Age dependence of homosynaptic non-NMDA mediated long-term depression in field CA1 of rat hippocampal slices

Det~elopmental Brain Research, 75 (1993) 253-260 © 1993 Elsevier Science Publishers B.V. All rights reserved 0165-3806/93/$06.00

253

BRESD 51696

Age dependence of homosynaptic non-NMDA mediated long-term depression in field CA1 of rat hippocampal slices Libor Veli~ek a, Solomon L. Mosh6 a,b,c and Patric K. Stanton ~,.b Departments of Neurology, h Neuroscience and ~ Pediatrics, Albert Einstein Colh'ge of Medicine, Bronx, NY 10461 (USA) (Accepted 25 May 1993)

Key words: Long-term depression; Hippocampus; N-Methyl-D-aspartate; Synaptic plasticity; Development: 2-Amino-5-phosphonopentanoic acid; Long-term potentiation: Ontogeny

It has been hypothesized that high levels of presynaptic activity that fail to activate postsynaptic N-methyl-o-aspartate (NMDA) receptors may lead to long-term depression (LTD). Therefore, we tested the ability of high-frequency (50 Hz) synaptic stimulation in the presence of a blocker of NMDA receptors to elicit homosynaptic LTD at Schaffer collateral-CA1 synapses in hippocampal slices from 15-, 30- and 60-day-old rats. In control slices, there were no developmental differences in the incidence of long-term potentiation (LTP) of either EPSP slope or population spike amplitude. However, while NMDA receptor blockade with the specific antagonist D-2-amino-5-phosphonopentanoic acid (AP5; 25 ~M) completely eliminated LTP in 30 and 60-day-olds, a significant number of slices from 15-day-old rats displayed some non-NMDA LTP of synaptic transmission. Moreover, under NMDA receptor blockade, the same high-frequency stimulation now induced homosynaptic LTD of population spike amplitude in a significant number of slices from 15- and 60-day-old rats (47% and 42%, respectively) but not in 30-day-olds (7%). LTD of population spike amplitude was most pronounced in 15-day-old slices (27 +_6% of baseline), whereas, in 60-day-old slices, LTD was 81 _+3% of baseline. LTD of EPSP slopes occurred in 44% of 15-day-olds, 13% of 30-day-olds, and 33% of slices from 60-day-old rats: the magnitude of EPSP was similar in 15 and 60-day-old slices (70_+9% versus 81 +_1% of baseline). Our findings indicate that: (i) substantial non-NMDA LTD can be unmasked when NMDA receptors are blocked in slices from 15- and 60-day-old rats; (ii) during development, non-NMDA LTD is largest in the immature (15-day-old) hippocampus; and (iii) a part of LTP in 15-day-old slices is also independent of NMDA receptor activation.

INTRODUCTION

Long-term potentiation (LTP) 4 is a long-lasting increase in synaptic strength elicited by brief, highfrequency bursts of synaptic activation, that is thought to play a role in learning and memory s and epileptic seizures 25. At many synapses (e.g. Schaffer collateralCA1 pyramidal cell synapses and perforant pathwaydentate granule cell synapses in the hippocampus), activation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor is required for induction of LTP. When N M D A receptors are blocked by an antagonist such as 2-amino-5-phosphonopentanoic acid (AP5), LTP cannot be induced at these synapses '~. N M D A receptors gate the influx of Ca R+ into neurons 1'2s'24, and increases in postsynaptic intracellular [Ca 2+] seem to be necessary to induce LTP 2z. At several synapses, LTP can be induced independent of

N M D A receptor activation (e.g. at mossy fiber-CA3 pyramidal ceil synapses~S); however this NMDA-independent LTP still seems to involve increases in intracellular free [Ca 2+] (presynaptic) by other means, such as by activating voltage-dependent calcium channels ~5 or metabotropic glutamate receptors 2. Long-term depression (LTD) of synapic strength would seem to form a natural counterpart to LTP. Homosynaptic associative LTD can be induced by pairing of low-frequency stimuli to one synaptic input with high-frequency bursts to a separate, second input, but only when they are applied out of phase to release glutamate at times when the postsynaptic neuron is relatively hyperpolarized and calcium influx is probably attenuated 6"3~. Homosynaptic LTD can also be induced with single-pathway stimulation in a frequency dependent manner. Prolonged low-frequency synaptic stimulation (1-5 Hz, 10-15 rain) has been shown to elicit

Correspondence: P.K. Stanton, Departments of Neuroscience and Neurology, Albert Einstein College of Medicine, Kennedy Center, Room B33, 1410 Pelham Parkway South, Bronx, NY 10461, USA. Fax: (1) (718) 824-3058. E-mail: STANTON(a,A,ECOM.Yu.EDU

254 LTD

in t h e C A I

region of hippocampal

slices Ih27'34.

