Brain-Derived Neurotrophic Factor Regulation of N-Methyl-D-Aspartate Receptor-Mediated Synaptic Currents inSuprachiasmatic Nucleus Neurons

Y.I. Kim1,*, H.-J. Choi1, and C.S. Colwell2,*

1Department of Physiology, Korea University College of Medicine, Seoul, Republic of Korea

2Department of Psychiatry and Biobehavioral Sciences, University of California—Los Angeles, Los Angeles,California

AbstractLight information reaches the suprachiasmatic nucleus (SCN) through a subpopulation of retinalganglion cells. Previous work raises the possibility that brain-derived neurotrophic factor (BDNF)and its high-affinity receptor TrkB may be important as modulators of this excitatory input into theSCN. To test this possibility, we used whole-cell patch-clamp methods to measure excitatory currentsin rat SCN neurons. These currents were evoked by electrical stimulation of the optic nerve. Wefound that the amplitude of the N-methyl-D-aspartate (NMDA) component of the evoked excitatorypostsynaptic currents (NMDA-EPSC) was increased by application of BDNF. The neurotrophin alsoincreased the magnitude of NMDA-evoked currents in SCN neurons. The BDNF enhancement ofthe NMDA-EPSC was blocked by treatment with the neurotrophin receptor antagonist K252a as wellas treatment with the soluble form of the TrkB receptor engineered as an immunoadhesin (TrkB IgG).Finally, the BDNF enhancement was lost in brain slices treated with the NR2B antagonist ifenprodil.The results demonstrate that BDNF and TrkB receptors are important regulators of retinalglutamatergic synaptic transmission within the SCN.

Keywordscircadian rhythms; NMDA; rat; TrkB; suprachiasmatic nucleus; SCN

During development, neurotrophic factors promote neuronal survival and differentiation(Lewin and Barde, 1996). Neurotrophins also modify the growth of developing axons (Alsinaet al., 2001; Frost, 2001) and through this mechanism are likely to be involved in theestablishment of neural circuits. Once the circuits are established in adult tissue, neurotrophinsappear to be utilized for other functions, including the regulation of cellular communicationand plasticity. In the adult nervous system, brain-derived neurotrophic factor (BDNF) and itshigh-affinity TrkB receptor are highly expressed in select regions, including the hippocampusand cortex (McAllister et al., 1999). In these regions, BDNF expression and release can berapidly regulated by neural activity. Mechanistically, BDNF can regulate transcription throughCREB activation (Finkbeiner et al., 1997). On a cellular level, BDNF can produce a wide rangeof effects including regulation of presynaptic release (Lessmann, 1998; Xu et al., 2000;Schinder et al., 2000), modulation of postsynaptic receptors (Levine et al., 1998; Lin et al.,

*Correspondence to: Y.I. Kim, Department of Physiol, Korea University College of Medicine, 126-1 Anam-dong 5-ga, Seoul 136-705,Republic of Korea. E-mail: yikim@korea.ac.kr; or C.S. Colwell, Department of Psychiatry and Behavioral Sciences, UCLA, 760Westwood Plaza, Los Angeles, CA 90024-1759. E-mail: ccolwell@mednet.ucla.edu

NIH Public AccessAuthor ManuscriptJ Neurosci Res. Author manuscript; available in PMC 2008 November 12.

Published in final edited form as:J Neurosci Res. 2006 November 15; 84(7): 1512–1520. doi:10.1002/jnr.21063.

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1998; Levine and Kolb, 2000), and direct activation of membrane channels (Blum andKonnerth, 2005).

Relatively less is known about the role of BDNF in the context of behaviorally relevant circuits.In mammals, one of the best-understood behavioral control circuits is found in thesuprachiasmatic nucleus (SCN). Neurons in this hypothalamic region are critical for thegeneration of circadian behaviors and their synchronization to light. SCN neurons gain accessto information about external lighting conditions, at least in part, through a specializedsubpopulation of light-sensitive retinal ganglion cells that contain the photopigmentmelanopsin (Van Gelder, 2005). These ganglion cells utilize glutamate and the neuropeptidepituitary adenylyl cyclase activating peptide (PACAP) as cotransmitters to communicate withthe SCN (Hannibal et al., 2000). A variety of evidence indicates that, within the SCN, the N-methyl-D-aspartate (NMDA) class of glutamate receptors plays a critical role in mediating thebehavioral effects of light (Colwell and Menaker, 1996).

