Developmental switch in the expression of NMDA receptors occurs in vivo and in vitro

Neuron,

Vol. 10, 267-278,

February,

1993, Copyright

0 1993 by Cell Press

Developmental Switch in the Expression of NMDA Receptors Occurs In Vivo and In Vitro ..

Keith Williams, Shari 1. Russell, Yu Min Shen, and Perry B. Molinoff Department University Philadelphia,

of

of Pharmacology Pennsylvania Pennsylvania

School of Medicine 19104-6084

The properties of many ligand-gated ion channels are altered during development. We have characterized a developmental switch in the sensitivity of NMDA receptors to the novel antagonist ifenprodil using ligand binding assays with rat brain membranes and voltage-clamp recording of Xenopus oocytes expressing NMDA receptors after injection of RNA from rat brain and from cloned subunits of the receptor. In neonatal rat brain, NMDA receptors have a uniformly high affinity for ifenprodil. During postnatal development, a second population of receptors having a IOO-fold lower affinity for ifenprodil is expressed and represents 50% of NMDA receptors in adult rat brain. This developmental change also occurred in cortical neurons maintained in primary culture. lfenprodil potently inhibited responses of homomerit NRl and heteromeric NRl/NRZB receptors but not NRllNR2A receptors expressed in oocytes, suggesting that inclusion of different NR2 subunits in native NMDA receptors can control the sensitivity to ifenprodil. Introduction The N-methyl-o-aspartate (NMDA) subtype of glutamate receptor is widely distributed in the central nervous system. This receptor is a ligand-gated ion channel complex that gates Nat, K+, and Ca2+ and contains distinct recognition sites for glutamate, glycine, Mg2+, Zn2+, and polyamines (Mayer and Westbrook, 1987; Collingridge and Lester, 1989). NMDA receptors play a pivotal role in the generation of various types of synaptic plasticity including the induction of some forms of long-term potentiation and theconsolidation of synaptic connections during development (Mayer and Westbrook, 1987; Collingridge and Lester, 1989; Bear et al., 1990; Constantine-Paton et al., 1990). MK-801 is one of several compounds, including phencyclidine and ketamine, that act as open-channel blockers of the NMDA receptor (Anis et al., 1983; Wong et al., 1986).As such, MK-801 labeled with either 3H or 125l is a useful tool to study the properties of the NMDA receptor and the interactions between various recognition sites on the receptor complex (Wong et al., 1986; Reynolds and Miller, 1988; Williams et al., 1991a). Binding of radiolabeled MK-801 is enhanced by glutamate, glycine, and some polyamines and is blockers inhibited by Mg2+, Zn*+, and open-channel (Foster and Wong, 1987; Reynolds and Miller, 1988;

Williamsetal.,1991a). IfenprodiI,originallydeveloped as an antihypertensive agent, is a novel noncompetitive antagonist of the NMDA receptorthat inhibits the binding of MK-801 (Carter et al., 1988; Reynolds and Miller, 1989), but its site and mechanism of action have not been defined. lfenprodil does not act at the recognition sites for amino acids or divalent cations, nor does it act as an open-channel blocker (Carter et al., 1988; Reynolds and Miller, 1989; Legendre and Westbrook, 1991). lfenprodil modulates some of the effects of polyamines on the NMDA receptor (Carter et al., 1989) but it does not appear to interact competitively at the polyamine recognition site (Reynolds and Miller, 1989; Williams et al., 1991b). It has been suggested that ifenprodil may stabilize an inactive or closed-channel conformation of the receptor (Reynolds and Miller, 1989) or promote a modal shift in channel gating properties (Legendre and Westbrook, 1991). The effects of ifenprodil have been reported to be biphasic in biochemical studies of NMDA receptors in adult rat brain and in electrophysiological studies of NMDA receptors on hippocampal neurons (Reynolds and Miller, 1989; Legendre and Westbrook, 1991), suggesting heterogeneity of NMDA receptors or the presence of two binding sites for ifenprodil on each receptor complex. Accumulating evidence suggests that the molecular properties of NMDA receptors are altered during development. NMDA receptors expressed in the visual cortex of neonatal rat and cat were reported to be more effective and to produce larger responses than receptors in adult animals (Tsumoto et al., 1987; Kato et al., 1991). Furthermore,theduration of postsynaptic NMDA receptor currents was longer in neonatal than in adult rat brain, probably as a result of differences in the channel kinetics of NMDA receptors (Hestrin, 1992). Developmental changes in the sensitivity of NMDA receptors to glycine and to Mg2+ have been reported (Ben-Ari et al., 1988; Morrisett et al., 1990; Kleckner and Dingledine, 1991; Williams et al., 1991a) and may contribute to the developmental differences in NMDA receptor-evoked synaptic responses. We have recently reported that the properties of the polyamine-binding site are also altered during postnatal development. In binding assays with [1251]I-MK-801, NMDA receptors expressed in neonatal rat brain were 7-fold less sensitive to spermine than receptors in adult brain. The increase in the apparent affinity for spermine occurred during the first 2 weeks of postnatal development (Williams et al., 1991a). NMDA receptors expressed on cortical neurons isolated from fetal rat brain exhibited a relatively low sensitivityto spermine, similar to that seen in neonatal rat brain. However, the developmental change in the sensitivity to spermine that was observed in vivo was not seen in cortical neurons maintained for upto5weeks in dissociated primary culture, indicating that some aspects

NlXltUl 268

Adult IC,,, = 0.65 pM (49%) = 76 pM (51%)

1O-3 [Ifenprodil] Figure 1. Inhibitory Neonatal and Adult

(M)

Effects of lfenprodil Rat Brain

The effects of ifenprodil on the binding determined using membranes prepared adult rat brain. Values are mean + SEM;

on NMDA

Receptors

of [‘“Ill-MK-801 from S-day-old n = 4.