L o w - f r e q u e n c y s t i m u l u s - e v o k e d t 1,27,34 ( b u t n o t a s s o c i a tive TM)L T D c a n b e b l o c k e d by A P 5 . B o t h N M D A - d e pendent

and NMDA-independent

forms of LTD can

b e o b s e r v e d at o n e set o f s y n a p s e s 7'35. T h e r e a r e rep o r t s o f f e r i n g d i r e c t e v i d e n c e f o r a r o l e for i n c r e a s e d i n t r a c e l l u l a r [Ca 2+] in i n d u c i n g L T D , s i n c e i n t r a c e l l u lar i n j e c t i o n o f c a l c i u m c h e l a t o r s also b l o c k t h e i n d u c t i o n o f L T D 5'27"37. T h u s , it has b e e n s u g g e s t e d t h a t i n t e r m e d i a t e i n c r e a s e s in i n t r a c e l l u l a r [Ca 2+ ] m a y c a u s e LTD, whereas L T P 5,22,27,37. There

larger

increases

are a few developmental

in

[Ca : + ]

elicit

s t u d i e s o n t h e in-

d u c i b i l i t y a n d e x p r e s s i o n o f L T P . L T P first a p p e a r s a f t e r h i g h - f r e q u e n c y s t i m u l a t i o n in f i e l d C A 1 in slices from immature

rat h i p p o c a m p u s

at 5 days o f age. By

a g e 7 to 8 days, s u b s t a n t i a l levels o f L T P h a v e b e e n o b s e r v e d , w i t h m a x i m a l L T P e x p r e s s e d at 15 days o f a g e t7'32. I n c o n t r a s t , L T P in n e o c o r t e x has b e e n vario u s l y r e p o r t e d to first a p p e a r at 6 - 1 0 days o f a g e 32, o r n o t u n t i l 21 days p o s t n a t a l 36. T h e r e is a r e p o r t o f an u n s u c c e s s f u l a t t e m p t to i n d u c e L T D in f i e l d C A 1 w i t h short bursts of high-frequency Schaffer collateral/comm i s s u r a l s t i m u l i (400 t r a i n s o f 5 p u l s e s / 1 0 0 in t h e p r e s e n c e o f t h e N M D A (50-200

/~M) TM. H o w e v e r ,

Hz) given

receptor blocker AP5

this s t u d y was p e r f o r m e d

o n l y o n slices f r o m a d u l t rats. T o d a t e , t h e r e h a v e b e e n no studies of the developmental

t i m e c o u r s e o f any

form of LTD during postnatal development. The purposes

o f this s t u d y w e r e

to d e t e r m i n e :

(1) if it is

p o s s i b l e to u n m a s k L T D a f t e r h i g h - f r e q u e n c y s t i m u l a t i o n if N M D A

r e c e p t o r s ( a n d , thus, L T P ) a r e b l o c k e d ;

(2) w h e t h e r n o n - N M D A

LTD may be age-dependent,

s i n c e l a r g e s t a m p l i t u d e L T P is f o u n d in 1 5 - d a y - o l d rats. Since LTP

is c h a n g i n g r a p i d l y d u r i n g

development,

L T D m a y also b e r e g u l a t e d in a b a l a n c i n g f a s h i o n . T h i s c o u l d s e r v e to m a i n t a i n stability o f p l a s t i c n e u r a l n e t w o r k s , as w e l l as b e an i m p o r t a n t f a c t o r in c o n t r o l l i n g t h e sensitivity o f t h e i m m a t u r e C N S to s e i z u r e s . MATERIALS AND METHODS We used male Sprague-Dawley rats aged 14-16 days (referred to as 15-day-old), 29-31 days old (referred to as 30-day-old) and 60-65 days old (referred to as 60-day-old). Fifteen-day-old pups were housed with their dams until the experiment. The rats were maintained on a 12 h dark/light cycle and had access to food pellets and water ad libitum. Rats were deeply anesthetized with ether vapors and decapitated. Brains were rapidly removed and the hippocampi dissected under ice-cold artificial cerebrospinal fluid (ACSF; composition in mM: NaCI 126, KCI 5, NaH2PO 4 1.25, MgCI 2 2, CaC12 2, NaHCO 3 26, glucose 10). Transverse hippocampal slices (400-450 ~m thick, n = 123) were cut using a vibratome (Campden Instruments), placed in an interface-style recording chamber 16 and perfused continuously with ACSF (2.5-3.0 ml/min). After one hour of preincubation, bipolar platinum wire stimulating electrodes (50 ~m tip diameter)