Recent work raises the possibility that BDNF is an important modulator of photic regulationin the circadian system. Both BDNF and its high-affinity TrkB receptor are expressed in theSCN (Liang et al., 1998; Allen and Earnest, 2005). Levels of BDNF fluctuate rhythmically inthis region, with peak protein levels found during the night, when exposure to light can alterthe phase of circadian oscillations (Liang et al., 1998). Functionally, both BDNF- and TrkB-deficient mice exhibit a reduction in the phase shifting effects of light on the circadian system(Liang et al., 2000; Allen et al, 2005). Thus, we became interested in exploring the possiblerole of BDNF as a regulator of synaptic-evoked NMDA currents in SCN neurons.

MATERIALS AND METHODSAnimals and Housing

Male Sprague-Dawley rats (n = 36; 100–250 g) purchased from Charles River LaboratoriesInternational (Wilmington, MA) were used in this study. The experimental proceduresconformed to the U.S. National Institutes of Health guidelines. Before being used forexperiments, the animals were housed in a temperature-controlled chamber (23–24°C) with a12:12 hr light/dark schedule for at least 1 week. By definition, zeitgeber time (ZT) 0 is the timeof lights-on and ZT 12 is the time of lights-off in the colony. All experiments were conductedduring the animal’s night based on the prior light/dark (LD) cycle.

Hypothalamic Slice PreparationRats were anesthetized with sodium pentobarbitone (100 mg/kg, i.p.) between ZT 10:30 and11:30, and the brains were quickly excised and submerged in ice-cold physiological saline(composition in mM: 127 NaCl, 2 MgCl2, 3 KCl, 1.25 NaH2PO4, 26 NaHCO3, 2 CaCl2, 10glucose; osmolality 300 mOsm/kg). After ∼1 min of chilling, the brains were trimmed to ablock containing the hypothalamus and optic nerves. With the use of a vibroslicer (modelNVSLM1; World Precision Instruments, Sarasota, FL), two parasagittal slices (400–550 μmthickness), each containing one optic nerve (2–4 mm) and an SCN, were cut from the tissueblock. In some cases, two coronal slices containing the SCN (450 μm thickness) were cut.During the slicing, the tissue was maintained in ice-cold physiological saline. One of the sliceswas transferred to a modified Hass-type gas interface recording chamber (Haas et al., 1979)and superfused continuously, at a rate of ∼0.7 ml/min, with physiological saline (pH 7.4)aerated with 95% O2/5% CO2. Air humidified by 95% O2/5% CO2 was also continuouslyblown over the slice to ensure further adequate oxygenation of the cells in the tissue. Theremaining slice was kept, for later use, in a storage chamber filled with physiological salineaerated by 95% O2/5% CO2.

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Whole Cell Voltage-Clamp RecordingAfter ∼1 hr of equilibration of the slice in the recording chamber, electrophysiologicalexperiments were begun. Micropipettes (tip diameter: 1.5–2.0 μm; 3–7 MΩ) pulled fromborosilicate tubing (od: 1.5 mm, id: 0.84 mm; World Precision Instruments) and filled with aninternal solution (composition in mM: 130 Cs-methanesulfate, 4 NaCl, 1 MgCl2, 3KCl, 3QX-314, 5 Mg ATP, 0.2 EGTA, 8 Na-HEPES, 1 GTP, 0.1 leupeptin, 10 phosphocreatine; pH7.2; osmolality 285 mOsm/kg) were used as recording electrodes. Whole-cell voltage-clamprecordings from SCN neurons were obtained, at room temperature, in continuous voltage-clamp mode with the use of Axoclamp-2A amplifier (Axon Instruments, Foster City, CA). Therecorded signals were filtered at 1 kHz and sampled at 50-μsec intervals with Digidata 1320Aand pClamp8.0 (Axon Instruments).