in were and

of “maturation” of the glutamate receptor system do not occur in culture as they do in situ (Williams et al., 1991a). Changes in the properties of NMDA receptors that occur during development in vivo may be due.to alterations in the subunit composition of the receptors, analogous to that described for nicotinic acetylcholine receptors at the neuromuscular junction (Mishina et al., 1986). Because of the reported interactions between polyamines and ifenprodil, we initially set out to investigate whether the change in sensitivity to polyamines influences the inhibitory effects of ifenprodil during development. In this paper, a developmental switch in the sensitivity of NMDA receptors to ifenprodil that is temporally and mechanistically distinct from the change in sensitivity to polyamines is described. In membranes prepared from neonatal rat brain, ifenprodil inhibited the binding of [1251]l-MK-801 in a monophasic manner. In adult animals, the inhibitory effects of ifenprodil were biphasic and could be described by high and low affinity components. There was a progressive increase in the proportion of the low affinity component during postnatal development. This may be due to the expression of two populations of NMDA receptors, one with a high affinity and the other with a low affinity for ifenprodil, with the low affinity population being expressed later in development. This developmental switch was also seen in cortical neurons maintained in primary culture. Thus, the appearance of a low affinity binding site for ifenprodil on the NMDA receptor occurs during development in vivo and in vitro. Furthermore, these results suggest that expression of NMDA receptors on neurons in primary culture is not entirely “locked in” to an embryonic or neonatal form. Results Effects of lfenprodil during Development In Vivo Compounds, including ifenprodil, that act as noncompetitive antagonists of the NMDA receptor inhibit

the binding of radiolabeled channel blockers IikeMK801. The effects of ifenprodil on the binding of [1251]1MK-801 to membranes prepared from 3-day-old and adult rat brain were determined (Figure 1). The binding of [1251]l-MK-801 was measured in the presence of 100 PM glutamate, glycine, and spermidine. This combination of modulators allows maximal activation of NMDA receptors in both adult and neonatal rat brain, and the binding of [1251]l-MK-801 reaches equilibrium within 2-3 hr (Williams et al., 1991a). In membranes prepared from 3-day-old rat brain, ifenprodil inhibited the binding of [1251]l-MK-801 in an essentially monophasic fashion with an lCsO of 0.22 PM (Figure 1). In some experiments there was a small (I%-2%) low affinity component of inhibition that was not well resolved. In contrast, the inhibition in adult brain was markedly biphasic, with half of the inhibitory effect showing a high affinity (ICso = 0.65 PM) for ifenprodil and half a low affinity (I& = 76 PM) (Figure 1). To define the time course of changes in the effects of ifenprodil, inhibition curves were carried out using membranes prepared from I- to 21-day-old rat brain (Figure 2A). There was a progressive increase in the proportion of the low affinity component between postnatal days 7 and 21. By day 21 the proportions of low and high affinity inhibition were similar to those seen in adult brain (Figure 2; Table 1). A small change in the I& of the high affinity component was seen during development, while ICSO values for the low affinity component were not significantly altered over time (Table 1). We have previously reported that the affinity of the NMDA receptor for [1251]l-MK-801, measured at equilibrium in the presence of glutamate, glycine, and spermidine, is not altered during development (Williams et al., 1991a). The binding of [1251]l-MK-801 measured under these conditions is thus directly proportional to the density of NMDA receptors. The density of NMDA receptors increased by approximately 7-fold during development, as assessed by the level of [1251]1MK-801 binding (Figure 2B, inset). Assuming that the high and low affinity components of the inhibitory effect of ifenprodil are accounted for by two separate populations of receptors, the number of high and low affinity ifenprodil sites was calculated. The number of both high and low affinity sites increased during postnatal days 7-21, with a progressive increase in the proportion of low affinity sites (Figure 2B). Receptors with a low affinity for ifenprodil were not detectable before postnatal day 7. The effects of modulators that alter channel opening of the NMDA receptor can also be studied by kineticanalyses of the binding of radiolabeled MK-801 (e.g., Reynolds and Miller, 1988; Williams et al., 1990). Glutamate, glycine, and spermidine, which act to increase channel opening and receptor function, accelerate the rates of association and dissociation of MK801, presumably by increasing access of the ion channel-binding site for MK-801. Some allosteric modulators that inhibit receptor activation have the

NMDA 269

Receptors

during

Development

Figure2. Change

0 7-Day-Old IO-Day-Old

V 14.Day-Old ‘I Pi-Day-Old

10-g

1 o-s

10-7

1o-5

10-s [Ifenprodil]

Low-affinity component

F 0.5 3 3 K

1 os

Inhibition by ifenprodil. q High-affinity component

f “’ I E

0.4

the

(M)

B 0.6

10’4

of

(A) The effects of ifenprodil on the binding of r251]l-MK-801 were determined using membranes prepared from I- toLl-day-old rat brain. Inhibition curves were fit to onesite (1 day) or two-site (7-21 day) binding isotherms. Values are mean from three or four determinations at each age; error bars (SEM < 10% of mean values) have been omitted for clarity. (B) The proportion of NMDA receptors accounting for the high and low affinity components of ifenprodil inhibition was calculated from the total specific binding of [1251]l-MK-801 (inset) and the percentage of the high and low affinity components (Table 1) at each age.

q I-Day-Old

.

Developmental Profile in Sensitivity to lfenprodil

TT

0 1

3

7

10

14

21

Adult

Age (days) 1

n 1

3

7

10

14

21

Adult

Age Ways)

opposite effect-they decrease the rates of association and dissociation of MK-801. In membranes prepared from 3-day-old or from adult rat brain, the rates of association and dissociation of [1251]l-MK-801 were biphasic under control conditions (in the presence of

Table Brain

1. High

and

Low Affinity High

Components

of Inhibition

glutamate, glycine, and spermidine). Both the fast and slow rates of association were45 times faster in adult than in 3-day-old brain (Figures 3A and 3B; Table 2). The effects of concentrations of ifenprodil that would affect predominantly the high affinity site (1 PM) or

of [‘251]l-MK-801

Affinity

Binding

by lfenprodil

in l-Day-Old

through

Low Affinity

Age

Go

(PM)

%

ICso (PM)

%

1 Day 3 Day 7 Day 10 Day 14 Day 21 Day Adult

0.26 0.22 0.26 0.41 0.60 0.67 0.65

k + k + * k f

100 99 92 86 67 53 49

133 106 77 67 76

0 I*1 8k3 14 + 33 * 47 + 51 &

0.06 0.04 0.06 0.04 0.15 0.18" 0.15"

Values are mean + SEM, n = 3-4 individual a p < 0.05 compared with l-day high affinity

f + f f + f

1 3 3 2 3 3

f + + + k

33 33 8 12 11

animals or groups of animals at each age. lCs0 (one-way ANOVA with post hoc Dunnett’s

test),

3 2 3 3

Adult

Rat

NWtD” 270

Adult

3-Day-Old

*

0

Control

1000 0””

0

no

--..