and glass extracellular recording electrodes filled wi~h 2 M NaCi (resistance 1-5 M.Q) were inserted into the slice. One stimulating electrode was placed in the Schaffer collateral commissural (stratum radiatum) on the CA3 side (with respect to the CA1 recording electrodes). Tiffs input was the one receiving high-frequency stimulation, The control input stimulating electrode was placed on the other side of the recording electrodes in stratum radiatum (closer to the subiculum). The two input pathways were shown to be independent by testing paired-pulse interactions in both directions (S1-$2 and $2-S1; 20-80 ms interstimulus interval) and verifying that there was no paired-pulse facilitation prior to the start of the experiment. One recording electrode was placed in the CAI pyramidal cell body layer (stratum pyramidale) to record population action potentials/population spikes), the other was in the apical dendritic zone of CA1 pyramidal cells (stratum radiatum) to record population excitato~ postsynaptic potentials (EPSP). The two Schaffer collateral inputs were alternately stimulated every 30 s. Only slices with population spike amplitudes > I mV and EPSP slopes > 1 V / s were used in these experiments. During the pretetanus baseline recording period, the stimulating current was adjusted to produce 60-70% of maximal responses (usually between 100-400/,A) to provide sufficient area on the input-output curve to assess the amplitude of LTD. After a stable baseline was recorded for at least 15 min, high-frequency stimulation was given to the Schaffer collateral inputs on the CA3 side (6 trains x 50 Hz/2 s; 15 s intertrain interval). The frequency used is considered to be midway between frequencies which are very unreliable at eliciting LTP (1-25 Hz) and frequencies that evoke maximal LTP (100-200 HzJ2). However, in very high frequencies the voltage-dependent calcium channels may open and contribute to the intracellular calcium load. To test for the presence of LTD under conditions of NMDA receptor blockade, the NMDA receptor antagonist D ( - )-2-amino-5phosphonopentanoic acid (A~; Cambridge Research Biochemicals) was added to the medium at a concentration of 25 /zM at least 15 rain before tetanization. Following high-frequency stimulation, evoked synaptic potentials were recorded each 30 s and saved on a computer data acquisition system (BrainWave, Inc.) for offline analysis. LTP of both population spike amplitude and EPSP slope was defined as an increase in response greater than 2 standard deviations (S.D.) over baseline averages, recorded 30 min post-tetanus. Conversely, LTD was considered to be present when responses were more than 2 S.D.s below pretetanus baselines. Population spike amplitude and maximal slope of the early EPSP were computed as described previously 3°. Baseline average and S.D. were computed from five subsequent values recorded immediately prior to tetanization and compared to the mean amplitudes from 29-31 min following tetanic stimulation. According to these criteria, both control and AP5 perfused groups of slices were further subdivided into three subgroups referred to as 'LTP'; 'LTD' and "no plastic change" for population spike amplitude and EPSP slope. For the evaluations of frequencies of occurrence of LTP versus LTD and 'no plastic change' within and between age groups, a Chi-square test was used (distribution of LTP, LTD and 'no plastic change' for either population spike amplitudes or EPSP maximal slopes in control versus AP5-perfused groups of slices). In untetanized inputs, we used an unpaired two-tailed Student's t-test to compare pooled averaged population spike amplitudes and EPSP maximal slopes in control versus AP5 perfused slices. The level of significance for all analyses was set to P < 0.05. RESULTS

Adults (60-day-olds) I n o u r initial e x p e r i m e n t s , w e t e s t e d t h e e f f e c t o f high-frequency

orthodromic

stimulation

on

Schaffer

c o l l a t e r a l s y n a p t i c t r a n s m i s s i o n in slices f r o m 60-dayo l d rats in t h e a b s e n c e

or presence

of the NMDA

r e c e p t o r a n t a g o n i s t A P 5 . P r i o r to h i g h - f r e q u e n c y stim-

255 ulation, mean amplitude of population spikes was 4.01 _+0.29 mV, and of EPSP slope was 2.27 +_0.2 V/s; at 60-70% of maximal response amplitudes. Immediately after tetanization, we observed both homo- and heterosynaptic depression in either control slices or those perfused with 25 /.tM AP5. In control slices, the homosynaptic depression was typically shorter in duration (< 2 min) than the heterosynaptic depression and, during perfusion with normal ACSF, transformed into LTP by 5 min post-tetanus. Heterosynaptic depression lasted longer (3-8 min) with slower recovery to baseline values for both population spikes and maximal EPSP slopes. In AP5 perfused slices, transient homosynaptic depression usually transformed into either 'no plastic change' or LTD. In 60-day-old control slices, thirty minutes after high-frequency stimulation (6 X 50 Hz/1 s), there was a significant enhancement in synaptic efficacy expressed as LTP of population spike amplitude (63% of slices; Fig. 1, 60-day-old CON, solid bar; n = 32) and LTP of EPSP slope (55% of slices; Fig. 2, 60-day-old, CON, solid bar; n = 11). Under control conditions, LTD of population spike amplitude or EPSP slope was observed rarely (12.5% and 9.1%, respectively; Figs. 1 and 2, 60-day-old CON, dashed bars). In this age group, AP5 treatment almost completely abolished LTP (Figs. 1 and 2, 60-day-old AP5, solid bars; pop spike n = 20, EPSP n = 9). As hypothesized, the incidence of

POP SPIKE 100

II

.=. 6o (O O

8

40

,,=, ,,=

[]

LTP

[ ] nochange [ ] LTD

CON

AP5

15 DAY OLD

CON

AP5

30 DAY OLD

CON

AP5

60 DAY OLD

Fig. I. The incidence of LTP, L T D and 'no change' of population spike amplitude after high-frequency stimulation of Schaffer collateral synapses ( 6 × 100 p u l s e s / 5 0 Hz) in hippocampal slices from 15-, 30- and 60-day-old rats. LTP = solid bars; L T D = hatched bars; No Change = open bars. LTP and LTD were defined as differences of more than 2 S.D.s from baseline average. Perfusion with the N M D A - r e c e p t o r antagonist AP5 (25 p~M) during and after highfrequency stimulation induced a conversion of substantial a m o u n t s of LTP into LTD in 15- and 60-, but not in 30-day-old slices. * marks significant differences in the distribution of LTP, LTD and "no plastic changes' between control (CON) and AP5 group within each age group ( P < 0.05; x2-test).

EPSP SLOPE

•

LTP

[ ] no change [ ] LTD

CON

AP5

15 DAY OLD

CON

AP5

30 DAY OLD

CON

AP5

60 DAY OLD

Fig. 2. The incidence of LTP, LTD and 'no plastic changes' of EPSP slope after high-frequency stimulation ( 6 x 1(10 p u l s e s / 5 0 Hz) of Schaffer collateral synapses in hippocampal slices from 15-, 30- and 60-day-old rats. Labels as in Fig. 1. EPSP slopes showed a similar distribution of LTP, L T D and 'no plastic changes' in control and AP5-perfused 30- and 60-day-old slices as population spike amplitudes. In 15-day-old slices perfused with AP5, there was a similar incidence of LTP to that in control slices, and also a substantial amount of n o n - N M D A receptor mediated LTD.