Optic Nerve StimulationThe optic nerve was stimulated with the use of a custom-made suction electrode; constantcurrent pulses (biphasic square wave) of 0.1–1.0 mA intensity and 0.5–2.0 msec duration wereapplied in 30-sec intervals.

DRUGSBicuculline methiodide (BIC; a GABAA receptor antagonist), 2-amino-5-phosphonopentanoicacid (APV; an NMDA receptor antagonist), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; anon-NMDA receptor antagonist), BDNF, NMDA, K252a (a neurotrophin receptor antagonist),anti-mouse TrkB receptor antibody, and ifenprodil (an NR2B NMDA receptor subunitantagonist) were used in this study. All the drugs were purchased from Sigma (St. Louis, MO),except the antibody TrkB receptor IgG (R&D Systems, Inc., Minneapolis, MN). BIC (25 μM),APV (50 μM), CNQX (20 μM), and ifenprodil (3 μM) were bath applied, and BDNF (200 ng/ml) and NMDA (0.5 mM) were applied directly to the SCN with the “Y-tube” method (Muraseet al., 1989). K252a was either bath applied (100 nM) or focally applied (200 nM) using the“Y-tube” method. TrkB receptor IgG (1 μg/μl) was applied directly to the SCN in the form ofdroplets (a total of 10 μl). Approximately 1 min prior to the TrkB receptor IgG application,bath perfusion was stopped, and it was resumed 2 min following the application.

Statistical AnalysesNumerical data are shown as mean ± SEM. Drug effects on the synaptic responses from opticnerve stimulation were estimated with the use of paired t-test. P < 0.05 was considered to besignificant.

RESULTSCharacterization of the NMDA Excitatory Postsynaptic Currents Evoked by Optic NerveStimulation

Experiments were conducted on >50 neurons located in the ventral region of the SCN in ratbrain slices recorded during the night. All of the neurons included in this study exhibited stable,short-latency excitatory currents in response to optic nerve stimulation. By this criterion, weconsidered these cells to be retinorecipient SCN neurons. For these experiments, NMDAreceptor-mediated currents in SCN neurons were isolated by recording responses evoked byoptic nerve stimulation in the presence of the non-NMDA receptor antagonist CNQX (20 μM)and the GABAA receptor antagonist BIC (25 μM). Because the NMDA currents are stronglyvoltage dependent and exhibit a reversal potential of about 0 mV, the neurons were held atpositive holding potentials (+30 to +80 mV). The NMDA excitatory postsynaptic currents(EPSCs) exhibited a 10–90% rise time of 20.3 ± 1.8 msec with a duration at half-maximalamplitude of 304 ± 16 msec (n = 9). As expected, the peak NMDA synaptic current in SCN

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neurons displayed a nonlinear current-voltage relationship with marked rectification atmembrane potentials more negative than -10 mV (Fig. 1). The NMDA-EPSCs were blockedby the NMDA antagonist APV (50 μM; Fig. 1).

Enhancement of NMDA Receptor-Mediated Response by BDNFThe effects of BDNF on the NMDA-EPSCs were examined in nine retinorecipient SCNneurons (Fig. 2). Overall, BDNF (200 ng/ml; 5 min) caused a rapid increase in the peakmagnitude of the NMDA-EPSC (20% ± 6% change; P = 0.02; n = 9). Enhancement of at least10% was found in six of nine neurons and fully reversed in three of these six cases. We nextexamined the effect of BDNF (200 ng/ml; 5 min) on the non-NMDA-EPSC. In theseexperiments, APV (50 μM) and BIC (25 μM) were added to the bath solution, and the neuronwas held at -60 mV. These synaptic evoked currents were completely blocked by CNQX.BDNF (200 ng/ml; 5 min) had no effect of the magnitude of these AMPA-EPSCs (0% ± 6%change; P > 0.48; n = 6). We then examined whether BDNF enhanced the inward currentsinduced by exogenously applied NMDA (0.5 mM; 3- or 5-sec pulse; delivered once every 120sec; Fig. 3). These NMDA currents were elicited in SCN neurons recorded in coronal slices,at holding potentials between -25 and -35 mV in the presence of BIC (25 μM) and CNQX (20μM). BDNF (200 ng/ml; 5 min) enhanced the NMDA currents significantly (24% ± 6% change;n = 8; P < 0.02; Fig. 3). Enhancement of at least 10% was found in six of eight neurons andfully reversed in four of these six cases.