’

:

500 1 uM ifenprodil 250

0

60

180

0

0'

60

420

0' 0

60

120’.

180

240

300

0

60

Minutes Figure

3. Effects

of lfenprodil

on the

120

180

240

300

Minutes Kinetics

of Binding

of [1~51]l-MK-801

The effects of 1 PM and 30 uM ifenprodil on the kinetics of association (A and 6) and dissociation (C and D) of binding of [1251]l-MK-801 were determined in membranes prepared from 3-day-old (A and C) and adult (B and D) rat brain. For dissociation experiments, samples were incubated for 180 min followed by the addition of unlabeled MK-801 (IO PM) with or without ifenprodil.

both the high and low affinity sites (30 PM) were examined. Addition of 1 uM or 30 uM ifenprodil decreased the rate of association of [1251]l-MK-801 in both 3-dayold and adult tissue (Figures 3A and 3B; Table 2). In 3-day-old brain, a fast component of association was not detected in the presence of ifenprodil, and the slow rate was decreased. In adult brain, ifenprodil decreased the fast and slow rates of association of [1251]l-MK-801 binding without altering the proportion of each rate (Table 2). lfenprodil abolished the fast component and decreased the rate of the slow component of dissociation in both adult and 3-day-old brain (Figures 3C and 3D; Table 3). A given concentration of ifenprodil had 2-to 3-fold greater effects on the rate of dissociation of [1251]l-MK-801 in neonatal than in adult brain (Table 3), similar to its effect on the rate of association. Zn2+ is a noncompetitive antagonist of the NMDA receptor that also decreases the rates of association

Table

2. Effects

of lfenprodil

on the Association

of Binding

and dissociation of MK-801 (Reynolds and Miller, 1988; Williams et al., 1990). Pentamidine, another atypical noncompetitive antagonist, has also been reported to decrease the rate of dissociation of MK-801 (Reynolds and Aizenman, 1992). To investigate the specificity of the developmental change in sensitivity to ifenprodil, inhibition curves for Zn2+ and pentamidine were measured in membranes prepared from 3-day-old and adult rat brain. There was no change in the sensitivity to Zn2+or to pentamidine during development (Figure 4). Thus, the switch in sensitivity to ifenprodil is not generalized to binding sites for other noncompetitive antagonists. NMDA Receptors Expressed in Experiments were carried out there are functional correlates changes observed in biochemical receptors and whether these

of [‘*51]l-MK-601

3-Day-Old kobr (minP

Control 1 uM lfenprodil 30 PM lfenprodil Values

are mean

from

two

Xenopus Oocytes to determine whether to the developmental assays of NMDA changes are a conse-

Adult x 103)

kabr (min-’

x 103)

Fast

Slow

% Fast

Fast

Slow

% Fast

18.5 -

6.3 3.5 2.0

79 0 0

101 26 19

25.5 5.5 3.5

47 42 47

experiments.

NMDA 271

Table

Receptors

3. Effects

during

Development

of lfenprodil

on the Dissociation

of Binding

of [1251]l-MK-801 Adult

3-Day-Old k,rr (min-’

Control 1 pM lfenprodil 30 uM lfenprodil Values

are mean

from

two

x 103)

k,rr (min-’

x 103)

Fast

Slow

% Fast

Fast

Slow

% Fast

15 -

2.4 0.4 0.1

30 0 0

20 -

5.1 1.7 0.7

41 0 0

experiments.

quence of alterations in mRNAs coding for the receptor subunits. In these experiments, the effects of ifenprodil on NMDA-induced responses were measured in Xenopus oocytes injected with RNA prepared from l-day-old, 21-day-old, and adult rat brain. In oocytes injected with rat brain RNA and voltage clamped at -70 mV, application of 100 j.rM NMDA produced inward currents of 20-200 nA when responses were measured in a Mg2+and Ca2+-free buffer containing Bach and 5 uM glycine (Figure 5A). Control responses were 2- to 3-fold larger in cells injected with 21-day or adult RNA (80 f 8 nA, mean f SEM, n = 32) than with l-day RNA (31 + 4 nA, n = 26). Ifenprodil, at concentrations up to 100 PM, did not induce currents or alter membrane resistance (data not shown). lfenprodil inhibited the response to NMDA in a concentration-dependent manner (Figure 5). Recovery from inhibition required 3-15 min; with concentrations of ifenprodil less than 30 PM the response to NMDA recovered to 90%-100% of values seen before application of ifenprodil (data not shown). Thus, in many oocytes it was possible to measure the effects of four to six concentrations of ifenprodil. A given concentration of ifenprodil produced a greater inhibition of steady-state NMDA currents in oocytes injected with RNA from l-day-old brain than in oocytes injected with RNAfrom adult brain (Figure 5). The concentration-response relationship in cells injected with l-day RNA was monophasic, with an I&, of 0.31 PM. In oocytes injected with 21-day or adult RNA, the concentration-response curve was biphasic, with high (I& = 0.47 PM, 76%) and low affinity (I& = 200 PM, 24%) components (Figure 5B). The I& values for ifenprodil measured in functional assays with Xenopus oocytes were similar to those seen in binding assays with [1251]I-MK-801. The presence of a low affinity component in cells injected with RNA from 21-day-old or adult brain, but not from l-day-old brain, is consistent with the delayed developmental appearance of a low affinity site as detected in ligand binding assays. Cloned NMDA Receptor Subunits Expressed in Oocytes A number of cDNAs coding for subunits of the NMDA receptor have recently been cloned and sequenced. These include the NMDARI (NRI) and NR2A, NR2B,

and NR2C genes isolated from rat brain (Moriyoshi et al., 1991; Monyer et al., 1992). The NRI subunit can form functional homomeric NMDA receptor/thannels after expression in Xenopus oocytes or mammalian cells, but the currents activated bythese receptors are very small. Much larger NMDA-evoked currents are seen after coexpression of NRI and NR2 subunits, suggesting that native NMDA receptors are composed of combinations of NRI and NR2 subunits (Moriyoshi et al., 1991; Monyer et al., 1992; Kutsuwada et al., 1992). To determine whether differences in NRl/ NR2 subunit composition may underlie differences in the sensitivity to ifenprodil, we measured the effects of ifenprodil on homomeric NRI and heteromeric NRl+NR2A and NRl+NRZB receptors expressed in Xenopus oocytes. NMDA or glutamate produced small inward currents in oocytes injected with the NRI subunit alone. In many cells, NMDA-induced currents could not be reliably measured in soultions containing BaCl2 rather than CaCb. Thus, for homomeric NRI receptors, we determined the effects of ifenprodil on currents induced by 100 FM glutamate, which produces larger responses than NMDA (Moriyoshi et al., 1991), in an extracellular solution containing CaC12. The shape of

25

tan”1 (W Figure 4. The Inhibitory Effects Altered during Development

of Zn2+ and Pentamidine

Are Not

The effects of Zn2+ (A) and pentamidine (6) on the binding of [1251]l-MK-801 to membranes prepared from 3-day-old and adult rat brain were determined. Values are mean f SEM, n = 3 (Zn*+) or mean f range, n = 2 (pentamidine).

