Functional Maturation of ExcitatorySynapses in Layer 3 Pyramidal Neuronsduring Postnatal Development of thePrimate Prefrontal Cortex

Guillermo Gonzalez-Burgos1, Sven Kroener2,3, Aleksey V.

Zaitsev1, Nadezhda V. Povysheva1, Leonid S. Krimer1,

German Barrionuevo2 and David A. Lewis1

1Department of Psychiatry and 2Department of Neuroscience,

University of Pittsburgh School of Medicine, Pittsburgh, PA

15261, USA3Current address: Department of Neurosciences, Medical

University of South Carolina, Charleston, SC, USA

In the primate dorsolateral prefrontal cortex (DLPFC), the density ofexcitatory synapses decreases by 40--50% during adolescence.Although such substantial circuit refinement might underlie theadolescence-related maturation of working memory performance,its functional significance remains poorly understood. The con-sequences of synaptic pruning may depend on the properties of theeliminated synapses. Are the synapses eliminated during adoles-cence functionally immature, as is the case during early braindevelopment? Or do maturation-independent features tag synapsesfor pruning? We examined excitatory synaptic function in monkeyDLPFC during postnatal development by studying properties thatreflect synapse maturation in rat cortex. In 3-month-old (earlypostnatal) monkeys, excitatory inputs to layer 3 pyramidal neuronshad immature properties, including higher release probability, lowera-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/N-methyl-D-aspartate (NMDA) ratio, and longer duration of NMDA-mediated synaptic currents, associated with greater sensitivity tothe NMDA receptor subunit B (NR2B) subunit--selective antagonistifenprodil. In contrast, excitatory synaptic inputs in neurons frompreadolescent (15 months old) and adult (42 or 84 months old)monkeys had similar functional properties. We therefore concludethat the contribution of functionally immature synapses decreasessignificantly before adolescence begins. Thus, remodeling of excit-atory connectivity in the DLPFC during adolescence may occur in theabsence of widespread maturational changes in synaptic strength.

Keywords: adolescence, EPSC, glutamate, maturation, NMDA, NR2B

Introduction

The extended period of adolescence in primates is character-

ized by both marked changes in emotion, cognition, and

behavior (Steinberg 2005) and a substantial refinement of

cortical circuits. In the neocortex of both monkeys and humans,

exuberant excitatory synapses produced by rapid synapto-

genesis during the perinatal period are eliminated during

adolescence when massive synaptic pruning occurs (Rakic

et al. 1986; Huttenlocher and Dabholkar 1997). In the monkey

dorsolateral prefrontal cortex (DLPFC), this developmental

trajectory in excitatory synaptic density is paralleled by similar

changes in the density of dendritic spines, the principal site of

excitatory inputs to cortical pyramidal cells (Anderson et al.

1995).

The substantial remodeling of excitatory connections in the

DLPFC during adolescence appears to contribute to the normal

functional maturation of this region. For example, structural

neuroimaging studies in humans demonstrated that adoles-

cence is associated with a substantial decrease in cortical gray

matter thickness (Giedd et al. 1999), a decrease usually inter-

preted to result from the massive synaptic pruning occurring

during this period (Gogtay et al. 2004). Neuropsychological

studies combined with functional neuroimaging showed that

the marked improvements in working memory function during

adolescence are accompanied by an increased recruitment

of DLPFC activity (Luna et al. 2004; Crone et al. 2006). In

macaque monkeys, the contribution of the DLPFC to behavioral

performance in tasks that require working memory becomes

fully mature near the end of adolescence (Goldman and

Alexander 1977).

Synaptic pruning during adolescence has also been linked to

neurodevelopmental models of schizophrenia because the

clinical symptoms, including working memory impairments,

typically arise near the end of adolescence, and early adulthood

(Lewis and Levitt 2002). The exuberant synapses present in the

DLPFC before adolescence have been hypothesized to com-

pensate for a dysfunction in excitatory transmission due to al-

terations in the expression of certain synaptic proteins (Mirnics

et al. 2001; Owen et al. 2005).

Understanding how reductions in excitatory synaptic density

during adolescence could contribute to such age- and disease-

related changes in the function of the DLPFC depends, in part,

on the knowledge of the functional properties of the synapses

present before adolescence-related pruning. One possibility is

that the eliminated synapses are functionally immature. For

example, during early brain development some synapses are

selectively stabilized by activity-dependent processes, which

increase their strength, whereas weak immature synapses that

are not strengthened are eliminated (Katz and Shatz 1996;

Waites et al. 2005; Le Be and Markram 2006). Consistent with

this view, the dendritic spines preferentially eliminated during

adolescence in mouse neocortex have immature morphological

features (Zuo, Lin, et al. 2005). Alternatively, it might be that the

functional maturation of most synapses in the DLPFC occurs

before adolescence and that another factor, such as the

neuronal source of the inputs, somehow marks functionally

mature synapses for elimination (Woo et al. 1997).

