Bidirectional Influence of Sodium Channel Activation on NMDA

Bidirectional inﬂuence of sodium channel activation on NMDA receptor–dependent cerebrocortical neuron structural plasticity

Joju Georgea, Daniel G. Badenb, William H. Gerwickc, and Thomas F. Murraya,1

aDepartment of Pharmacology, Creighton University School of Medicine, Omaha, NE 68178; bCenter for Marine Science, University of North Carolina, Wilmington, NC 28409; and cCenter for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California at San Diego, La Jolla, CA 92093

Edited by William A. Catterall, University of Washington School of Medicine, Seattle, WA, and approved October 16, 2012 (received for review July 31, 2012) Neuronal activity regulates brain development and synaptic plastic- NMDA receptors (NMDARs) and downstream signaling path- ity through N-methyl-D-aspartate receptors (NMDARs) and calcium- ways (9, 10). Previous studies have indicated that changes in in- + + dependent signaling pathways. Intracellular sodium ([Na ]i)also tracellular sodium concentration ([Na ]i) produced in soma and + exerts a regulatory influence on NMDAR channel activity, and [Na ]i dendrites as a result of neuronal activity may play a role in ac- may, therefore, function as a signaling molecule. In an attempt to tivity-dependent synaptic plasticity. Synaptic stimulation causes fl + mimic the in uence of neuronal activity on synaptic plasticity, we [Na ]i increments of 10 mM in dendrites and of up to 35–40 mM used brevetoxin-2 (PbTx-2), a voltage-gated sodium channel (VGSC) in dendritic spines (11). In hippocampal neurons, intracellular fi + + gating modi er, to manipulate [Na ]i in cerebrocortical neurons. [Na ] increments greater than 5–10 mM have been demonstrated The acute application of PbTx-2 produced concentration-dependent to increase NMDAR-mediated whole-cell currents and single- + 2+ increments in both intracellular [Na ] and [Ca ]. Pharmacological channel activity by increasing both channel open probability and 2+ evaluation showed that PbTx-2–induced Ca influx primarily in- mean open time (12). volved VGSC activation and NMDAR-mediated entry. Additionally, + In the present study, we used brevetoxin-2 (PbTx-2), a voltage- PbTx-2 robustly potentiated NMDA-induced Ca2 influx. PbTx-2–ex- + gated sodium channel (VGSC) gating modifier, to manipulate [Na ]i posed neurons showed enhanced neurite outgrowth, increased in immature cerebrocortical neurons in an effort to mimic neuronal fi dendritic arbor complexity, and increased dendritic lopodia den- activity. Brevetoxins interact with neurotoxin site 5 on the α subunit of fl sity. The appearance of spontaneous calcium oscillations, re ecting VGSCs (13, 14) and augment sodium influx by shifting the activation synchronous neuronal activity, was accelerated by PbTx-2 treat- potential to more negative values and inhibiting channel inactivation ment. Parallel to this response, PbTx-2 increased cerebrocortical neu- (15). We have shown previously that in cerebrocortical neurons, ron synaptic density. PbTx-2 stimulation of neurite outgrowth, den- + PbTx-2 elevates [Na ]i and augments NMDAR whole-cell currents dritic arborization, and synaptogenesis all exhibited bidirectional by increasing both channel open probability and mean open time concentration–response profiles. This profile paralleled that of – without affecting the resting membrane potential (16). PbTx-2 NMDA, which also produced bidirectional concentration response treatment affects neurite outgrowth in day in vitro (DIV)-1 cere- profiles for neurite outgrowth and synaptogenesis. These data are brocortical neurons in a bidirectional pattern (16). We hypothesized consistent with the hypothesis that PbTx-2–enhanced neuronal plas- that the effects of PbTx-2 on structural plasticity in cerebrocortical ticity involves NMDAR-dependent signaling. Our results demon- neurons may be mediated by NMDAR-dependent mechanisms strate that PbTx-2 mimics activity-dependent neuronal structural + + subsequent to an increase in [Na ]i. Here, we show that acute plasticity in cerebrocortical neurons through an increase in [Na ]i, + 2+ + application of PbTx-2 causes Na and Ca influx, with the [Na ]i up-regulation of NMDAR function, and engagement of downstream fi Ca2+-dependent signaling pathways. These data suggest that VGSC increment being suf cient to up-regulate NMDAR function. fi Furthermore, we demonstrate that PbTx-2 treatment enhances gating modi ers may represent a pharmacologic strategy to regu- fi late neuronal plasticity through NMDAR-dependent mechanisms. dendritic arborization, lopodia formation, and synaptogenesis. We also demonstrate that PbTx-2 exposure engages downstream 2+ neuronal connectivity | ion flux activity-dependent mechanisms involving Ca -calmodulin kina- ses (CaMKs), ERK, cAMP response element binding protein (CREB), and BDNF signaling pathways. Taken together, our evelopment of the nervous system involves both a pre- results indicate that PbTx-2 mimics activity-dependent synaptic Ddetermined genetic program that controls the general or- plasticity by potentiating NMDAR function with downstream + ganization of the brain and activity-dependent mechanisms that engagement of Ca2 -dependent signaling mechanisms. modulate several developmental processes, including dendritic elaboration, spine formation, synapse formation, and synapse Results elimination (1). Neuronal activity is one of the essential extra- fi fl PbTx-2, a Sodium Channel Gating Modi er, Increases Intracellular cellular signals that in uences dendritic arborization (2). During Sodium and Calcium Influx in DIV-1 Cerebrocortical Neurons. To development, dendrites are first decorated by filopodia (3) that + determine the extent to which PbTx-2 can elevate [Na ]i in im- are considered to be precursors of spines (4, 5). During initial mature DIV-1 cerebrocortical neurons, we imaged individual development, synapses emerge at contacts between axons and dendritic filopodia (6, 7) that may mature into excitatory spine