NW"J" 272

A

l-Day

RNA

Adult RNA NMDA

[ifenprodil] (pM)

NMDA+ Ifen 0.1

0.3

1.0

3.0

WL

1ml”

the currents was more complex than that seen in the effects of ifenpresence of Ba *+ , but the inhibitory prodil on steady-statecurrentscould be reliablyquantified (Figure 6). lfenprodil inhibited responses of homomeric NRI receptors in a monophasic fashion with an affinity (I& = 0.28 PM) similar to that seen with receptors from neonatal rat brain (Figure 6). Currents activated by heteromeric NRl+NR2 receptors were S- to 15-fold larger than those seen after expression of NRI alone in the same batch of oocytes. Therefore, for NRl+NRZ receptors, NMDA-induced currents were measured in the presence of Ba2+ (Figure 7A). Similar conditions were used in experiments with oocytes injected with brain RNA. lfenprodil potently inhibited responses of heteromeric NRl+NR2B receptors (Figure 7). The degree of inhibition seen with 0.3 uM and 3 uM ifenprodil was similar to that seen with oocytes expressing receptors from neonatal rat brain RNA (Figure 7B). In contrast, ifenprodil was only a very weak antagonist of currents activated by heteromeric NRl+NRZA receptors (Figure 7).

Effects of lfenprodil

10-7

10-s

[Ifenprodil] Figure 5. Effects Xenopus Oocytes

of lfenprodil

10-s

(M)

on NMDA-Induced

Currents

in

(A) Representative pen-recorder traces of NMDA-induced currents in oocytes injected with RNA prepared from l-day-old (left panel) or adult (right panel) rat brain. Oocytes were voltage clamped at -70 mV. The extracellular solution contained 5 pM glycine and 1.8 mM Bach. NMDA (100 PM), in the absence or presence of the indicated concentrations of ifenprodil, was applied during the times shown by the horizontal bars, which are not corrected for dead space in the perfusion system (3-5 s). Inward currents are downward. Arrowheads in the top traces indicate where steady-state currents were measured in the presenceof NMDAor NMDA+ ifenprodil. Note thatthe rateatwhich block develops increases with increasing concentrations of ifenprodil at both ages, but a larger inhibition of NMDA currents is seen in cells injected with l-day-old than with adult RNA. Corresponding tracesforoocytes injected with l-day-old or adult RNA are from experiments carried out on the same day with oocytes from the same donor frog. (6) Inhibitory effects of increasing concentrations of ifenprodil on NMDA-induced currents in oocytes injected with RNA from l-day-old (open circles) and 21-day or adult (closed circles) rat brain. Results obtained with 21-day RNAwere indistinguishable from thoseobtained with adult RNA, and data from theseexperimentswerecombined.Solidlinesshowthefitstoone-site(l-day) or two-site (21-day/adult) binding isotherms. Values are mean + SEM of results obtained with three batches of RNA at each age, with a total of 5-13 cells at each concentration. Where error bars are not shown they are within the size of the symbol. Broken lines are the fitted curves for [1251]l-MK-801 binding in l-day-old and adult rat brain, which are replotted from Figure 1 and Figure 2 for comparison.

during

Development

In Vitro

Experiments were carried out to determine whether the developmental switch in the sensitivity to ifenprodil also occurs in vitro. For these experiments, cortical neuronswere prepared from embryonic rat brain and maintained in primary culture. Neurons, in coculture with glia, were maintained for 7-28 days in vitro. NMDA receptors appear to be expressed exclusively on neurons in such cultures, and the receptors have

1O-7

10-6

[Ifenprodil]

(M)

Figure 6. Effects of lfenprodil pressed in Xenopus Oocytes

on Homomeric

NRI

Receptors

Ex-

The effect of increasing concentrations of ifenprodil on the response to 100 uM glutamate was determined in oocytes injected with the NRI subunit alone. Oocytes were voltage clamped at -70 mV.Theextracellularsolution contained 1.8 mM CaC&. Data are expressed as a percentage of control currents measured in the absence of ifenprodil. Values are mean f SEM from 4-7 cells at each concentration. (Inset) Representative glutamate-induced currents. L-Glutamate (100 PM; with 10 PM glycine), in the absence or presence of 3 PM ifenprodil, was applied during the times shown by the horizontal bars. Inward currents are downward.

NMDA 273

Receptors

during

Development

A

B 100

NMDA + 3 pM ifen

NMDA --

__________~~____________________ I3 NRl

t

NRI + NRPA =F

75

+ NRPA

0 1-Day rat brain

2= 00 ub $S NMDA + 3 uM ifen

NMDA

NRl + NRPB

sr” co l@

50

’

25

NMDA

+

NMDA

0.3 PM lfenprodil Figure

7. Effects

of lfenprodil

on Cloned

NMDA

Receptors

Expressed

in Xenopus

+

3 PM lfenprodil Oocytes

(A) Representative NMDA-induced currents in cells expressing heteromeric NRl+NWAand NRl+NR2B receptors. Oocyteswerevoltage clamped at -70 mV. The extracellular solution contained 1.8 mM BaCIZ. NMDA (100 PM; with 10 RM glycine), in the absence or presence of 3 PM ifenprodil, was applied during the times shown by the horizontal bars. Inward currents are downward. (B) Effects of ifenprodil (0.3 and 3 PM) on NMDA-induced currents in oocytes injected with NRl+NR2A and NRl+NR2B subunits. Data are expressed as a percentage of the control current measured in the absence of ifenprodil. Values are mean + SEM from 5 cells at each concentration. Data for oocytes injected with l-day-old rat brain RNA (open bars) are replotted from Figure 5 for comparison.

