single synaptic vesicles in a extracellular Ca2ϩ (2 mM), PPRs for the TTX- and NBQX- low external Ca2ϩ (0.5 mM) saline (14, 27, 28). Blocking effects treated PCs were significantly smaller than for the vehicle- of PDA were the same for the TTX-, NBQX-, and vehicle- treated PCs (Fig. 3F). Previous studies show that postsynaptic treated PCs at all PDA concentrations tested (Fig. 3 A and B), receptors are saturated in extracellular Ca2ϩ Ͼ1 mM (28). The

Fig. 3. No changes in quantum size and release probability of CFs. (A and B) Glutamate transients were estimated in low external Ca2ϩ (0.5 mM) saline in which one-site-one-vesicle release occurred from each CF terminal. Specimen traces (A) and summary graphs (B)(n ϭ 7 for each group) with increasing concentrations of PDA. (C and D) Glutamate transients were estimated in the normal (2.0 mM) and low (0.5 mM) external Ca2ϩ saline for the TTX-treated (C)(n ϭ 7) and NBQX ϩ (D)(n ϭ 7) PCs. *, P Ͻ 0.05; comparison between data in the normal (2.0 mM) and low (0.5 mM) external Ca2 saline in the TTX-treated PCs (C) by Student t test. ϩ (E and F) Paired-pulse plasticity tested in 0.25 mM, 0.5 mM, and 2.0 mM external Ca2 (n ϭ 6 for vehicle, n ϭ 5–13 for TTX, and n ϭ 7–14 for NBQX). *, P Ͻ 0.05; **, P Ͻ 0.01; comparison between the TTX- or NBQX-treated PCs and the vehicle-treated PCs by Student t test.

19182 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0504359103 Kakizawa et al. Downloaded by guest on September 29, 2021 Fig. 4. Diminished CF innervation in NBQX-treated mice. (A–D) Wiring of anterogradely labeled CF (red) onto PC dendrites (green) in the vehicle- (A and C) and NBQX- (B and D) treated mice. Black and white arrowheads indicate the distal and proximal edges of CF innervation, respectively. [Scale bars, 20 ␮m(A and B) and 10 ␮m (C).] (E–J) Electron micrographs of the vehicle- (E, G, and I) and NBQX- (F, H, and J) treated mice. Asterisks indicate PC spines contacting PF (G and H)orCF(I and J) terminals. PCD, shaft dendrites of PC. [Scale bars, 1 ␮m(E and G).] (K and L) Quantitative analysis for the number of spines per 100 ␮m of shaft dendrites (Ͼ2 ␮m in caliber) (K) and the density of PF–PC synapses per 100 ␮m2 of the neuropil area (L). Mean Ϯ SD, n ϭ 3, Student’s t test.

postsynaptic-receptor saturation significantly affects PPR (14, and NBQX-treated mice. By anterograde labeling with dextran 28, 29). Thus, the present result suggests that, in normal Texas red, we showed that CFs ascended along calbindin- extracellular Ca2ϩ, the extent of postsynaptic-receptor satu- immunostained PC dendrites to the border between shaft ration at CF–PC synapses is smaller for the TTX- or NBQX- dendrites and spiny branchlets in the vehicle-treated mice (Fig. treated PCs. This finding is consistent with the results that the 4 A and C), as in untreated control mice (31, 32). By contrast, size of glutamate transient in the synaptic cleft for the CF innervation territory was diminished in the NBQX-treated TTX-treated (Fig. 1) or NBQX-treated PCs (Fig. 2) is smaller mice (Fig. 4 B and D). The CFs of NBQX-treated mice often than that for the vehicle-treated PCs. Taken together, these stopped innervation in the midst of shaft dendrites, leaving the results indicate that chronic TTX or NBQX treatment induces remaining distal part free of CF innervation (Fig. 4 B and D). significant reduction of functional release sites without affect- CF innervation was sometimes lost from the basal regions of ing release probability. shaft dendrites (Fig. 4 B and D). The decreased