synapses in later development. Previous studies have demon- Author contributions: J.G. and T.F.M. designed research; J.G. performed research; D.G.B. strated that neuronal activity and activity-dependent gene tran- and W.H.G. contributed new reagents/analytic tools; J.G. and T.F.M. analyzed data; and scription have profound effects on synaptogenesis (8). J.G. and T.F.M. wrote the paper. The N-methyl-D-aspartate (NMDA) type of glutamate receptor The authors declare no conﬂict of interest. mediates many of the effects of neuronal activity on structural This article is a PNAS Direct Submission. plasticity. The development and structural remodeling of den- 1To whom correspondence should be addressed. E-mail: [email protected]. drites and spines depend on actin cytoskeletal reorganization, and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cytoskeletal dynamics are regulated by calcium inﬂux through 1073/pnas.1212584109/-/DCSupplemental.

19840–19845 | PNAS | November 27, 2012 | vol. 109 | no. 48 www.pnas.org/cgi/doi/10.1073/pnas.1212584109 Downloaded by guest on October 4, 2021 + (Fig. 1C). To delineate the Ca2 -influx pathways triggered by 2+ PbTx-2, a pharmacological evaluation of the [Ca ]i response to 100 nM PbTx-2 was performed. Neurons were pretreated with the antagonists TTX (VGSC), amino-5-phosphonopentanoic acid (D-APV) (NMDAR), or nifedipine (L-type voltage-gated calcium channel) before PbTx-2 challenge (Fig. 1D). TTX (1 μM) completely blocked the response to PbTx-2, whereas D-APV (100 μM), but not nifedipine (1 μM), significantly reduced PbTx-2–in- + duced Ca2 influx. A pharmacological evaluation of PbTx-2 in- + duced Ca2 influx at the population level using neurons loaded with Fluo3-AM yielded similar results (Fig. S3). These experi- + ments indicate that PbTx-2–induced Ca2 influx requires VGSC activation and primary cell entry through the calcium-permeable NMDAR. Given the ability of PbTx-2 exposure to augment NMDAR channel function (12, 16), we next evaluated the ability of + PbTx-2 treatment to affect NMDA-induced Ca2 influx at the single-cell level in DIV-1 neurons. NMDA alone produced a con- 2+ centration-dependent elevation of [Ca ]i (Fig. 1E) with an EC50 of 444.1 nM (95% CI, 24 nM to 8.2 μM). In the presence of a concentration of PbTx-2 (30 nM) that alone produced a modest 2+ 2+ transient increase in [Ca ]i, the NMDA-induced Ca influx was robustly potentiated. The NMDA concentration–response curve displayed a leftward shift [NMDA plus PbTx-2: EC50, 12 nM (95% Fig. 1. PbTx-2, a sodium channel gating modifier, increases intracellular CI, 4.7–30.2 nM)] and an increased maximum response (Emax)in sodium and calcium levels in DIV-1 cerebrocortical neurons. (A) PbTx-2 = – + fl the presence of PbTx-2 [NMDA: Emax 0.013 (95% CI, 0.011 concentration-dependent elevation of [Na ]i.SBFI uorescence data are = – expressed as maximum changes in (340/380) ratio divided by the baseline 0.016); NMDA plus PbTx-2: Emax 0.025 (95% CI, 0.022 0.028)]. + fi fi ratio (ΔR/R0). Right ordinate depicts the calibrated [Na ]i corresponding to These ndings con rm the ability of PbTx-2 treatment to en- SBFI fluorescence ratio. Data shown are from a representative experiment hance NMDAR function in cerebrocortical neurons. (n = 20–30 cells) repeated three times. (B) Pretreatment with TTX (1 μM) blocked 100 nM PbTx-2–induced increase in SBFI fluorescence. TTX and PbTx- PbTx-2–Enhanced Neurite Outgrowth Exhibits a Bidirectional Profile + 2 addition indicated by arrows. A representative trace is shown. (C)Ca2 Similar to that of NMDA. We have shown previously that the effect imaging with Fura-2 AM showed PbTx-2 concentration-dependent incre- of PbTx-2 on neurite outgrowth exhibited a bidirectional concen- 2+ fl ments in [Ca ]i. Data are expressed as maximum change in uorescence – fi 2+ tration response pro le and that this effect was primarily de- ratio (340/380) divided by baseline ratio. Right ordinate indicates [Ca ]i fl pendent on NMDARs (16). Lipton and Nakanishi (18) have derived from calibration of the Fura-2 uorescence. PbTx-2 EC50 value: 360 fi nM (95% CI, 133-969 nM). Data shown are