LTD increased markedly for both population spike amplitude (to 42%) and EPSP slope (33%; Figs. 1 and 2). A substantial amount of LTP was also transformed to the 'no plastic changes' group for both population spike amplitudes and EPSP slopes in AP5-treated slices (Figs. 1 and 2, 60-day-old, open bars). These differences in the frequency of occurrence of plastic changes between control and AP5-perfused group were statistically significant (x2-test; P < 0.05). The average amplitude of LTP in 60-day-old control slices (LTP group) was 136_+ 4% and 132_+ 6% of baseline for population spike amplitude and EPSP slope, respectively (Figs. 3 and 4; 60 days, solid bars). In contrast, LTD in AP5-perfused slices averaged 81 _+ 3% of baseline for population spike amplitudes and 81 _+ 1% for EPSP slopes (Figs. 3 and 4; 60 days, hatched bars). The normalized time course of experiments (in percent of pre-tetanus baseline) in the 60day-old LTD subgroup are shown in Fig. 5. There was no difference in the time course of homosynaptic posttetanic depression between population spike amplitudes and EPSP slopes. There was an initial robust homosynaptic depression. Thereafter, both population spikes (open circles) and EPSP slopes (closed circles) recovered to 80% of baseline values. Thirty minutes after high-frequency stimulation, LTD of both population spike amplitudes and EPSP slopes had stabilized. It should be noted that after initial heterosynaptic depression, the untetanized input population spikes recovered and there were no long-lasting changes in

256

EPSP SLOPE

POPULATION SPIKE 10o]

CONTROL LIP

(16) []

AP5 LTD

50 (9)

W g. ¢/}

II

ul

~

ul u~ In

o.

AP5 LTD

,,-I, 2o if) II]

(1)

O

~

(8)

~ g

-50

o

w z z

~ Z

(s)

S Q40 ¢9 Q. uJ UJ

(2O)

CONTROL LTO

[]

(5) -3-

In

B

-2o (3)

(.~ -40

~k (O) -10o

15 days

30 days

60 days

-60

15 days

30 days

AGE Fig. 3. Population spike amplitudes in LTP and L T D groups in the slices perfused with normal A C S F and A C S F containing 25 /zM AP5. Solid bars = LTP amplitude in control groups; hatched bars = LTD amplitude in AP5-treated slices. % differences from pre-tetanus baseline are plotted (mean_+S.E.M.). * denotes significant differences compared to the same treatment group of slices from 60-day-old rats ( A N O V A with post-hoc Fisher PLSD test, P < 0.05). L T D in slices from 15-day-old rats was significantly greater than in 60-day-otds.

synaptic potentials at untetanized inputs in either control (101.5 _+ 5.8% of baseline) or AP5-treated (100.8 + 9.6% of baseline) slices. EPSP slopes were similarly unchanged at control untetanized synapses (92.3 _+ 7.3% and 93.3_+ 5.5% in control and AP5-perfused slices, respectively). Thus, the n o n - N M D A LTD we observed was purely homosynaptic.

60 days

AGE Fig. 4. EPSP slopes in LTP and L T D groups in slices perfused with A C S F and A C S F plus 25/~M AP5, respectively. Details as in Fig. 3. All m e a n s shown are significantly different from their respective pre-tetanus baselines.

(6.3 and 0%, respectively; Figs. 1 and 2, 30-day-old CON, hatched bars). In AP5-perfused slices, LTP was observed sparsely (14% for population spike amplitude and 0% for EPSP slopes, Figs. 1 and 2, 30-day-old AP5, solid bars; pop spike n = 14; EPSP n = 8). In contrast to 60-day-old slices, the incidence of non-

60 120

AP5

DAY

()l,I)

(25 /~M)

50Hz/1

;

6i

s

30-day-olds The average amplitudes of evoked potentials (pop spike = 4.31 + 0.34 mV and EPSP = 2.34 _+ 0.2 V / s ) and the time course and pattern of immediate poststimulation homo- and heterosynaptic depression in slices from 30-day-old rats were similar to those obtained from 60-day-old rats. In 30-day-old control slices, thirty minutes after high-frequency stimulation (6 × 50 H z / 1 s), we observed significant LTP of both population spike amplitude and EPSP slope (56% of slices; Figs. 1 and 2, 30-day-old, CON solid bars; pop spike n = 16; EPSP n = 9). Under control conditions, LTD of population spike amplitude or EPSP slope was rarely observed

.~

60 40

e pop'spike aPmp

2O 0 0

i0

i

i

20

30

Time

40

50

(min)

Fig. 5. Time course of normalized population spike amplitude and EPSP slope in the L T D group of slices from 60-day-old rats perfused with ACSF containing 25 /zM AP5 (each point ks m e a n + S . E : M . ) . Solid circles = EPSP slope (n = 3); open circles = population spike amplitude (n = 8). After a short post-tetanic depression, LTD of both EPSP slope and population spike amplitude was established at about 80% of pre-tetanus baseline.