TrkB Receptors Mediate the Effects of BDNFTo determine whether the effect of BDNF on NMDA currents was mediated by activation ofneurotrophin receptors, we utilized the tyrosine kinase inhibitor K252a (100–200 nM). Wefound that, by itself, K252a (200 nM; focal application) attenuated the NMDA componentssignificantly (-27% ± 5% change; n = 8; P < 0.001). In the presence of K252a (100 nM),application of BDNF (200 ng/ml; 5 min) did not enhance the NMDA-EPSCs (-18% ± 12%change; n = 7; P > 0.23; Fig. 4A,B). Bath application of K252a also blocked the BDNF-inducedenhancement of exogenous NMDA-induced currents (3% ± 5% change; n = 7; P > 0.8; Fig.4C). Finally, we used a soluble form of the TrkB receptor engineered as an immunoadhesin(TrkB-IgG) to block TrkB ligands. We found that pretreatment with the TrkB receptor IgG (1μg/μl; 10 μl) prevented BDNF enhancement of the NMDA-EPSC (-5% ± 5% change; n = 6;P > 0.25; Fig. 5).

Treatment With the NR2B Receptor Blocker Ifenprodil Prevented BDNF EnhancementFinally, we examined the role of the NR2B subunit of the NMDA receptor in mediating theeffects of BDNF on NMDA-EPSC in SCN neurons. By itself, bath application of thenoncompetitive NR2B antagonist ifenprodil (3 μM, 5 min) significantly reduced the magnitudeof the NMDA-EPSC (-40% ± 7% change; n = 7; P < 0.05). With the conditions under whichthese experiments were performed, the inhibition by ifenprodil did not wash out within 30 min.For comparison, similar treatment with the competitive NMDA antagonist APV (50 μM, 5min) completely suppressed the NMDA-EPSC (Fig. 1). In the presence of ifenprodil (3 μM),the application of BDNF (200 ng/ml, 5 min) was no longer able to enhance the NMDA-EPSC(-7% ± 6% change; n = 6; P > 0.2; Fig. 6).

DISCUSSIONThe circadian system is largely responsible for the temporal organization of an organism’sphysiology and behavior. The cells within the SCN are responsible for the generation of thedaily rhythms as well as their regulation by light. The light information is detected byspecialized light-sensitive retinal ganglion cells that utilize the photo-pigment melanopsin(Peirson and Foster, 2006). The axons of these retinal ganglion cells form a fiber tract

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(retinohypothalamic tract or RHT) that terminates in the SCN. Previous work has establishedthat these axon terminals utilize glutamate and the neuropeptide PACAP as transmitters(Hannibal et al., 2000). Postsynaptically, the NMDA receptor plays a critical role in transducingthe light information signaled by the release of glutamate to a behavioral response of thecircadian system. Among other findings, blocking NMDA receptors prevents the effects oflight on the circadian system (Colwell et al., 1990), whereas activation of these receptors canmimic the effect of light (Mintz et al., 1999). The SCN is made up of functionally distinct cellpopulations (Antle and Silver, 2005) with only a subset of SCN neurons in the ventral regionreceiving these direct innervations from the RHT (Morin et al., 2006). Previous data raise thepossibility that BDNF may play a role in regulation of this light-input pathway. For example,a recent anatomical study found fluorescent immunostaining for the TrkB receptor throughoutthe SCN with a substantial overlap in distribution of TrkB immunoreactivity and RHTinnervation (Allen and Earnest, 2005). The present study uses physiological tools to examinethe hypothesis that BDNF modulates excitatory communication at the RHT/SCN synapticconnection in rats.