In order to begin to discriminate between these 2 alter-

natives, we studied during postnatal development the physio-

logical properties of excitatory synapses in a living slice

preparation of the monkey DLPFC, focusing on properties

that reflect glutamate synapse maturation in rat cortex. We

found that early in postnatal development, a significant pro-

portion of excitatory inputs have immature properties. In

addition, the contribution of immature synapses declines

significantly before the onset of adolescence. Consequently,

before synaptic pruning begins, the functional properties of

excitatory synaptic inputs are indistinguishable from those

observed in neurons from adult monkeys. Our results therefore

indicate that the changes in DLPFC circuits associated with

Cerebral Cortex March 2008;18:626--637

doi:10.1093/cercor/bhm095

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the maturation of working memory performance during

adolescence involve a decrease in excitatory synapse number

in the absence of substantial maturational changes in synaptic

strength.

Methods

Brain Slice PreparationThese experiments were carried out with tissue obtained from 14

female rhesus monkeys (Macacamulatta) supplied by the University of

Pittsburgh Primate Research Center. Housing and experimental proce-

dures were conducted in accordance with United States Department of

Agriculture and National Institutes of Health guidelines and with

approval of the University of Pittsburgh’s Institutional Animal Care and

Use Committee. All animals 42 months of age or less were bred at this

facility. As described previously (Cruz et al. 2003), animals were housed

with their mothers until 6 months of age when they were placed by

groups in the same social setting. Older animals were housed either in

pairs or in single cages in the same setting. All animals were

experimentally naive at the time of entry into this study.

To determine the functional properties of excitatory synapses, we

prepared brain slices at 4 postnatal time points. The first group of

animals (n = 5 animals and 5 hemispheres) was studied at 3 months of

age, when the period of rapid synaptogenesis is close to complete and

the densities of excitatory synapses and layer 3 pyramidal cell spines are

at peak values (Rakic et al. 1986; Anderson et al. 1995). The second

group of animals (n = 3 animals and 6 hemispheres) was studied at 15

months of age, just prior to the onset of the period of rapid synaptic

elimination, when synapse and spine densities are not appreciably

different from those present at 3 months of age. The third group of

animals (n = 4 animals and 8 hemispheres) was studied at 42 months of

age, when the period of adolescence-related pruning of excitatory

synapses and spines is essentially complete. A fourth group of animals (n

= 2 animals and 3 hemispheres) was studied at 84 months of age to

determine whether the functional properties of excitatory synapses are

stable across early adulthood.

Tissue blocks containing portions of DLPFC areas 9 and 46 were

obtained from one or both hemispheres of each animal. Each of the

3-month-old and one of the 84-month-old animals were deeply anesthe-

tized and perfused transcardially with cold artificial cerebrospinal fluid

(ACSF) solution of the following composition (in mM): sucrose 210.0,

NaCl 10.0, KCl 1.9, Na2HPO4 1.2, NaHCO3 33.0, MgCl2 6.0, CaCl2 1.0,

glucose 10.0, and kynurenic acid 2.0; pH 7.3--7.4 when bubbled with 95%

O2--5% CO2, as previously described (Gonzalez-Burgos et al. 2004). The

brain was then rapidly removed and bilateral DLPFC blocks were

prepared. For all other animals, an initial tissue block was removed

from one hemisphere using a previously described surgical procedure

(Gonzalez-Burgos et al. 2004) and then a second DLPFC tissue block was

removed 1--2 weeks later following the transcardial cold ACSF perfusion

procedure described above. When 2 tissue blocks were removed per

animal in separate surgical procedures, the locations of the blocks were

offset in the rostral--caudal axis, so that nonhomotopic portions of the

DLPFC were studied from each hemisphere. Previous studies have

shown that the first procedure does not alter the physiological or

anatomical properties of the neurons and local circuits present in the

tissue obtained in the second hemisphere (Gonzalez-Burgos et al. 2000).

Cortical slices (350 lm thick) were cut in the coronal plane using

a vibrating microtome (VT1000S, Leica Microsystems, Nussloch, Ger-

many) in ice-cold ACSF. Immediately after cutting, slices were trans-

ferred to an incubation chamber maintained at room temperature and

filled with a solution of the following composition (in mM): NaCl 126.0,

KCl 2.0, Na2HPO4 1.2, glucose 10.0, NaHCO3 25.0, MgCl2 6.0, and CaCl21.0; pH 7.3--7.4 when bubbled with 95% O2--5% CO2.

Electrophysiological RecordingsFor recording, slices were submerged in a chamber superfused at a rate