characteristics brain

with

similar regard

to their

to

their

counterparts

for [1251]l-MK-801 and glycine (Williams

affinity

in

rat and et

their sensitivity to glutamate al., 1991a, 1992). There was an increase in the density of NMDA receptors on neurons maintained in culture for 7-28 days (Figures, inset). lfenprodil inhibited the binding of [1251]l-MK-801 to membranes prepared from cultured neurons.At all time points examined, the inhibitory effects of ifenprodil were biphasic (Figure 8). lCSO values for the high and low affinity components of inhibition were similar to those seen with NMDA receptors in rat brain. The proportion of the low affinity component of inhibition increased over time in culture, similar to the increase seen in vivo. In 7-day cultures, the low affinity component accounted for only 12% of the inhibition by ifenprodil. The proportion increased progressively to 29% by 21-28 days in vitro (Figure 8). Discussion The structural and functional properties of a number of ligandand voltage-gated ion channels have been found to change during development. The bestdocumented example involves changes in the properties of nicotinic acetylcholine receptors expressed on skeletal muscle. In neonatal, noninnervated muscle fibers, pentameric acetylcholine receptors contain a y subunit. Receptors expressed at the motor end plate in innervated adult muscle have an E subunit that replaces they subunit (Mishina et al., 1986). The switch

in subunit composition (from y to E) is responsible for differences in the gating properties of fetal as compared with adult receptors (Mishina et al., 1986). Another developmental change in the expression of acetylcholine receptor genes has been reported. Only an al, subunit is expressed in Xenopus oocytes and early stage embryos, while al, and alb subunits are coexpressed in late stage embryos and adult Xenopus muscle (Hartman and Claudio, 1990). In the central nervous system, developmental changes in the affinity of glycine receptors for strychnine are due to the expression of different forms of the a subunit of these receptors in neonatal and adult animals (Kuhse et al., 1990). Changes in the levels and distribution of mRNAs coding for subunits of neuronal acetylcholine receptors, y-aminobutyric acidA receptors, and a-amino-3hydroxy-5-methyl4isoxazolepropionic acid (AMPA) receptors have also been observed during development and may be involved in the expression of receptors having different functional and pharmacological properties (Bettler et al., 1990; Daubas et al., 1990; Killisch et al., 1991; Monyer et al., 1991; Poulter et al., 1992). Thus, alteration in the expression and molecular composition of ligand-gated ion channels during development appears to be a general phenomenon and may be related to defining the correct connectivityandfunctioningof synapsesandthecharacteristics of synaptic plasticity. Developmental changes in the sensitivity of NMDA receptors to Mg2+, glycine, and polyamines have been reported (Ben-Ari et al., 1988; Morrisett et al., 1990; Kleckner and Dingledine, 1991; Williams et al., 1991a).

NeUrO”

274

c

2oo,

[Wll-MK-801

'o-5

'o-4

binding

25

0 1O-g

10'8

10.' I,knpro~~l-~M)

Figure 8. Inhibitory Effects of lfenprodil Expressed on Cultured Neurons

on

NMDA

1o-3

Receptors

The effects of ifenprodil on the binding of [‘251]1-MK-801 were determined using membranes prepared from cortical neurons maintainedfor7,14,21,or28daysin dissociated primary culture. Inhibition curves were fit to two-site binding isotherms. There was no difference between the inhibition seen in 21-and 28-day cultures, and data from these experiments were pooled. The percentage of the low affinity component of inhibition was 12%, 15%, and 29% in 7-, 14, and 21-28-day cultures, respectively. lCSO values were 0.13 PM, 0.25 PM, and 0.40 PM for the high affinity component, and 100 PM, 72 PM, and 63 PM for the low affinity component in 7-, 14, and 21-28-day cultures, respectively. Values are mean from three to six determinations at each time. Broken lines, which show the fitted curves for ifenprodil inhibition in 3-day-old and adult rat brain, are replotted from Figure 1 for comparison. (Inset)The level of [‘251]l-MK-801 binding (fmol/ mg protein) measured in the absence of ifenprodil is shown at various times in culture.

In this paper we have characterized another developmental alteration in the properties of NMDA receptors. The major observation from this work is that ifenprodil interacts with high affinity at a single class of NMDA receptors in neonatal rat brain, while the inhibition in adult brain is biphasic, with high and low affinity components. The proportion of the low affinity component increased progressively during postnatal days 7-21. The most likely explanation for these results is the existence of two populations of NMDA receptors, one having a high affinity binding site for ifenprodil and another, expressed later in development, with a low affinity binding site. An alternative interpretation is that NMDA receptors expressed in neonatal rat brain contain a single ifenprodilbinding site while receptors in adult brain each have two binding sites with different affinities for ifenprodil. In addition to the change in the proportion of receptors having a low affinity for ifenprodil, there was also a small, progressive change in the ICso values for ifenprodil during development. The model used to determine I&, values assumes two independent classes of binding sites for ifenprodil on separate populations of receptors. If, however, two ifenprodil sites are expressed on each receptor, there may be interactions between the two sites that could influence their affinities. Another possibility is that the properties of the