CF innervation was conspicuous at the very regions near the Elvax implanta- No Perceptible Functional Alteration in PF-to-PC Synapses and Inhib- tion (see Fig. 10, which is published as supporting information itory Interneuron-to-PC Synapses. We found no perceptible on the PNAS web site). We found that the diminished inner- changes in blocking effects of PDA on PF-EPSCs or paired-pulse vation by anterogradely labeled CFs did not result from excess plasticity (see Fig. 9, which is published as supporting informa- wiring by other CFs of different origins (i.e., anterogradely tion on the PNAS web site). Furthermore, the amplitude of unlabeled CFs) onto these vacant parts of shaft dendrites (see quantal PF-EPSCs were unchanged (Fig. 9). As for inhibitory Fig. 11, which is published as supporting information on the interneuron-to-PC synapses, we found no perceptible changes in PNAS web site). As for Bergmann glia, immunohistochemical PPR of inhibitory postsynaptic currents evoked by stimulating analysis for glial fibrillary acidic protein and a glial glutamate putative basket͞stellate cell axons (data not shown). These transporter GLAST revealed no perceptible abnormalities results suggest that a few days’ blockade of neuronal activity or (see Fig. 12, which is published as supporting information on AMPA receptors induces no detectable functional alteration in the PNAS web site). PF–PC synapses or inhibitory interneuron-to-PC synapses. At the electron-microscopic level, the surface of shaft den- Diminished CF Innervation Territory After Chronic NBQX Treatment. drites appeared to be smooth (Fig. 4 E and F), with no significant The results in Fig. 3 indicate that a decrease in the number of difference in the spine density along shaft dendrites (Fig. 4K). In functional release sites per synapse underlies the reduced the molecular layer, both mice had numerous synapses (Fig. 4 G frequency of quantal EPSC, smaller glutamate concentration and H), most of which were identified as PF–PC synapses. transient at the synaptic cleft, and decreased amplitude of Occasionally, characteristic CF–PC synapses with large termi- evoked CF-EPSC. However, if the chronic NBQX treatment nals containing densely packed synaptic vesicles were encoun- causes morphological changes and decreases the number of CF tered (Fig. 4 I and J). In NBQX-treated mice, basic morpholog- synapses, it may also lead to the reduced frequency of quantal ical features of PF–PC (Fig. 4H) and CF–PC (Fig. 4J) synapses NEUROSCIENCE CF-EPSC and the decreased amplitude of evoked CF-EPSC. appeared normal, and their synaptic clefts were sealed tightly Previous morphological studies indicate that chronic TTX with lamellate processes of Bergmann glia. Furthermore, no treatment in the adult cerebellum results in retraction of CF significant difference was seen in the density of PF–PC synapses territory and reciprocal expansion of PF territory to proximal (Fig. 4L). All these findings indicate that the NBQX treatment dendrites (9, 30). We thus examined whether chronic NBQX caused rather selective reduction of CF innervation at shaft treatment caused any morphological changes (Fig. 4). At the dendrites. Thus, in addition to changes in presynaptic function of light-microscopic level, arborization of PC dendrites was well CFs, this morphological alteration contributes to the reduced developed (Fig. 4 A–D), and the surface of shaft dendrites frequency of quantal EPSC and decreased amplitude of evoked appeared to be smooth (Fig. 4 C and D) in both the vehicle- CF-EPSC.