from a representative experiment similarly described an inverted-U pro le for the relationship be- (n = 20–30 cells) repeated four times. (D) Pharmacological evaluation of 100 tween NMDAR activity and neuronal survival, with too little or too + nM PbTx-2–induced Ca2 influx examined using TTX (1 μM), D-APV (100 μM), much NMDAR activity resulting in diminished growth or neuronal or nifedipine (1 μM). TTX abrogated the response to PbTx-2, whereas D-APV, death. We reasoned that PbTx-2 and NMDA may share common but not nifedipine, significantly reduced PbTx-2–induced Ca2+ influx. Rep- downstream signaling pathways and, therefore, directly compared resentative traces are shown; data were collected from cells treated with their effects on neurite outgrowth in DIV-1 neurons. The PbTx-2 either 100 nM or 1 μM PbTx-2 (n = 20–30 cells per condition per experiment – + and NMDA concentration response curves for neurite outgrowth and repeated thrice). (E) PbTx-2 potentiation of NMDA-induced Ca2 influx. fi – 2+ fl both exhibited bidirectional pro les consistent with a common NMDA concentration response effects on Ca in ux in the absence and NMDAR-dependent mechanism (Fig. 2 A and B). presence of 30 nM PbTx-2. Data shown (means ± SEM) are from a representative experiment (n = 30 cells) performed in sister coverslips repeated in three independent cultures. PbTx-2 Enhances Dendritic Arbor Development and Stimulates Filopodia Formation. NMDARs play a critical role in activity-regulated ex- + pansion of dendritic arbors through Ca2 -dependent signaling cas- + neurons loaded with the Na -sensitive fluorescent dye sodium- cades (17, 19). Inasmuch as PbTx-2 augments NMDAR function binding benzofuran isophthalate-acetoxymethyl ester (SBFI-AM). and enhances neurite outgrowth, we determined the effect of PbTx- As depicted in Fig. 1A, PbTx-2 produced a concentration-de- 2 on the development of dendritic arbors and filopodia. In DIV-4 + control neurons, there is a gradual increase in branch complexity pendent elevation of [Na ]i. The PbTx-2 concentration–response moving away from the soma reaching a maximum of 7.5 ± 0.6 PHARMACOLOGY data revealed an EC50 of 12.7 nM [95% confidence interval (CI), 5–33 nM] (Fig. 1A). An in situ SBFI calibration (Fig. S1) revealed intersections per neuron and then progressively declining beginning + ∼10 μmfromthesoma(Fig.2C and D). PbTx-2 (30 and 100 nM) a basal [Na ]i of 9 ± 3 mM in DIV-1 neurons, and PbTx-2 (1 μM) + produced robust increases in dendritic complexity and a rightward produced a maximum [Na ]i of 60 ± 10 mM. To confirm the in- + shift in the Sholl plot compared with control neurons (Fig. 2D)(20). volvement of VGSCs in the observed elevation of [Na ] ,we i An area under the curve (AUC) analysis of these Sholl data showed treated neurons with tetrodotoxin (TTX), a VGSC pore blocker. asignificant increase in dendritic complexity following either 30 or TTX (1 μM) treatment completely blocked the 100 nM PbTx-2– + 100 nM PbTx-2 compared with control (ANOVA, P < 0.01; Dun- induced elevation of [Na ]i, establishing VGSCs as the source of nett’s post hoc test, P < 0.05) (Fig. 2E). The PbTx-2 effect on den- + B Na entry (Fig. 1 ). dritic complexity again displayed a bidirectional profile, with the Given that activity-dependent neuronal development and 2+ 300 and 1,000 nM concentrations showing progressively smaller structural plasticity primarily engage Ca -dependent signaling responses (Fig. 2E). Using selective pharmacological inhibitors, we 2+ pathways (17), we next assessed whether PbTx-2 affects Ca dy- next explored the signaling mechanisms underlying the enhanced namics in these neurons. PbTx-2 produced a rapid and concen- dendritic arborization produced by PbTx-2. As depicted in Fig. 2 F 2+ C tration-dependent increase in [Ca ]i (Fig. 1 and Fig. S2). and G, coincubation of TTX (1 μM) or (+)-5-methyl-10,11-dihydro- 2+ Calibration of Fura-2 fluorescence showed a basal [Ca ]i of 60 ± 5H-dibenzo[a, d]cyclohepten-5,10-imine maleate (MK-801) (1 μM) 2+ 20 nM, and 1 μM PbTx-2 produced a [Ca ]i of 1,320 ± 413 nM with 30 nM PbTx-2 markedly attenuated (ANOVA, P < 0.01;