ifenprodil-binding site are affected by changes in the efficacy of glycine during development, since the affinityfor ifenprodil is dependent in part on the activation state of the receptor complex (Reynolds and Miller, 1989; Legendre and Westbrook, 1991). Furthermore, part of the mechanism of action of ifenprodil may involve noncompetitive antagonism of theeffects of glycine (Legendre and Westbrook, 1991). The mechanism of action of ifenprodil at the NMDA receptor has not been clearly defined, but ifenprodil may act as a “closed-channel blocker.” In the present study, ifenprodil decreased the association and dissociation of binding of [12s1]I-MK-801, consistent with results of a previous report that ifenprodil slows the dissociation of [3H]MK-i301 (Reynolds and Miller, 1989). These results are consistent with a model in which ifenprodil promotes or stabilizes a closed-channel conformation of the receptor, such that the ion channel-binding site for MK-801 is less accessible. Interactions between polyamines and ifenprodil at NMDA receptors have been reported (Carter et al., 1989), but ifenprodil does not appear to interact competitively at the positive allosteric polyamine site (Reynolds and Miller, 1989; Legendre and Westbrook, 1991; Williams et al., 1991b). The affinity of the NMDA receptor for polyamines is altered during postnatal development, and it is conceivable that this change may be responsible in part for the change in the proportion of receptors having a low affinity for ifenprodil. However, several observations suggest that this is not the case. First, all of the biochemical experiments in the present report were carried out in the presenceof aconcentration of spermidine (100 FM) that causes maximal enhancement of [1251]l-MK-801 binding in both neonatal and adult animals. Second, the developmental time course of the change in sensitivity to ifenprodil is different from that of polyamines. The increase in affinity forspermineoccursduringthefirst2weeksofpostnatal life and is almost complete by postnatal day 10 (Williams et al., 1991a). In contrast, the increase in the number of low affinity ifenprodil sites occurs later and does not reach adult levels until day21. Finally, a change in the apparent affinity of NMDA receptors for spermine was not observed in cortical neurons maintained for up to 5 weeks in dissociated primary culture (Williams et al., 1991a), whereas a change in the inhibitory profile of ifenprodil occurred during days 7-21 in vitro in such cultures. These results suggest that developmental changes in the effects of ifenprodil are not related directly to changes in the sensitivity to polyamines and that different molecular mechanisms are probably responsible for the two phenomena. These data also reinforce the conclusion that ifenprodil does not interact competitively at the polyamine recognition site. The Xenopus oocyte expression system is useful for studying the pharmacology and function of NMDA receptors expressed from brain RNA (Verdoorn et al., 1987). A functional correlate to the results observed in

NMDA 275

Receptors

during

Development

ligand binding experiments was seen in studies of NMDA receptors expressed in Xenopus oocytes. In oocytes injected with RNAfrom adult rat brain, NMDA receptors having a low and a high affinity for ifenprodil were seen. Receptors expressed from l-day-old rat brain RNA had only a high affinity site for ifenprodil. The affinities measured in this paradigm were similar to those determined in binding assays with [1251]l-MK-801 and to those reported in patch-clamp studies of cultured hippocampal neurons (Legendre and Westbrook, 1991). The expression of NMDA receptors differing in sensitivity to ifenprodil presumably reflects the presence of different molecular forms of the NMDA receptor-ion channel complex after injection of oocytes with RNA prepared from neonatal or adult rat brain. The most likely explanation for this is the existence of different mRNA species coding for subunits of the NMDA receptor during development. This would be analogous to the developmental changes in the levels of mRNAs coding for subunits of acetylcholine, y-aminobutyric acid, glytine, and AMPA receptors. Native NMDA receptors are thought to be heterooligomeric complexes composed of four or five interacting subunits. The subunit composition of brain NMDA receptors has not yet been defined, but molecular cloning has identified two families of rat brain NMDA receptor subunits termed NRI and NR2 (Moriyoshi et al., 1991; Monyer et al., 1992). The NR2 family includes NRZA and NR2B, which are expressed widely in the forebrain of adult rats, and NR2C, which is expressed almost exclusively in the cerebellum (Monyer et al., 1992). Recently, the existence of multiple isoforms of NRI, which probably arise through alternative splicing of RNA, has been reported (Anantharam et al., 1992; Sugihara et al., 1992). Differences in NR2 subunit composition may underlie differences in the affinity of receptorsforglycineand Mg*and the properties of cerebellar versus forebrain NMDA receptors (Monyer et al., 1992; Kutsuwada et al., 1992). In the present work we found that homomeric NRI receptors have a high affinity for ifenprodil, similar to that of receptors expressed from neonatal brain RNA. This suggests either that the high affinity ifenprodil site is localized on the NRI subunit, or that the interaction of two or more NRI subunits is sufficient to form a functional, high affinity ifenprodil site. Heteromeric NRl/NR2B receptors have a sensitivity to ifenprodil similar to that of homomeric NRI receptors, while NRlINR2A receptors have a very low apparent affinity for ifenprodil. These data suggest that NRllNR2B subunits interact to form a high affinity site, whereas NRl/ NR2A subunits interact to form a low affinity site. Alternatively, the inclusion of NR2A or NR2B in heteromerit NRI-NR2 complexes may alter the affinity of an ifenprodil-binding site localized on the NRI subunit. It is reasonable to propose that inclusion of the NR2A subunit in native NMDA receptors is responsible for the generation of receptors having a low affinity for

ifenprodil. Changes in the levels of expression or the receptor stoichiometry of NR2A and NR2B subunits may underlie developmental changes in the sensitivity of NMDA receptors to ifenprodil. The developmental change in the sensitivity of NMDA receptors that occurred in vivo was also seen in vitro. A progressive increase in the proportion of the low affinity ifenprodil site was seen in cortical neurons maintained for 7-28 days in dissociated primary culture. The change in sensitivity to ifenprodil occurs during a period of neuronal maturation and at a time when the density of NMDA receptors expressed on the neurons is increasing. It is possible that neurons that mature early in development express only NMDA receptors having a high affinity ifenprodil site, while later in development, NMDA receptors having the low affinity site are expressed on another population of neurons. Alternatively, individual neurons may express both forms of the NMDA receptor, with the low affinity form being expressed only after day 7 in vitro. The observation that the properties of NMDA receptors expressed on neurons in vitro are altered over time suggests that this change is mechanistically similar to the developmental change observed in vivo. In contrast to the switch in sensitivity to ifenprodil, a change in affinity for polyamines occurs in vivo but not in cultured cortical neurons (Williams et al., 1991a). Thus, different mechanisms, possibly involving changes in expression of NMDA receptor subunits or isoforms, are responsible for these two phenomena. These observations suggest that the normal developmental sequence of NMDA receptor expression is arrested at different stages in vitro. Cultured neurons thus provide a useful model system for investigating factors that control the developmental switch in the properties of NMDA receptors. It is possible that neurons are”preprogrammed” to express different forms of the NMDA receptor at different stages of maturation. Alternatively, these -changes may be due to neuralor glial-derived factors, neuronal activity, or growth factors in the culture medium. The difference between the l&s of the high and lowaffinitycomponentsof ifenprodil inhibition islOOto 300-fold. Furthermore, the low affinity site is virtually undetectable in neonatal rat brain while the ratio of high to low affinity sites in adult brain is 1:l. These changes are thus relatively large compared with changes in the effects of glycine and Mg2’(2to cfold; Kleckner and Dingledine, 1991; Williams et al., 1991a). NMDA receptors having either a high or a low affinity for ifenprodil can be characterized with relative ease in binding assays with [1251]1-MK-801 and in electrophysiological assays of receptors expressed in Xenopus oocytes. lfenprodil is thus a useful tool for fingerprinting different molecular forms of the NMDA receptor-ion channel complex using a variety of experimental approaches. lfenprodil has been shown to be neuroprotective