BDNF Regulation of NMDA-EPSCsWe focused our analysis on SCN neurons that receive monosynaptic excitatory input from theRHT based on two criteria. First, stimulation of the optic nerves evoked EPSCs in the SCNwith low stimulus intensity, and the amplitude of the EPSCs was relatively fixed. Second, thelatency of the response to stimulation was consistent and short (<25 msec). These types ofcriteria are widely used to identify monosynaptic connections (Berry and Pentreath, 1976). Forthe retinorecipient SCN neurons, we found that BDNF increased, whereas the neurotrophinreceptor antagonist K252a decreased, the amplitude of synaptic-evoked NMDA currents.These changes in the amplitude of the NMDA-EPSC could reflect a modulation in presynapticrelease of glutamate or postsynaptic changes in receptor sensitivity or a change in ionic drivingforce. However, our finding that the AMPA-EPSC was not similarly enhanced by BDNF arguesagainst BDNF working presynaptically to increase the release of glutamate in the presentpreparation. This selectivity to NMDA receptors was a surprise to us, insofar as we havepreviously found evidence for BDNF enhancement of both AMPA and NMDA currents in themouse (Michel et al., 2006b). It is not clear whether the difference lies in the species used, theSCN cell populations measured, or some other variable. Regardless, in the present study, wedirectly demonstrate that application of BDNF enhances the magnitude of the currents evokedby application of NMDA in rat SCN neurons. Although these results do not rule out apresynaptic modulation of glutamate release, they do demonstrate that BDNF can regulate thepostsynaptic NMDA receptor in the SCN.

Underlying MechanismsTo clarify the mechanisms underlying BDNF modulation of NMDA-EPSCs in the SCN, weundertook three sets of experiments. First, we found that the Trk-signaling pathway inhibitorK252a prevented the effects of BDNF. Although the inhibitor K252a is the standardpharmacological agent used to assess the role of neurotrophin receptors, K252a is not selectivefor TrkB receptor (Knusel and Hefti, 1992). It is also possible that some of the effects of K252aalone on the NMDA-EPSC amplitude are due to an inhibition of the endogenous actions ofPACAP. This neuropeptide can activate Trk neurotrophin receptors (Lee et al., 2002; Rajagopalet al., 2004), and we have recently found physiological evidence that PACAP regulates NMDAcurrents in SCN neurons (Michel et al., 2006a). Next, to explore specifically the role of theTrkB receptors, we made use of a TrkB receptor antibody (TrkB IgG). This antibody competeswith native TrkB receptors to sequester BDNF and prevent TrkB receptor activation (Davis etal., 1994; Shelton et al., 1995). Because of the expense of applying this antibody through thebath, we applied small drops of the treatment directly onto the SCN. Our finding that the

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microdrops of TrkB-IgG blocked the effect of BDNF gives us confidence that the TrkBreceptors specifically mediate the effects of BDNF.

The NMDA receptor is composed of different subunits (Cull-Candy et al., 2001), and previouswork indicates that blocking the NR2B subunit prevents BDNF action on NMDA currents(Crozier et al., 1999; Levine and Kolb, 2000). To explore the role of the NR2B subunit in theNMDA-EPSCs recorded in the SCN, we used the non-competitive antagonist ifenprodil(Williams, 2001; Neyton and Paoletti, 2006). At 3 μM, ifenprodil almost completely blocksNR2B-containing NMDARs but does not affect those containing NR2A. We found that, byitself, ifenprodil significantly reduced the magnitude of the NMDA-EPSCs, demonstrating arole for the NR2B receptor sub-type in the SCN. In the presence of ifenprodil, BDNF no longerenhanced the NMDA current, suggesting that the BDNF regulation of NMDA-EPSCs mightwork through the NR2B subunit. Interestingly, TrkB is an intrinsic component of thepostsynaptic density in neurons, as are NMDA subunits, including NR2B (Kennedy, 1997;Aoki et al., 2000). This physical proximity raises the possibility that a localized TrkB-mediatedphosphorylation cascade modulates NMDA receptors in SCN neurons. This model is consistentwith previous findings in the hippocampus, where BDNF enhances the phosphorylation of theNR2B subunit (Lin et al., 1998) and increases the open probability of NMDA channels (Levineand Kolb, 2000). An alternative explanation is that BDNF could drive changes in the surfaceexpression of NMDA receptors in the SCN. There is growing evidence that NMDA receptorsubunits can undergo rapid cycling in and out of the membrane (Barria and Malinow, 2002;Nong et al., 2004; Lavezzari et al., 2004). Regulation of synaptic strength by phosphorylationof NMDA receptors and changes in subcellular localization are not mutually exclusive, andboth mechanisms could be at work in the SCN.