George et al. PNAS | November 27, 2012 | vol. 109 | no. 48 | 19841 Downloaded by guest on October 4, 2021 filopodia, indicating that filopodia maturation is a process dependent on neuronal activity (3). TTX and APV treatment, but not nimodipine, antagonized the PbTx-2–induced dendritic arbor complexity and increase in filopodial density (Fig. 3A). These data suggestthatthePbTx-2influence on NMDAR signaling accelerates the development of cerebrocortical neurons by stimulating dendritic arbor elaboration and both filopodia formation and maturation.

PbTx-2 Enhances Neuronal Connectivity As Revealed by an Acceleration of the Appearance of Spontaneous Calcium Oscillations and Synapse Formation. Next, we examined the influence of PbTx-2 on sponta- + neous Ca2 oscillations that report the degree of neuronal connectivity in a population of cerebrocortical neurons. Spontaneous synchronized depolarization, detectable as intracellular calcium oscillations, reflect typical network activity characteristic of cere- + brocortical neuron cultures (21). These spontaneous Ca2 oscillations become prominent from DIV-6 onward in both amplitude [3,391 ± 173.4 fluorescence units (FU)] and frequency (11 ± 1.5/ 2+ 300 s) (Fig. S4). The presence of spontaneous Ca oscillations was assessed from DIV-2 onward at 12-h intervals (Fig. 4). PbTx-2 (30 + nM) accelerated the emergence of spontaneous Ca2 oscillations by + 24 h (DIV-5), shifting leftward the Ca2 oscillation time–response curves for both amplitude and frequency (Fig. 4 B and C). As + depicted in Fig. 4A, control Ca2 oscillations did not become prominent until 148 h (DIV-6), at which time, the oscillation amplitudes reached values of 3,000 FU. In contrast, PbTx-2–exposed neurons displayed greater oscillation amplitudes 24 h earlier at 124 h (DIV-5) (124 h: 4,209.6 ± 692 FU). Analysis of oscillation Fig. 2. PbT-2–enhanced neurite outgrowth and dendritic arborization exhibit – – fi frequency (number of spikes/300 s) in the PbTx-2 treated cultures a bidirectional concentration response pro le. (A) Representative images of also showed a significant increase from 124 h onward compared NMDA-induced neurite outgrowth. (Scale bar: 10 μm.) (B)Quantification of with control [control vs. PbTx-2: 4.8 ± 2vs.10.5± 1.8(124h),4.8± neurite outgrowth 24 h after plating. PbTx-2 or NMDA were added to culture at ± ± ± C 3 h postplating. Each value represents mean ± SEM of 120 neurons. (C)Repre- 2.2 vs. 13 2.5 (136 h), and 9 1.1 vs. 15.8 2.7 (148 h)] (Fig. 4 ). sentative images of 3′-tetramethylindocarbocyanine (DiI)-loaded cerebrocortical Given the strong association between spontaneous calcium neurons at DIV-4. (D) Sholl analysis to quantify arbor complexity. (E) Area under oscillations and maturation of synapses and pattern-generating thecurve(AUC)analysisofSholldata.PbTx-2at30and100nMsignificantly circuits, we reasoned that the accelerated appearance of sponta- enhanced dendritic complexity. The experiment was performed thrice, and each neous calcium oscillations produced by PbTx-2 may reflect an point represents the mean derived from the analysis of 30–40 neurons (ANOVA, increased rate of synaptogenesis. Considering that synchronized P < 0.01; Dunnett’s post hoc test, P < 0.05). (F) Pharmacological evaluation of 30 2+ – Ca oscillations represent a functional assay for synaptogenesis, nM PbTx-2 induced dendritic arborization. The response to 30 nM PbTx-2 ex- we directly assessed the effect of PbTx-2 on synapse density in posure was examined in the presence of TTX (1 μM), MK-801 (1 μM), or nimodipine (1 μM) beginning at 3 h after plating. Each data point represents mean ± cerebrocortical neuron cultures. Antibodies against synaptophysin SEM of 50–60 neurons. (G) AUC analysis of the data shown in F.PbTx-2–enhanced dendritic arbor complexity was significantly attenuated by MK-801 and TTX. (ANOVA, P < 0.01; Bonferroni post hoc test, *P < 0.05).