257 N M D A mediated LTD was minimal in this age group: 7% for population spike amplitude and 13% EPSP slope (Figs. 1 and 2). In contrast, the majority of the LTP was transformed by N M D A receptor blockade into the 'no plastic changes' group for both population spike amplitudes (79%) and EPSP slopes (88%; Figs. 1 and 2; 30 days old, open bars). The differences in the incidence of plastic changes between control and AP5perfused slices in the 30-day age group were also statistically significant (,v2-test, P < 0.05). The average amplitude of LTP 30 min after highfrequency stimulation in control slices (LTP group) was 128_+ 6% (population spike) and 142_+ 10% (EPSP slope) of pre-tetanus baseline (Figs. 3 and 4; 30 days, solid bars). In contrast to slices from 60- and 15-dayolds, LTD was not seen often enough to evaluate its magnitude (Figs. 3 and 4). At untetanized input synapses, there were no longlasting changes in population spikes in either control (102.9 + 8.0% of baseline) or AP5-treated (106.0 _+ 7.5% of baseline) slices following tetanization, indicating that slices were not showing non-specific decline over the recording period. Likewise, EPSP slopes stabilized at 108.3 + 6.1% of baseline in control and 94.1 _+ 5.0% in APS-perfused slices.

15-day-olds There were no significant differences in average response amplitude (pop spike = 3.3 _+ 0.31 mV; EPSP = 2.21 _+ 0.4 V/s), or in the duration of rapid homoand heterosynaptic poststimulation depression, in slices from 15-day-old rats compared to 30 and 60-day-olds. In control slices (pop spike n = 24; EPSP n = 12) thirty minutes after high-frequency stimulation (6 × 50 H z / 1 s), there was a marked LTP of population spike amplitude in 67% of slices and EPSP slope in 42% of slices (Figs. 1 and 2, 15-day-old CON, solid bar). In contrast, LTD of population spike amplitude or EPSP slope was rarely, if ever, observed in controls (13% and 0%, respectively), similar to both older age groups (Figs. 1 and 2; 15-day-old CON, hatched bars). In AP5-perfused slices from 15-day-old rats (pop spike n = 17; EPSP n = 9) there were two significant findings: (1) in contrast to the older age groups, a significant proportion of non-NMDA LTP was observed (29% of slices for population spikes and 44% of slices for EPSP slopes; Figs. 1 and 2, 15 day old AP5, solid bars); (2) in contrast to 30-day-old slices, but similar to 60day-olds, the incidence of LTD was increased by N M D A receptor blockade. Forty-seven percent of slices exhibited LTD of population spike amplitudes, and 44% LTD of EPSP slopes (Figs. 1 and 2). As in the other age groups, the differences in the incidence of

15 DAY OLD 140

120

%

60

e

40

[- AP5

i

(e5~M)

1

• e p s p s,op~,

6x 50Hz/ls

o pop spike e~mp

2O 0 l 0

10

20

30

40

fJ0

Time (rnin~ Fig. 6. Normalized population spike amplitude (n = S) and EPSP slope ( n = 4 ) in the LTD group of slices from 15-day-old rals perfused with ACSF containing 25 ktM AP5 {each point m e a n + S.E.M.). Details as in Fig. 5. EPSP slopes displayed a short posttetanic depression, which rapidly stabilizcd as LTD at approximately 60-70% of pre-tetanus baseline. Population spike amplitudes recovered much more slowly and only to the level of 20G of pre-telanus baseline, showing marked homosynaptic LTD.

long-term plastic changes between control and AP5perfused groups were statistically significant (~e-test; P < 0.05). In control slices, LTP of synaptic transmission (LTP group) averaged 155+_ 13% of baseline population spike amplitude and 129 _+ 15e/~ of baseline EPSP slope (Figs. 3 and 4; 15 days, solid bars). In contrast, blockade of NMDA receptors with AP5 converted half the slices to exhibiting LTD of population spike amplitudes to 27_+ 6% of baseline, and LTD of EPSP to 70_+ 9% of baseline values (Figs. 3 and 4; 15 days, hatched bars). The normalized time course data for the LTD subgroup are shown in Fig. 6. There was an interesting difference in the time course of post-tetanic homosynaptic depression between population spike amplitudes, that showed a rapid drop to significantly more reduced values than older age groups (20% of baseline; open circles), while EPSP slope recovered to levels of LTD similar to those in 6(l day old sliccs (approx. 70% of baseline; closed circles). Thirty minutes after high-frequency stimulation, LTD of both population spike amplitudes and EPSP slopes had stabilized to their final values. Following an initial heterosynaptic depression, untetanized control input population spikes recovered to pre-tetanus baseline and there were no differences between slices in control and AP5-containing ACSF (116.2 + 13.3% and 81.1 _+ 12.2% of baseline, respectively). Likewise, EPSP slopes showed no long-term changes in synaptic efficacy at the untetanized control