As with other neurotrophins and neuropeptides, BDNF is synthesized as a precursor (pro-BDNF), which is proteolytically cleaved to form BDNF. Recent work suggests that the pro-BDNF interacts preferentially with the low-affinity receptor p75NTR, which in turn triggersintracellular signaling pathways (Lee et al., 2001; Huang and Reichardt, 2003). Endogenouspro-BDNF is thought to be secreted from neurons and may influence synaptic physiology (see,e.g., Woo et al., 2005) in addition to its better-described role promoting apoptosis. Thep75NTR receptors are expressed at the afferent pathways innervating the SCN, and targetedlesions of the p75NTR containing cells with the immunotoxin 192 IgG-saporin impactscircadian rhythms in the rat (Beaule and Amir, 2002; Erhardt et al., 2004). Although thephysiological effects of pro-BDNF and p75NTR activation at the RHT/SCN synapse are notknown, it is quite possible that pro-BDNF as well as BDNF will play a role in the regulationof the SCN and circadian rhythms.

Functional SignificanceOur results indicate that BDNF alters the ratio of the NMDA/AMPA currents evoked in therat SCN in response to optic nerve stimulation. The magnitude of this effect on amplitude ofthe NMDA-EPSC was modest (∼20%). However, the long time course of these NMDAcurrents (∼300 msec duration at half-maximal amplitude) indicates the potential for BDNF tohave a larger impact on the integration of synaptic input in the SCN neurons than would bepredicted by amplitude alone. Interestingly, computational models suggest that changing theNMDA/AMPA current ratio can have a profound effect on the processing of afferent input(Wolf et al., 2005). Thus, BDNF’s modulation of dendritic integration may be particularlyimportant in SCN neurons in which synaptic inputs have to be translated into intracellularprocesses to cause phase shifts of the circadian system. Calcium levels are critical for mediatingthe transduction of membrane events into transcriptional regulation (see, e.g., Finkbeiner etal., 1997), and a calcium influx has been shown to be critical for phase shifting circadianoscillators in nonmammalian species (Colwell et al., 1994). Because the activation of NMDA

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receptors containing the NR2B subunit results in slower and larger EPSCs (Monyer et al.,1994; Barria and Malinow, 2002), BDNF enhancement of the NR2B subunit would result inmore current carried by the NMDA receptor and more calcium entry. Previous studies haveshown that blocking the NR2B subunit causes a major reduction in NMDA-induced calciumchanges in neurons (Sobczyk et al., 2005). There is also evidence that the NR2B subunit isphysically associated with Ras (Krapivinsky et al., 2003) and can play an important role in theregulation of the Ras-ERK pathway (Kim et al., 2005). This signaling pathway is thought tobe a critical component of the mechanisms underlying light-induced phase shifts of thecircadian system (Butcher et al., 2005). Therefore, we propose that BDNF regulates the effectsof light on the circadian system through the modulation of the NR2B subunit.

Previous work of Earnest and colleagues found that BDNF- or TrkB-deficient mice exhibitreduced magnitude of light-induced phase shifts (Liang et al., 2000; Allen et al., 2005). In bothcases, the mice homozygous for these mutations do not survive to adulthood, so the effects ofthe complete loss of BNDF/TrkB signaling on circadian behavior could not be evaluated. Inaddition, with a systemic knockout of the gene, the site of action of the loss of BDNF or TrkBreceptors on circadian behavior cannot be established, although it can be presumed to be in theSCN or the light-input pathway to the SCN. Nevertheless, this work strongly suggests a rolefor BDNF signaling in light- and glutamate-induced phase shifts of the circadian system. Ourdata provide support for the behavioral studies and indicate that the site of action of BDNF ison NMDA receptors within the SCN. Together, the data indicate that neurotrophins areimportant modulators of light’s actions on the circadian system.

ACKNOWLEDGMENTSY.I. Kim and H.-J. Choi were supported by the Brain Korea 21 Project in 2005.