Bonferroni post hoc test, P < 0.05) the enhancement of dendritic complexity. In contrast, nimodipine (1 μM), an L-type calcium channel blocker, did not produce a statistically significant reduction in the enhancement of dendritic arborization produced by PbTx-2 (P > 0.05). These results suggest that PbTx-2 activation of sodium channels with attendant enhancement of NMDAR signaling is re- sponsible for the expansion of dendritic arbors. On DIV-5, 30 nM PbTx-2–treated neurons displayed a dramatic increase in the density of dendritic protrusions [32.4 ± 3.3 protrusions per 50 μm of dendrite vs. control (9.6 ± 1.1)] (Fig. 3). Morphological analyses of these protrusions with a defined algo- rithm for length and head diameter showed that most of the pro- – fi trusions in PbTx-2 treated neurons were long lopodial-like Fig. 3. PbTx-2 enhances filopodia formation. (A) Representative images of structures (Fig. 3 A and B). On DIV-6, PbTx-2–exposed neurons DiI-labeled neurons at DIV-5. Depicted are PbTx-2 concentration-dependent revealed a shift in the distribution of protrusions from pre- effects on protrusion density (protrusions/50 μm neurite length) and phar- dominantly long filopodial-type (12.5 ± 3.04) to stubby filopodial- macological evaluation of PbTx-2–induced (30 nM) protrusion density. (Scale type protrusions (20 ± 3.5) (Fig. 3 C and D). Elongated filopodia are bar: 5 μm.) (B and D) Representative images and analysis of long and stubby referred to as immature or exploratory-type filopodia principally filopodial protrusions. PbTx-2 significantly enhanced total protrusion density δ < ’ looking for contact partners, and stubby/club-shaped filopodia are on DIV-5 and -6 compared with control ( P 0.05, Student s t test), and on DIV-6, PbTx-2–exposed neurons showed a significant shift from pre- referred to as mature filopodia (3). This indicates that PbTx-2 en- fi < ’ fi dominantly long to stubby lopodial protrusions (*P 0.05, Student s t test). hanced both the formation and maturation of lopodia in cere- Each bar represents the mean ± SEM of 20–30 neurons. (C) Representative brocortical neurons. Blocking neuronal activity by acute treatment images of Dil-labeled neurons at DIV-6 showing PbTx-2 (30 and 100 nM) with TTX has been shown to shift mature filopodia to exploratory enhancement of filopodial maturation.