258 inputs in control (99.5 + 4.9%) and AP5-treated (91.8 + 3.7%) slices. Ocerall det,elopmental differences in neuronal plasticity To summarize, in control slices perfused with normal ACSF, there were no significant differences in the incidence of plastic changes in synaptic strength across age groups. Furthermore, neither the amplitudes of LTP of the population spike, nor the EPSP slopes were significantly different in the three age groups. In contrast, in AP5-treated slices we did observe significant differences in the occurrence of plastic changes. The low incidence of LTP in 30-day-old slices in AP5 and low incidence of 'no plastic changes' in slices from 15-day-old rats with N M D A receptor blockade were the most important contributors to these differences (X e test with post-hoc cell contribution; P < 0.05). Moreover, LTD of population spike amplitude was much greater in the LTD group of 15 day old slices compared to 30 and 60-day-olds (ANOVA with post-hoc Fisher PLSD test; F2,14 = 34.28; P < 0.05). It should be emphasized that LTP and LTD for pop spikes and EPSP slopes were evaluated separately. Nevertheless, the coincidence of LTD of pop spike amplitude and LTP of EPSP slope occurred only once in control slices in the 30-day-old group. DISCUSSION Our findings demonstrate that non-NMDA receptor mediated long-term depression (LTD) of synaptic strength can be induced by high-frequency afferent stimulation in hippocampal slices from 15- and 60-dayold rats, but only rarely in slices from 30-day-old rats. LTD was observed when slices were perfused with ACSF containing AP5 to block LTP, and thus unmask presumed non-NMDA receptor mediated LTD. The magnitude of LTD was largest in slices from 15-day-old rats, and somewhat smaller in slices from 60-day-old rats. At Schaffer collateral commissural synapses in field CA1 of control slices, LTP could be induced with high-frequency stimulation throughout development. A surprising finding was a relatively high incidence of LTP that persisted even when N M D A receptors were blocked in slices from 15-day-old rats. LTP is considered to be induced by an increase in intracellular free [Ca2+] 22. However, there are a number of ways to increase intracellular [Ca 2÷] concentration, including: (1) through N M D A receptor channels; (2) through voltage-dependent calcium channels; and (3) by release of Ca 2 + from intracellular stores by a variety of transmitters, including norepinephrine and glutamate 2s. In our experiments, we blocked N M D A receptors with

the specific antagonist AP5. It has been reported that stimulation at significantly higher frequencies (2(/0 Hz) than we employed can induce an NMDA-receptor independent LTP at Schaffer collateral synapses, which is thought to be mediated by voltage-dependent calcium channel opening l-s. While 50 Hz is typically too low a frequency to activate voltage-dependent calcium channels, it is possible, that, early in development, these channels may be activated at lower frequencies of stimulation (50 Hz), especially at higher stimulus intensity. Alternatively, another source of release of intraceltular Ca 2+ may be supporting this LTP. In slices from adult rats (7 weeks old), it has been reported that the late phases of LTP ( > 1(10 min) can be abolished by pretetanus application of the metabotropic glutamate receptor antagonist DL-2-amino-3phosphonopropionic acid 2 (AP3; see also39). In addition, there is a report showing high metabotropic receptor sensitivity early in development in kitten visual cortex"~. Therefore, it is also possible that metabotropic receptors may contribute more to LTP early in development. However, it has also been reported that metabotropic receptors can play a role in induction of LTD 29. While our experiments demonstrate that pharmacologically blocking N M D A receptor-dependent LTP can uncover non-NMDA LTD, we cannot say whether this is a true unmasking of an activity-dependent depression that is always present but usually masked by LTP or if preventing NMDA-gated Ca 2÷ influx actually alters the balance of ions and intracellular messengers such that LTD is newly evoked. Likewise, the extent to which either messengers or targets of synaptic modification are shared by the two phenomena is unknown. Because we affected only one of many routes of Ca 2+ entry, it is possible that remaining mechanisms might induce some LTP in slices from 15-day-old rats. It is, however, extremely unlikely that the concentration of AP5 we used (25 p.M) was too low to sufficiently block N M D A receptors for two reasons. First, it has been shown that 25 p.M o - ( - ) - A P 5 blocks > 97% of pure NMDA synaptic EPSPs at Schaffer collateral synapses in hippocampal slices from adult rats z4. Second, we should also address the possibility that a higher density of N M D A binding sites known to be present in 15-day-old rats 2°'33 could explain the nonN M D A LTP as simply an artifact of insufficient blockade. By the law of mass action, a large excess volume of perfusate with 25 /.~M AP5 ensures that, even if there is an increased density of N M D A receptors, they will be blocked to the same degree. Only a change in receptor affinity can alter the percentage of receptors blocked, and numerous studies have shown that N M D A

259 receptor number, but not affinity, changes during development 2°'33. Therefore, we conclude that insufficient blockade of N M D A receptors is unlikely to explain the n o n - N M D A LTP we observed. The findings presented here demonstrate that there are developmental differences in the distribution and amplitude of n o n - N M D A receptor mediated LTD. These effects are probably caused by the differential development of multiple mechanisms that can participate either in the induction a n d / o r expression of LTD. Currently, several forms of LTD have been described. Low-frequency stimulus-induced L T D can be blocked by AP5 t 1,27,34. Other types of L T D a p p e a r to be N M D A receptor independent 3j, although they may be dependent on glutamate metabotropic receptor activation 2~34 or voltage dependent calcium channel function ~5. In cerebellar neurons, L T D requires concomitant activation of both ionotropic (AMPA) and metabotropic glutamate receptors 21. Other studies have demonstrated that hyperpolarization paired with concomitant 5 - 1 0 Hz stimulation can elicit L T D 31'37 and also that the levels of extracellular [Ca 2+ ] can determine whether LTP (high levels) or LTD (intermediate levels) is evoked by the same stimulus 27'3s. So far, it is unclear whether multiple second messenger cascades can trigger LTD, if aforementioned various mechanisms of LTD induction a p p e a r at different stages in development, or what is the developmental involvement of Ca 2+ ions. There is a hypothesis that the level of intracellular [Ca 2+] increase determines whether LTP (large increases) or L T D (mild increases) is induced 5'2737. This is supported by experiments where the intracellular injection of a calcium chelator converted a stimulus-induced LTP into one that elicited L T D 3s, as well as studies showing that intracellular Ca 2+ chelators can block the induction of lowfrequency LTD 27. At the very least, it appears that the very young hippocampus exhibits the most robust nonN M D A receptor activity-dependent LTD. Since we were blocking N M D A receptor activation, our studies do not reveal whether there is also a pure N M D A receptor-mediated LTD which may be similarly robust early in development. T h e r e is one report of an unsuccessful attempt to induce LTD during N M D A - r e c e p t o r blockade t4. That study used rats between 150-220 g, roughly corresponding to 45-50 days of age. It is, therefore, possible that at this age in their preparations, slices were exhibiting the very small amount of LT'D we observed in the 30-day-old group. The aforementioned study also employed 100 Hz stimulation (2000-5000 pulses) to induce LTD, although this frequency of stimulation is probably the most reliable in producing LTP 12. Both