NeUKNl 276

in models of focal cerebral &hernia and to possess anticonvulsant activity (Gotti et al., 1988; Carter et al., 1991). It is possible that the neuroprotective effects of ifenprodil are due to its action as an antagonist at the NMDA receptor. However, ifenprodil can also interactwith high affinityat aI-adrenergic receptors, some types of voltage-dependent Ca2+ channels, and socalled “o-sites” (reviewed in Carter et al., 1991; Williams et al., 1991b); antagonism at these sites cannot be excluded as a possible locus for the neuroprotective effects of ifenprodil. Nonetheless, it is possible that ifenprodil, or related compounds, will be clinically useful by virtue of their antagonist effects at NMDA receptors. lfenprodil does not share the behavioral and motor side effects associated with some competitive NMDA receptor antagonists and openchannel blockers (Carter et al., 1991). The identification of two developmentally regulated ifenprodilbinding sites associated with the NMDA receptor has implications for the design and use of novel drugs targeted toward NMDA receptors and for understanding the mechanism of action of novel allosteric antagonists and the structural features of different subtypes of NMDA receptor. Experimental

Procedures

Primary Culture of Cortical Cells Neuron-glia cocultures were prepared from neocortex of embryonic rats at 17 days gestation as previously described (Williams et al., 1991a, 1992). Cells were plated at a density of l.O1.3 x IO* cells per cm* in 100 mm plastic dishes that had been precoated with poly-o-lysine (20 Bglml). Cells were maintained in culture medium (Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum, 10% Ham’s F-12 nutrient mixture, 50 U/ml penicillin, and 50 pg/ml streptomycin) at 37OC in a humidified atmosphere containing 7% C02. Medium was replaced after 1 day and subsequently replaced every 3-5 days. Under these conditions, neurons grow predominantly in a dispersed monolayer on a layer of dividing glial cells. After 6-7 days in culture, when theglialcell layer had becomeconfluent, cytosinearabinoside (IO BM) was added for 24 hr to prevent further proliferation of nonneuronal cells. Binding Assays with [‘251]1-MK-B01 A washed membrane fraction was prepared from the brains (minus cerebellum and brain stem) of I- to 21-day-old or adult (1416 weeks old) Sprague-Dawley rats, or from cultured cortical neurons as previously described (Williams et al., 1991a). Membranes were stored at -8OOC until assayed (within 7 days). Aliquots of brain membranes or membranes from cultured neurons were thawed, diluted in assay buffer (20 mM K-HEPES, 1 mM K-EDTA [pH 7.0]), washed twice by incubation at 32OC for 30 min, centrifuged (34,000 x g, 30 min), and resuspended in assay buffer. Binding assays with [‘251]1-MK-801 (0.2 nM; specific activity, 2200 Ci/mmol) were carried out as previously described (Williams et al., 1991a)with brain membranes (5-15 pg of protein) or membranes prepared from cultured neurons (20-40 ug of protein). All assays contained 100 uM glutamate, 100 uM glycine, and 100 PM spermidine. This combination of modulators produces maximal enhancement of binding of [‘“l]l-MK-801 in membranes from both neonatal and adult animals (Williams et al., 1991a). Duplicate samples were incubated at 32OC for 3 hr. Nonspecific binding (
to 7 hr). For studies of the dissociation of [‘Z51]l-MK-801, membranes were incubated for 3 hr with [‘251]l-MK-801 in a total volume of 12 ml, followed by the addition of unlabeled MK-801 (IO ~1; 10 trM final concentration) with or without ifenprodil (1 or 30 uM final concentration). Aliquots (200 ~1) were removed at various times. Data from kinetic analyses were fit to a single exponential or to the sum of two exponentials to determine values for the observed rates of association (kobr) and the dissociation rate constants (k,tt). Data from inhibition curves were analyzed by leastsquares nonlinear regression curve fitting to obtain values for the lCSoof the high and low affinity components and the proportion of each component. Data were fit to a one-site or a two-site binding isotherm using the following equations: (one

site)

(two

site)

where B is the specific binding of [‘Z51]l-MK-801, BIFhlgh and BIFlaw are the fractions of the high and low affinity components of inhibition, I&,s are the corresponding IC5,,values for ifenprodil, and [IF] is the concentration of ifenprodil. Preparation of RNA Total RNA was prepared from rat brain by a modification of the method of Auffrayand Rougeon (1980).Tissuewas homogenized in 6 M urea, 3 M LiCl containing 10 mM NaOAc and 0.1% SDS, and the homogenate was allowed to stand at 4°C for 12-18 hr. Homogenates were centrifuged, and RNA in the pellet was extracted three times against phenol, chloroform, isoamyl alcohol (25:24:1) and twice against chloroform, isoamyl alcohol (241). RNA was precipitated, dissolved in water (3-5 bglpl), and stored in aliquots at -8OOC. The plasmid pN60,containingthefulI-length coding sequence for NRI (Moriyoshi et al., 1991), was a gift from Dr. S. Nakanishi (Kyoto University, Japan). This clone corresponds to the NRIA splice variant (Sugihara et al., 1992). Plasmids containing the NR2A and NRLB clones (Monyer et al., 1992) were a gift from Dr. P. H. Seeburg (University of Heidelberg, Germany). In vitro synthesis of RNA was carried out using protocols described by Promega Corporation (Promega Protocols and Applications Guide, second edition, 1991). After linearization with Notl (NRI), EcoRl (NR2A), and EcoRV (NR2B), capped RNA was synthesized from each of thecDNA templates usingT7(NRl)orT3(NR2Aand B) RNA polymerase in the presence of m7GG’)ppp(S’)G. RNAasefree DNAasel was then added to remove the template DNA, and RNA transcripts were isolated and purified. RNA was dissolved in water and stored in aliquots at -8O’C. Electrophysiology on Xenopus Oocytes AdultfemaleXenopus laevis (Xenopus I,Ann Arbor, Ml or Nasco, Fort Atkinson, WI) were anesthetized by immersion in tricaine (0.17%), and ovarian lobes were removed through a small incision in the abdomen. Lobes were opened with forceps, and the follicular layer was softened by incubation with collagenase (2 mglml; 2-3 hr) in a saline solution (82 mM NaCI, 2.5 mM KCI, 1 mM MgCb, 5 mM HEPES [pH 7.61). The remaining follicle cells were then removed manually, and the oocytes werewashed and placed in fresh saline solution (96 mM NaCI, 2 mM KCI, 1 mM MgCl?, 1.8 mM CaCI,, 5 mM HEPES, 2.5 mM sodium pyruvate, 50 Bg/ml gentamycin [pH 7.61). StageVor VI oocytes (Dumont, 1972) were injected in the vegetal pole with brain RNA (50 nl; 150-250 ng) or with cRNA prepared from cDNA clones (IO-25 ng of NRI and 12-25 ng each of NRl+NRZA or NRlfNR2B) and maintained at 18OC for 3-5 days before recordings were made. For some experiments on the expression of brain RNA, oocytes were maintained in saline solution supplemented with 5% horse serum (Quick et al., 1992). Oocytes were transferred to a serum-free solution 3-12 hr before recording. Addition of horse serum improved the viability of the cells and their resting membrane po-