19842 | www.pnas.org/cgi/doi/10.1073/pnas.1212584109 George et al. Downloaded by guest on October 4, 2021 a marked increase in phosphorylation of CREB at Ser133, and this response is abrogated by TTX (1 μM), D-APV (100 μM), or the CaMKK inhibitor 1,8-naphthoylene benzimidazole- 3-carboxylic acid (STO609) (2.6 μM) (P < 0.05). However, nimodipine (1 μM); 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f] quinoxaline-2,3-dione (NBQX) (5 μM), an AMPA receptor + blocker; and ω-conotoxin (1 μM), an N-type Ca2 channel blocker, all were without effect on PbTx-2–induced activation of CREB (Fig. S5 C and D). These data are consistent with a previous study + (23) showing that Ca2 influx through NMDARs, rather than L- type VGCCs, leads to robust CREB (Ser133) phosphorylation in + neurons. Our results indicate that NMDAR-mediated Ca2 influx is required for PbTx-2–induced CREB phosphorylation. BDNF is an activity-dependent trophic factor that mediates many aspects of neuronal plasticity and is regulated by CREB-dependent transcription (24). The activity-dependent transcription of BDNF mRNA is a rapid response (25). We, therefore, assessed the influence of PbTx-2 on BDNF transcription with conventional RT-PCR in DIV-1 cerebrocortical neurons. BDNF mRNA ex- + Fig. 4. PbTx-2 exposure accelerates the appearance of spontaneous Ca2 pression level increased in a time- and concentration-dependent oscillations. (A) Effect of PbTx-2 (30 nM) on Ca2+ oscillations. PbTx-2 was manner following PbTx-2 exposure (Fig. S5E). added in alternate wells of 96-well plates beginning 3 h postplating, and The BDNF-tropomyosin-related kinase B (TrkB) receptor sig- 2+ plates were scanned for Ca oscillations at the indicated time points. Data naling complex exerts neurotrophic effects in both the developing are from a representative experiment performed in quadruplicate and re- and mature nervous system, and TrkB receptor surface expression peated twice. (B and C) Quantification of data shown in A.(B and C) Mean ± + SEM of peak amplitude (B) and frequency (C)ofCa2 oscillations (number of is regulated by neuronal activity (26). Using a biotinylation assay in spikes per 300 s). PbTx-2 accelerated the appearance of spontaneous Ca2+ DIV-1 cerebrocortical neurons, we have found that PbTx-2 expo- oscillations by 24 h (DIV-5) and produced a significant leftward shift in the sure affects surface expression of TrkB receptors. Cell surface ex- time–response relationship for both oscillation amplitude and frequency (*P < pression of TrkB receptors (control, 23.6 ± 3.4% of total) was 0.05, Student’s t test comparison vs. control at respective time points). increased substantially within 30 min of PbTx-2 exposure (PbTx-2:

(presynaptic marker) and postsynaptic density 95 (PSD-95) (postsynaptic marker) were used to quantify synapse density, as indicated by colocalized fluorescent puncta. We exposed neurons to PbTx-2 beginning 3 h postplating, and synapse density was assayed on DIV-5. Concentration–response analysis of the influence of PbTx-2 on synaptic density indicated that PbTx-2 at concentrations of 30 and 100 nM produced a significant (ANOVA, P < 0.01; Dunnett’s post hoc test, P < 0.05) increase in synapse formation (number of puncta per 20 μm) compared with control cultures. Separate analysis of pre- and postsynaptic puncta showed an increase in PSD-95, but not synaptophysin, puncta following PbTx-2 exposure. Higher concentrations of PbTx-2 (300 and 1,000 nM) produced a progressive decline in synaptic density [control, 0.95 ± 0.2 puncta per 20 μm of dendrite; PbTx-2 (30 nM), 3.99 ± 0.24; PbTx-2 (100 nM), 3.8 ± 0.34; PbTx-2 (300 nM), 2.53 ± 0.24; PbTx-2 (1,000 nM), 0.79 ± 0.27] (Fig. 5 A and B). We theorized that if the influence of PbTx-2 on synaptic density was the result of augmentation of NMDAR function, then NMDA should also display a bidirectional concentration–response profile on synaptic density. Consistent with this hypothesis, NMDA produced a bidirectional concentration–response profile for synaptogenesis (Fig. PHARMACOLOGY 5 C and D), suggesting that common NMDAR-dependent mechanisms are operative in these effects of PbTx-2 and NMDA.

PbTx-2 Exposure Engages Activity-Dependent Signaling Mechanisms That Drive Structural Plasticity. Inasmuch as activity-dependent 2+ neuronal development primarily involves Ca -mediated signal- Fig. 5. Effect of PbTx-2 on formation of synapses in cerebrocortical cultures. ing events including CaMKs downstream from NMDARs, we (A) Representative images of double-immunostained cerebrocortical neurons 2+ have examined whether the increase in [Ca ]i evoked by PbTx-2 at DIV-5. PbTx-2 was added to cultures 3 h after plating. Antibodies against also engages these signaling pathways. Acute exposure to PbTx-2 synaptophysin (presynaptic marker/green) and PSD-95 (postsynaptic marker/ (100 nM) does produce a rapid and sustained phosphorylation of red) were used to quantify synapse density, as indicated by colocalized fluorescent puncta (yellow). Boxed regions are shown in higher magnification CaMK1 (Thr177) and CaMKII (Thr286), whereas stimulation μ fi of phospho-ERK 1/2 is phasic (Fig. S5 A and B). In contrast, separately and overlaid. (Scale bar: 2 m.) (B)Quanti cation of colocalized fluorescent puncta (each bar represents n = 3 independent experiments with PbTx-2 treatment does not activate either CaMKIV (Thr196) or duplicate samples; ANOVA, P < 0.01; Dunnett’s post hoc test, *P < 0.05). (C and Akt (Ser473). The transcription factor CREB has been implicated D) Representative images and quantification of NMDA concentration–response in activity-dependent neuronal development, where it is regu- effect on synaptogenesis (n = 3 independent experiments with duplicate lated by CaMKs and MAPKs (22). PbTx-2 exposure produces samples; ANOVA, P < 0.01; Dunnett’s post hoc test, *P < 0.05). (Scale bar: 2 μm.)