developmental stage and frequency of tetanization could explain differences between our findings and theirs. The amplitudes of LTP and L T D we observed in slices from 60-day-old rats are consistent with recent data on the induction of LTP with prolonged highfrequency stimulation 13'15'~ and on low-frequency-induced L T D 1~. The larger L T D seen in slices from 15-day-old rats suggests that there is more LTD at this age. This may be a counterbalance to larger LTP also observed at this age w. A balanced ratio of LTP and LTD may be necessary both for maximizing information extracted from time-varying signals 3~, as well as limiting the susceptibility of the immature brain to hyperexcitability and seizures 2". We have recently found that the amount of LTD elicited by lowfrequency stimuli in slices from adult animals is greater at previously tetanized synapses displaying LTP 34. This fuels the speculation that dynamic regulation of LTD threshold to dynamically stabilize synaptic strength (a 'sliding threshold rule') 3 may also be present early in development. A final interesting observation from our experiments in slices from 15-day-old rats is the markedly larger amount of LTD of the population spike not accounted for by L T D of the EPSP. This suggests that, in immature hippocampal pyramidal neurons, there may be an additional form of LTD of E P S P - a c t i o n potential ( E S) coupling that can be activated by synaptic stimulation, but also does not require N M D A receptor activation. While we do not yet know if this E - S coupling LTD requires orthodromic stimulation, it does appear to be homosynaptic, like EPSP LTD. Summarizing the developmental differences we observed, slices from 15-day-old rats showed the largest n o n - N M D A receptor-mediated LTD and significant amounts of n o n - N M D A receptor-mediated LTP, as well. In contrast, in 30-day-old slices there was little n o n - N M D A mediated LTD and UFP was completely N M D A - d e p e n d e n t . In 60-day-old slices, we again observed significant LTD, and LTP was also largely N M D A - d e p e n d e n t . These developmental changes may underlie different capabilities for learning and retrieval of information, as well as influencing the changes in seizure susceptibility, occuring during development. Acknowledgements. This work was supported by an American Epilepsy Society fellowship with support from the Milken Family Medical Foundatkm to L.V., NIH Grant NS20253 to S.L.M., and NIH Grant MH45752 and an Office of Naval Research grant to P.K.S.

REFERENCES 1 Ascher. P. and Nowak. L., The role of divalent cations in the N-methyl-~-aspartate responses of mouse central neurones in culture, J. Physiol., 399 (1988) 247-266.

260 2 Behnish, T., Fjodorow, K. and Reymann, K.G., Dl.-2-amino-3phosphonopropionate blocks late synaptic long-term potentiation, NeuroReport, 2 (1991) 386-388. 3 Bienenstock, E.L., Cooper, L.N. and Munro, P.W., Theory for the development of neuron selectivity: orientation specificity and binocular interaction in visual cortex, J. Neurosci., 2 (1982) 32-48. 4 Bliss, T.V.P. and l~mo, T., Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path, J. Physiol.. 232 (1973) 331-356. 5 Brocher, S., Artola, A. and Singer, W., lntracellular injection of Ca 2+ chelators blocks induction of long-term depression in rat visual cortex, Proc. Natl. Acad. Sci. USA, 89 (1992) 123-127. 6 Chattarji, S., Stanton, P.K. and Sejnowski, T., Commisural synapses, but not mossy fiber synapses, in hippocampal field CA3 exhibit associative long-term potentiation and depression, Brain Res., 495 (1989) 145-150. 7 Christie, B.R. and Abraham, W.C., Priming of associative longterm depression in the dentate gyrus by 0 frequency synaptic activity, Neuron, 9 (1992) 79-84. 8 Collingridge, G.L., The role of NMDA receptors in learning and memory., Nature, 330 (1987) 604-605. 9 Collingridge, G.L., Kehl, S.J. and McLennan, H., Excitatory amino acids in synaptic transmission in the Schaffer collateralcommissural pathway of the rat hippocampus, J. Physiol., 334 (1983) 33-46. 10 Dudek, S.M. and Bear, M.F., A biochemical correlate of the critical period for synaptic modification in visual cortex, Science, 246 (1989) 673-675. 11 Dudek, S.M. and Bear, M.F., Homosynaptic long-term depression in area CAI of the hippocampus and effects of N-methyl-Daspartate receptor blockade, Proc. Natl. Acad. Sci. USA, 89 (1992) 4363-4367. 12 Dunwiddie, T.V. and Lynch, G., Long-term potentiation and depression of synaptic responses in the rat hippocampus: localization and frequency dependency, J. Physiol., 276 (1978) 353-367. 13 Fujii, S., Saito, K., Miyakawa, H., Ito, K. and Kato, H., Reversal of long-term potentiation (depotentiation) induced by tetanus stimulation of the input to CA1 neurons of guinea pig hippocampal slices, Brain Res., 555 (1991) 112-122. 14 Goldman, R.S., Chavez-Noriega, L. and Stevens, C.F., Failure to reverse long-term potentiation by coupling sustained presynaptic activity and N-methyl-D-aspartate receptor blockade, Proc. Natl. Acad. Sci. USA, 87 (1990) 7165-7169. 15 Grover, L.M. and Teyler, T.J., Two components of long-term potentiation induced by different patterns of afferent activation, ?v2~ture, 347 (1990) 477-479. 16 Haas, H.L., Schaerer, B. and Vosmansky, H., A simple perfusion chamber for the study of nervous tissue slices in vitro, ,L Neurosci. Methods, 1 (1979)323-325. 17 Harris, K.H. and Teyler, T.J., Developmental onset of long-term potentiation in area CA1 of the rat hippocampus, J. Physiol., 346 (1984) 27-48. 18 Harris, E.M. and Cotman, C.W., Long-term potentiation of guinea pig mossy fiber responses is not blocked by N-methyI-D-aspartate antagonists, Neurosci. Lett., 70 (1986) 132-137. 19 Huang, Y.-Y., Colino, A., Selig, D.K. and Malenka, R.C., The influence of prior synaptic activity on the induction of long-term potentiation, Science, 255 (1992) 730-733. 20 Insel, T.R., Miller, R.P. and Gelhard, R.E., The ontogeny of excitatory amino acid receptors in rat forebrain - I. N-methyl-oaspartate receptors and quisqualate receptors, Neuroscience, 35 (1990) 31 ~43.