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Fig. 1.Isolation of NMDA receptor-mediated synaptic currents in SCN neurons. Left: Example ofthe time courses of the EPSCs in an SCN neuron evoked by optic nerve stimulation. The holdingpotential (Vh) is indicated to the right of each current trace. The stimulus artifacts are indicatedwith arrowheads. Each of the current traces in this and the following figures is an average ofthree to eight individual traces. The top panel illustrates that NMDA-EPSCs were blocked bythe bath application of the NMDA receptor antagonist APV (50 μM, 5 min). The bottom panelshows the EPSCs measured at six different holding potentials. Right: Relationship betweenholding potential and the peak NMDA-EPSC current in the neuron from the left panel. Thetime of peak current measurement occurred between 30 and 80 msec poststimulation. As ischaracteristic of NMDA currents, the current-voltage relationship is not linear, with significantrectification seen at the hyperpolarized voltages. Depending on the cell, the peak inward currentoccurred with Vh between -20 and -40 mV.

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Fig. 2.BDNF enhanced the magnitude of NMDA-EPSC. Left: Example of the time courses of theNMDA-EPSCs evoked by optic nerve stimulation before and during treatment with BDNF(200 ng/ml; 5 min). The two traces are superimposed at the bottom to make the comparisoneasier. These recordings were made with Vh at +50 mV. Right: The peak amplitudes of theNMDA-EPSCs measured in each SCN neuron before and during BDNF treatment are plotted.In this and the other figures, lines connect data from a single neuron before and during BDNFtreatment. Solid circles indicate data from those cells whose response increased with BDNFtreatment.

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Fig. 3.BDNF enhanced the magnitude of NMDA currents in SCN neurons. The whole-cell patch-clamp recording technique was used to measure directly currents evoked by NMDA in ventralSCN neurons during the night. To isolate the postsynaptic response better, BIC (25 μM) andCNQX (20 μM) were added to the bath. In these experiments, the inward currents evoked bythe focal application of NMDA (0.5 mM; 3-sec or 5-sec pulse; applied once every 120 sec)were measured with Vh = -30 mV. The magnitudes of the NMDA-evoked currents weremeasured before, during, and after treatment with BDNF (200 ng/ml, 5 min). Left: An exampleof the NMDA-evoked currents recorded using this protocol before, during and after treatmentwith BDNF. Right: The peak amplitudes of the NMDA-evoked currents measured in eachSCN neuron before and during the BDNF treatment are plotted. Application of BDNF increasedthe magnitude of the NMDA currents. Solid circles indicate data from those cells whoseresponse increased with BDNF treatment.

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Fig. 4.The neurotrophin receptor antagonist K252a inhibited the effects of BDNF on SCN neurons.A: Example showing the time courses of the NMDA-EPSCs evoked before and during BDNFtreatment (focal application; 200 ng/ml, 5 min) in the presence of K252a in the bath (100 nM).B: The peak amplitudes of the NMDA-EPSCs measured in each SCN neuron before and duringBDNF treatment are plotted. C: The peak amplitudes of the NMDA-evoked currents measuredin each SCN neuron before and during BDNF treatment are plotted. For both B and C, solidcircles indicate data from those cells whose response increased with BDNF treatment in thepresence of K252a.

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Fig. 5.TrkB receptor IgG prevented the BDNF enhancement of the NMDA-EPSC evoked by opticnerve stimulation. Left: Example showing the time courses of the NMDA-EPSCs evokedbefore and during BDNF treatment (200 ng/ml, 5 min) in a slice pretreated with TrkB IgG (1μg/μl; 10 μl). Right: The peak amplitudes of the NMDA-EPSCs measured in each SCN neuronbefore and during BDNF treatment are plotted. Solid circles indicate data from those cellswhose response increased with BDNF treatment in the presence of TrkB IgG.

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Fig. 6.BDNF failed to enhance the magnitude of the NMDA-EPSC when the NR2B subunit of theNMDA receptor was inhibited. Left: Example showing the time courses of the NMDA-EPSCsevoked before and during treatment with BDNF (200 ng/ml, 5 min) in the presence of ifenprodil(3 μM). Right: The peak amplitudes of the NMDA-EPSCs measured in each SCN neuronbefore and during BDNF treatment are plotted. Solid circles indicate data from those cellswhose response increased with BDNF treatment in the presence of ifenprodil.

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