Kakizawa et al. PNAS ͉ December 27, 2005 ͉ vol. 102 ͉ no. 52 ͉ 19183 Downloaded by guest on September 29, 2021 Discussion inputs. Whereas TTX blocks both excitatory and inhibitory Chronic TTX or NBQX Treatment Induces Functional Weakening of CF synaptic inputs, NBQX selectively blocks excitatory inputs. If Synapses. It has been demonstrated morphologically that chronic excitatory and inhibitory inputs have opposite effects on the infusion of TTX into the rat cerebellum results in retraction of maintenance of CF synapses, the effect of NBQX would be larger CF innervation territory on PC dendrites (9, 30). We have not than that of TTX. only described electrophysiological changes in CF–PC synapses after chronic TTX treatment but also found two important Possible Mechanisms of AMPA Receptor-Mediated Maintenance of CF results. First, blockade of postsynaptic AMPA receptors by Synapses. We have reported that CF-innervation territory is NBQX caused essentially the same changes as nonspecific significantly diminished to the proximal portion of PC den- ϩ blockade of neuronal activity by TTX, suggesting the existence drites in mutant mice deficient in P͞Q-type Ca2 channel ␣1A of some retrograde signaling mechanism from postsynaptic subunit (31, 32). The diminished CF innervation of ␣1A neuron to presynaptic terminals. Second, chronic TTX or NBQX knockout mice is similar to that observed in NBQX-treated treatment induced not only morphological retraction of CF mice, in which CFs often stop innervation in the midst of shaft innervation but also clear reduction in the number of functional dendrites, leaving the remaining distal part free of CF inner- release sites per active zone at individual CF terminals. Thus, this vation. Although there is no direct evidence, the similar study has made significant advances over the morphological phenotype in these two mouse models suggests that the same ϩ observation reported in refs. 9 and 30). defect in Ca2 -dependent signaling in PCs may underlie the diminished CF innervation. AMPA Receptor-Mediated Neuronal Activity Maintains Mature Prop- The present results, that presynaptic functions are main- erties of CF Synapses. We have shown that immature CFs in tained by postsynaptic AMPA receptor-mediated activity, developing cerebella have lower probability of multivesicular suggest that some transsynaptic retrograde signaling mecha- release (14). We show here that the TTX or NBQX treatment nism must exist to inform presynaptic terminals of postsynaptic in the mature cerebellum induced significant weakening of the activity. One possibility would be that some diffusible retro- CF–PC synaptic strength, with decreased probability of multive- grade messengers are released from PCs and act on CF sicular release, similar to the properties of immature CFs (14). terminals. There is increasing evidence that PCs release These results suggest that AMPA receptor-mediated activity is endocannabinoids and͞or glutamate which act as retrograde required for the maintenance of mature properties of glutamate messengers and modulate excitatory synaptic transmission release from CFs in the adult cerebellum. NBQX treatment not onto PCs (36–39). These molecules may play a role also in the only reduces PC excitability due to blockade of AMPA receptor- maintenance of CF synapses. Cytokines are other candidate mediated excitatory drive but also may reduce PF activity by molecules. We have shown that insulin-like growth factor 1 silencing granule cells. However, CF-EPSC is not attenuated by supports CF–PC synapses in the developing cerebellum during reduced PF activity caused by chronic blockade (from P17–P24) P8–P12. The effects are not observed after P12 (15, 16, 19), of NMDA receptors at mossy-fiber-to-granule-cell synapses and, therefore, insulin-like growth factor 1 itself may not be (15). Furthermore, in several mouse models, including GluR␦2- involved in the maintenance of CF synapses in the mature deficient mice (31, 33), impairment of PF–PC-synapse formation cerebellum. Nevertheless, other cytokines that have similar and resultant reduction of PF drive of PCs causes expansion of effects on CF synapses may play a role in the mature cerebel- CF territory. Therefore, the retraction of CF territory in the lum. Another possibility would be that molecules that bridge NBQX-treated mice is not likely to result from reduced PF pre- and postsynaptic sites may mediate retrograde signals by activity. protein–protein interaction. For example, certain cell- The NBQX treatment may also block Ca2ϩ-permeable AMPA adhesion molecules might be maintained in a manner that receptors on