21 Linden, D.J.. Dickinson, M.H., Smeyne, M. and Connor, J.A., A long-term depression of AMPA currents in cultured ccrebellar Purkinje neurons, Neuron, 7 (199t) 81-89. 22 Lynch, G,, Larson, J., Kelso, S., Barrionuevo, G. and Schottlcr. F., Intracellular injections of EGTA block induction of long-term potentiation, Nature, 305 (1983) 719-721. 23 MacDermott. A.B., Mayer, M.L.. Westbrook, G.L., Smith, S.J. and Barker. J.L., NMDA-receptor activation increases cytoplasmic calcium concentration in cultured spinal cord neurones, Nature, 321 (1986) 519-522. 24 Mayer, M.L., MacDermott, A.B., Westbrook, G.L., Smith, S.J. and Barker, J.L., Agonist- and voltage-gated calcium entry in cultured mouse spinal cord neurons under voltage clamp measured using arsenazo Ill, J. Neurosci.. 7 (1987) 3230-3244. 25 Morrell, F., Kindling and Synaptic Plasticity. The Legacy ~[" Graham Goddard, Birkh~iuser, Boston, (1991). 26 Mosh~, S i . , Epileptogenesis and the immature brain, Epilepsia, 28 Suppl. (1987) $3-S15. 27 Mulkey, R.M. and Malenka, R.C., Mechanisms underlying induction of homosynaptic long-term depression in area CAI of the hippocampus, Neuron, 9 (1992) 967-975. 28 Sladeczek, F., Pin, J.P.. Recasens, M., Bockaert, J. and Weiss, S., Glutamate stimulates inositol phosphate formation in striatal neurones, Nature, 317 (1985) 719-719. 29 Stanton, P.K., Chattarji, S. and Sejnowski, T.J., 2-Amino-3-phosphonopropionic acid, an inhibitor of glutamate-stimulated phosphoinositide turnover, blocks induction of homosynaptic longterm depression, but not potentiation, in rat hippocampus, Neurosci. Lett., 127 (1991) 61-66. 30 Stanton, P.K., Jones, R.S.G., Mody, 1. and Heinemann, U., Epileptiform activity induced by lowering extracellular [Mg 2 + ] in combined hippocampal-entorhinal cortex slices: modulation by receptors for norepinephrine and N-methyl-D-aspartate, Epilepsy Res.. 1 (1987)53-62. 31 Stanton, P.K. and Sejnowski, T.J., Associative tong-term depression in the hippocampus induced by hebbian covariance, Nature, 339 (1989) 215-218. 32 Teyler, T.J., Perkins, A.J. and Harris, K.M., The development of king-term potentiation in hippocampus and neocortex, Neuropsychologia, 27 (1989) 31-40, 33 Tremblay, E., Roisin, M.P., Represa, A., Charriaut-Marlangue, C. and Ben-Aft, Y., Transient increased density of NMDA binding sites in the developing rat hippocampus, Brain Res.. 461 (1988) 393-396. 34 Wexler, E,M. and Stanton, P.K., Priming of homosynaptic Mngterm depression in hippocampus by previous synaptic activity, NeuroReport, 4: 591-594. 35 Wickens, J.R. and Abraham, W.C., The involvement of L-type calcium channels in heterosynaptic long-term depression in the hippocampus, Neurosci. Lett., 130 (1991) 128-132. 36 Wilson, D.A. and Racine, R.J., The postnatal development of post-activation potentiation in the rat neocortex, Det,. Brain Res,, 7 (1983) 271-276. 37 Xie, X., Berger, T.W. and Barrionuevo, G., Isolated NMDA receptor-mediated synaptic responses express both LTP and LTD, J. Neurophysiol., 67 (1992) 1009-1013. 38 Yoshimura, Y., Tsumoto, T. and Nishigori, A., Input-specific induction of long-term depression in CaZ+-chelated visual cortex neurons, NeuroReport, 2 (1991) 393-396. 39 Zheng, F. and Gallagher, J.P., Metabotropic glutamate receptors are required for the induction of long-term potentiation, Neuron, 9 (1992) 163-172.