George et al. PNAS | November 27, 2012 | vol. 109 | no. 48 | 19843 Downloaded by guest on October 4, 2021 30 nM, 62.9 ± 7.8%; 100 nM, 40.3 ± 10.6%; 1 μM, 62.7 ± 14.7% The effects of PbTx-2 on dendritic arborization and synapto- compared with the control fraction; Fig. S5 F and G). Given the genesis displayed bidirectional concentration–response profiles. predominance of GluN2B subunit–containing NMDARs in im- Moreover, PbTx-2 effects on calcium influx, dendritic arboriza- mature neurons (27) and the role of GluN2B subunits in the reg- tion, and filopodia formation were dependent on NMDARs, ulation of NMDAR cell surface expression (28), we also evaluated underscoring the role of NMDAR signaling in the influence of the influence of PbTx-2 on GluN2B cell surface expression. The PbTx-2 on neuronal structural plasticity. Additional evidence for control level of GluN2B subunit surface expression was 18.1 ± NMDAR involvement in the effects of PbTx-2 on structural 5.25%, and PbTx-2 exposure significantly increased the surface plasticity was the similar bidirectional patterns shown by NMDA expression of GluN2B subunits (PbTx-2: 30 nM, 26.7 ± 4.35%; 100 and PbTx-2 on both neurite outgrowth and synaptogenesis. An nM, 39.9 ± 1.2%; 1 μM, 49.2 ± 10.02% compared with control; Fig inverted-U model describes the relationship between NMDAR S5 F and G). PbTx-2 exposure, therefore, acutely increases both activity and neuronal survival and growth (18). This inverted-U TrkB and NMDAR surface expression in cerebrocortical neurons. NMDA concentration–response relationship has primarily, but + not exclusively, been regressed to intracellular Ca2 regulation. + Discussion An optimal window for [Ca2 ] is required for activity-dependent + i Na influx is fundamental to electrical signaling in the nervous neurite extension and branching, with low levels stabilizing system and is essential for the upstroke of action potentials (29). In growth cones and high levels stalling them, in both cases pre- + murine neurons, VGSCs appear as early as embryonic day 12 (30). venting extension (37). Given that the major source of Ca2 in- Given the role of VGSCs in controlling action potential generation, flux following PbTx-2 exposure is through NMDAR channels, we they serve as candidates for mediating forms of synaptic plasticity suggest that similar mechanisms are operative in the bidirectional that depend on neuronal firing (31). Neuronal activity exerts pro- effects of NMDA and PbTx-2 on structural plasticity. found effects on brain development and the structural plasticity of Trafficking and surface expression of NMDARs are also reg- dendritic arbors and spines (17, 32). Toward the goal of mimicking ulated by neuronal activity (38) and phosphorylation of the activity-dependent phenomena, we used a VGSC gating modifier to + GluN2B subunit (39). The GluN2B subunit is a primary de- elevate neuronal [Na ]i and potentiate NMDAR function (12, 16). terminant of the surface mobility of NMDARs (28), and acute An interesting feature of our results is that the influence of PbTx-2 PbTx-2 exposure was found to enhance the surface expression of on neuronal structural plasticity followed a bidirectional pattern this subunit. PbTx-2 was also capable of activating neuronal ac- similar to that of NMDA. This result is consistent with a role for + tivity-dependent signaling targets such as CaMKs, ERK, and 2+ [Na ]i in enhancing NMDAR function with engagement of down- CREB. Activity-dependent increases in intracellular Ca have stream mechanisms involved in the regulation of neuronal plasticity. + been shown to enhance ERK signaling, with subsequent CREB Previous reports suggested that [Na ]i may act as a signaling phosphorylation downstream of NMDARs in cerebrocortical molecule to influence NMDAR function through Src-induced neurons (40). Consistent with this earlier report, we found that phosphorylation of the receptor (12). High firing rates that are ca- PbTx-2 stimulation of CREB phosphorylation was NMDAR- pable of inducing long-term potentiation (LTP) are associated with + dependent. CREB has been shown to mediate activity-de- elevated [Na ]i and a Src-induced up-regulation of NMDAR pendent dendrite lengthening (36), and CREB stimulation leads function (13). To unambiguously confirm the enhancement of to an activation of BDNF gene expression. BDNF increases NMDAR function by PbTx-2, we have previously recorded single- dendritic complexity in neurons and blockade