Bergmann glia. Alteration of glial AMPA receptors depends on the postsynaptic Ca2ϩ level. Blockade of AMPA to Ca2ϩ-impermeable ones results in retraction of glial processes receptor-mediated postsynaptic activity and the resultant de- and multiple CF innervation of PCs (34). However, the normal crease in Ca2ϩ influx might degrade the integrity of such morphology and synapse enwrapping of Bergman glia and the molecules, leading to retraction of CF terminals from PC lack of multiple CF innervation in the NBQX-treated mice dendrites. indicate that the changes in CF’s presynaptic function was not Although a number of previous studies indicate that activity- caused by reduced Ca2ϩ influx to Bergman glia through AMPA dependent refinement of synaptic connection is manifest receptors. Therefore, we conclude that the changes in the during postnatal development, such change can occur in NBQX-treated mice resulted mainly from blockade of excitatory mature animals as well. For example, somatotopic maps in the postsynaptic activity in PCs. sensory cortex can change dynamically after partial depriva- tion of sensory inputs or after change in the pattern of sensory Apparent Difference in the Effects of TTX and NBQX. It has been inputs (40–42). NMDA receptor-mediated neuronal activity reported that chronic infusion of TTX to the mature rat cere- appears to be required for reorganization of somatosensory bellum causes significant regression of CF innervation, induces maps in the adult cortex (43). The present findings that AMPA hyperspiny transformation of PC soma and proximal dendrites, receptor-mediated postsynaptic activity maintains presynaptic and permits proximal expansion of PF-synapse territory by functions have disclosed a new aspect of activity-dependent innervating the ectopic spines (9, 30, 35). Although the chronic modification of adult neural circuitry. Further elucidation of NBQX application to the mature mouse cerebellum via Elvax the mechanisms of the NMDA receptor-dependent and caused apparent regression of CF innervation, the application AMPA receptor-dependent mechanisms will deepen our un- had no perceptible effect on the morphology of PF–PC synapses derstanding of how synaptic connections are maintained in the and spine formation at the somatodendritic domain. We assume mature nervous system. that blockade of both pre- and postsynaptic activity by TTX might be required for clear morphological changes in PF–PC Methods synapses and for the hyperspiny transformation. Experiments were conducted according to the guidelines of the The effect of chronic NBQX treatment appears to be stronger Animal Welfare Committee of Kanazawa University. To rule than that of TTX. We assume that the difference may be partly out possible experimenter bias, all experiments have been done because of the possible contribution of inhibitory synaptic blinded to the treatments of the mice.

19184 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0504359103 Kakizawa et al. Downloaded by guest on September 29, 2021 Implantation of Elvax. To chronically block neuronal activity or Morphological Examination. Under deep anesthesia, excitatory postsynaptic activity in the cerebellum, we applied a vehicle- and NBQX-treated mice were perfused transcardially voltage-gated Naϩ channel blocker, TTX, or a selective AMPA- with 4% paraformaldehyde in 0.1 M sodium phosphate buffer receptor antagonist NBQX locally to the mouse cerebellum by (pH 7.2) at 1 week after Elvax implantation (i.e., P31). One week continuous infusion from Elvax implants (18). We implanted before the Elvax implanting (i.e., P17), some mice were injected Elvax to the cerebella of mice (C57BL6͞J) at P24. Preparation with dextran Texas red (3,000 MW, Molecular Probes) for of Elvax pieces and their implantation into mouse cerebella are anterograde labeling of CFs, as described in refs. 31 and 32. detailed in Supporting Text. Then, light and electron microscopic examinations were per- formed as detailed in Supporting Text. Electrophysiology. Mice were killed by cervical dislocation under deep anesthesia. Parasagittal cerebellar slices of This work was supported by Grants-in-Aid for Scientific Research ␮ 17023021 and 17100004 (to M.K.) and 17023001 (to M.W.) and Special 200- m thickness were prepared and incubated as described in Coordination Funds for Promoting Science and Technology from the refs. 11, 12, and 21. Recording, stimulation, and data acquisition Ministry of Education, Culture, Sports, Science and Technology of Japan were performed as detailed in Supporting Text. (to M.K.).

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