NMDA 277

Receptors

during

Development

tential but had no qualitative effect on the response to NMDA or the inhibition of NMDA responses by ifenprodil. For recording, oocytes were positioned in a small perspex chamber and continuously superfused (5-10 ml/min) with a Mg2+-free saline solution (96 mM NaCI, 2 mM KCI, 1.8 mM BaCI,, 5 mM HEPES [pH 7.61). Unless otherwise indicated, the recording solution contained BaClz rather than CaCI, to minimize the Caz+-activated Cl- current (Leonard and Kelso, 1990). Ba2+ has a slightly higher permeability than Ca2+ through the ion channel of NMDA receptors, but does not activate the endogenous Ca*+-sensitive Cl- current in Xenopus oocytes (Leonard and Kelso, 1990). NMDA and ifenprodil were applied in solutions containing 5 or 10 PM glycine. For studies on cells injected with brain RNA, all solutions contained 5 PM glycine. Currents were recorded from oocytes using a two-electrode voltage clamp with an OC-725 Ooclamp amplifier (Warner Instruments, Hamden, CT). Electrodes, pulled from borosilicate glass and filled with 3 M KCI, had resistances of l-3 MD. Cells were voltage clamped at a holding potential of -70 mV. Data were recorded on a chart recorder or digitized using a MacPaq-100 interface with AcqKnowledge software (Biopac Systems, Coletta, CA; World Precision Instruments, Sarasota, FL) and stored on the hard disk of a Macintosh computer. Signals were low pass filtered @-pole Bessel) at a corner frequency of IO Hz and digitized at 25-35 Hz. In some experiments, data were digitally refiltered (low pass Bessel, 2.5-5 Hz) before analysis. Materials (+)[3-‘251]l-MK-801 (specific activity, 2200 Ci/mmol) was purchased from New England Nuclear-Du Pont (Boston, MA). (+)MK-801 was a gift from Merck, Sharp and Dohme Division of Merck and Co., Inc. (West Point, PA). lfenprodil was a gift from Synthelabo Recherche (Bagneux, France). NMDA was purchased from Cambridge Research Biochemicals (Wilmington, DE). Pentamidine isethionate and t-glutamate were purchased from Sigma Chemical Co. (St. Louis, MO). Molecular biology reagents and restriction enzymes were purchased from Pharmacia (Piscataway, NJ), Boehringer Mannheim (Indianapolis, IN), and Promega (Madison, WI). Other chemicals and tissue culture reagents were from sources as previously described (Williams et al., 1991a, 1992). Acknowledgments This work was supported by grants from the United States Public Health Service (NS 30000 and GM 34781), ICI Americas, and the Epilepsy Foundation of America. Wearegrateful to Dr. S. Nakanishi for the NRI cloneand Drs. P. H. Seeburg and D. Pritchett for the NR2 clones. Correspondence should be addressed to Dr. Keith Williams. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisemem” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

September

3, 1992; revised

November

11, 1992.

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Sugihara, H., Moriyoshi, K., Ishii,T., Masu, M., and Nakanishi, S. (1992). Structures and properties of seven isoforms of the NMDA receptor generated by alternative splicing. Biochem. Biophys. Res. Commun. 185, 826-832. Tsumoto, T., Hagihara, K., Sato, H., and Hata, Y. (1987). NMDA receptors in the visual cortex of young kittens are more effective than those of adult cats. Nature 327, 513-514. Verdoorn, T. A., Kleckner, N. W., and Dingledine, R. (1987). Rat brain N-methyl-o-aspartate receptors expressed in Xenopus oocytes. Science 238, 1114-1116. Williams, K., Dawson, V. L., Romano, C., Dichter, M. A., and Molinoff, P. B. (1990). Characterization of polyamines having agonist, antagonist, and inverse agonist effects at the polyamine recognition site of the NMDA receptor. Neuron 5, 199-208. Williams, K., Hanna, J. L., and Molinoff, P. B. (1991a). Developmental changes in the sensitivity of the N-methyl-o-aspartate receptor to polyamines. Mol. Pharmacol. 40, 774-782. Williams, K., Romano, C., Dichter, M. A., and Molinoff, P. B. (1991b). Minireview: modulation of the NMDA receptor by polyamines. Life Sci. 48, 469-498. Williams, regulation neurons 147-151.

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Note

Added

in Proof

A recent report has shown that mRNA encoding the ~2 (NRLB) subunit of the NMDA receptor is expressed in fetal through to adult mouse brain, whereas the &I (NRLA) subunit is expressed later, beginning during postnatal days I-7(Watanabe, M., Inoue, Y., Sakimura, K., and Mishina, M. 1993. Developmental changes in distribution of NMDA receptor subunit mRNAs. NeuroReport, in press). These observations are consistent with the hypothesis that adelayed developmental expression of the NRZA subunit in rat brain is responsible for the late developmental appearance of NMDA receptors having a low affinity for ifenprodil. We are grateful to Dr. M. Mishina for communicating these results prior to publication.