of TrkB receptors channel currents from cell-attached patches on cerebrocortical abrogates this effect (41). The ability of PbTx-2 treatment to neurons; PbTx-2 treatment increased both the mean open time and increase bdnf mRNA expression and TrkB surface localization fl open probability of NMDARs in the absence of an in uence on demonstrates the influence of neuronal activity on these phe- resting membrane potential (16). These results were extended in the nomena. VGSC gating modifiers may accordingly represent a present study by showing that PbTx-2 treatment enhanced NMDA- pharmacologic strategy to regulate neuronal plasticity through an 2+ fl fi induced Ca in ux in cerebrocortical neurons. These ndings are NMDAR-dependent mechanism. Recent studies have shown that fl + consistent with a regulatory in uence of Na on NMDAR function. neuronal rewiring following a stroke event occurs as a result of NMDAR stimulation promotes dendritic arbor growth, whereas heightened plasticity in the periinfarct cortex, with enhanced ax- pharmacological blockade of NMDARs reduces dendritic growth onal sprouting, synaptogenesis, and turnover of dendritic spines (33). The NMDA-induced increase in dendritic arbor complexity (42). Because the processes involved in rewiring and recovery after is suppressed by blockers of voltage-gated calcium channels or stroke are similar to those regulating development, it is feasible inhibitors of intracellular calcium mobilization (34), indicating fi 2+ that VGSC gating modi ers that augment developmental den- that NMDAR activation recruits Ca signaling pathways to reg- dritogenesis and synaptogenesis may also improve stroke recovery. ulate dendritic growth (19). Consistent with these reports, we 2+ found that exposure to PbTx-2 increased neuronal [Ca ]i, with Materials and Methods 2+ fl NMDAR channels constituting the primary source of Ca in ux Brevetoxin-2 (PbTx-2) was isolated and purified from Karenia brevis cultures in cerebrocortical neurons. at the Center for Marine Sciences at the University of North Carolina (Wilmington, The effects of exposure to PbTx-2 on dendritic arbor complexity NC). Fluo-3 AM, SBFI-AM, and Pluronic acid were purchased from Invitrogen. are consistent with a previous demonstration that neuronal activity affects structural plasticity in vivo through NMDAR-triggered in- Cell Culture. Primary cultures of cerebrocortical neurons were prepared from tracellular signaling events (35). In accordance with earlier reports embryos of Swiss–Webster mice on embryonic day 16 as described (16). The for activity-dependent plasticity, we found that PbTx-2 treatment Creighton University Institutional Animal Care and Use Committee approved increased neurite outgrowth, dendritic arborization, and filopodia all animal use protocols. formation and maturation. The PbTx-2–enhanced dendritic arborization was mediated by NMDAR-dependent mechanisms: conso- Calcium and Sodium Imaging. Intracellular calcium and sodium concentration fl nant with a report of activity-dependent dendritic arborization in neurons were assessed using the respective uorescent dyes Fura-2 and SBFI. Additional details are provided in SI Text. requiring NMDAR activation in DIV-9 hippocampal neurons (36). The exposure to PbTx-2 also enhanced synaptogenesis. As a conse- fl 2+ Double Immuno uorescence and Image Acquisition and Colocalization Analysis. quence of this latter action, the appearance of spontaneous [Ca ]i Double immunostaining with synaptophysin (green), a presynaptic marker, and oscillations was accelerated. These observations implicate synaptic PSD-95 (red), a postsynaptic marker, were used to quantify colocalized puncta mechanisms in the observed PbTx-2 acceleration of the development (yellow) as an indication of the presence of a synapse. Additional details are of neuronal connectivity in cerebrocortical neuron cultures. provided in SI Text.

19844 | www.pnas.org/cgi/doi/10.1073/pnas.1212584109 George et al. Downloaded by guest on October 4, 2021 Diolistic Labeling and Quantiﬁcation of Dendritic Branching and Filopodial ACKNOWLEDGMENTS. This work was supported by National Institute of Protrusion Density. The details of diolistic neuron labeling and confocal im- Neurological Disorders and Stroke Grant NSO53398 (to W.H.G. and T.F.M.) and aging acquisition are described in SI Text. Nebraska EPSCoR National Science Foundation Grant EPS-1004094 (to T.F.M.).

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