of 2--3 ml/min with a solution that was bubbled with 95% O2/5% CO2

and maintained at 30--32 �C. The superfusion solution had the following

composition (in mM): NaCl 126.0, KCl 2.5, Na2HPO4 1.2, Na2HCO3 25.0,

glucose 10.0, CaCl2 2.0, MgCl2 1.0, and bicuculline methiodide 0.02. In

some experiments, gabazine (0.02 mM) was used instead of bicuculline,

to block c-aminobutyric acid A (GABAA) receptor channels. Using

infrared differential interference contrast video microscopy (Stuart

et al. 1993), whole-cell recordings were obtained from visually identified

pyramidal neurons in layer 3 of DLPFC areas 9 and 46. Recording

micropipettes pulled from borosilicate glass had a resistance of 3--5

MOhm when filled with a solution of the following composition (in

mM): CsGluconate 120.0, NaCl 10.0, ethyleneglycol-bis(2-aminoethy-

lether)-N,N,N9,N9-tetraacetic acid 0.2, 4-(2-hydroxyethyl)-1-piperazinee-

thanesulfonic acid 10.0, MgATP 4.0, NaGTP 0.3, NaPhosphocreatine

14.0, and biocytin 0.5%. The pH of the pipette solution was adjusted to

7.2--7.3 using CsOH. Recordings were performed using Axoclamp 200A,

Axopatch 1C, or Axopatch 1D amplifiers (Axon Instruments, Union City,

CA) operating in voltage-clamp mode without series resistance com-

pensation. Signals were low-pass filtered at 3 kHz, digitized at 10 or

20 kHz, and stored on disk for off-line analysis. Data acquisition and

analysis were performed using customer-made programs written in

LabView (National Instruments, Austin, TX).

The ability to voltage clamp the postsynaptic membrane at steady-

state potentials was assessed in each cell by recording the excitatory

postsynaptic currents (EPSCs) at different somatic holding potentials.

The reversal potential of the EPSCs typically had positive values close to

0 mV. If the EPSC reversal potential had values positive to +15 mV, the

recordings were not considered for data analysis. During the experi-

ment, the series resistance was continuously monitored measuring the

current evoked by a short voltage step. If the series resistance increased

more than 20%, the recordings were excluded from data analysis.

Focal Extracellular StimulationTo elicit a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)

receptor--mediated EPSCs (AMPA EPSCs) or N-methyl-D-aspartate

(NMDA) receptor--mediated EPSCs (NMDA EPSCs), focal extracellular

stimulation was applied using theta glass pipettes (1.5 mm outside

diameter, Warner Instruments Corporation, Hamden, CT) pulled to a tip

diameter of 2--3 lm and filled with freshly oxygenated extracellular

solution. Chlorided silver wires placed inside each compartment of the

theta glass were connected to a stimulus isolation unit (Model A350D-A,

World Precision Instruments, Sarasota, FL) to apply bipolar focal

stimulation. The stimulation electrodes were placed in layer 3 at ~100lm above (or below) and ~50--100 lm lateral to the soma of the

recorded neurons, by means of motorized micromanipulators (Model

MP-285, Sutter Instruments Co., Novato, CA). Stimulation current

parameters and electrode position were adjusted to elicit small

amplitude responses resembling monosynaptic EPSCs, as described

previously (Gonzalez-Burgos et al. 2000). Stimuli were applied at

a baseline frequency of 0.1 Hz.

Pharmacological CompoundsGABAA receptor--mediated currents were blocked by application of

bicuculline methiodide or gabazine (20 lM) to the extracellular

solution. To block the NMDA component of the EPSCs, 100 lM of D,L

(-)-2-amino-5-phosphonopentanoic acid (D,L-AP5) was used. AMPA

receptor--mediated EPSCs were blocked by application of 20 lM6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). To compare the proba-

bility of glutamate release across age groups, we examined the rate of

blockade of NMDA EPSCs by 25 lM of (+)-5-methyl-10,11-dihydro-5H-

dibenzo(a,d)cyclohepten-5,10-imine maleate (MK801). All reagents

were obtained from Sigma-Aldrich (St. Louis, MO).

Analysis of the AMPA/NMDA Ratio of EPSCsTo determine the AMPA/NMDA ratio of EPSCs evoked by extracellular

stimulation, cells were initially held at –80 mV and then the holding

potential was changed in +20 mV increments until reaching +40 mV. At

least 20 sweeps were collected at each holding potential and averaged

for data analysis. After recording EPSCs at +40 mV, the holding potential

was returned to –80 mV and the EPSCs recorded at –80 mV at the

beginning and end of the voltage-clamp protocol were compared. At the

baseline stimulation frequency of 0.1 Hz employed in our experiments,

no evidence of induction of synaptic plasticity was observed after

voltage-clamping neurons at positive membrane potentials. These

results are in agreement with previous findings showing that induction

of plasticity by pairing presynaptic stimulation with postsynaptic

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depolarization requires a stimulation frequency higher than 0.1 Hz

(Isaac et al. 1997; Rumpel et al. 1998; Montgomery et al. 2001; Barria and

Malinow 2005). Changes of more than 10% in the EPSC amplitude

recorded at –80 mV were exclusively due to changes in the series

resistance. Such recordings were excluded from data analysis in the

present study. The EPSCs recorded at –80 mV were readily blocked by

application of the AMPA receptor antagonist CNQX and were not

affected by application of the NMDA antagonist D,L-AP5. Thus, the peak

amplitude of EPSCs recorded at –80 mV was used to estimate the AMPA

receptor contribution to the EPSCs. To determine the NMDA receptor

contribution, in individual neurons the amplitude of the current

recorded at a holding potential of +40 mV was measured over a time

period when the AMPA EPSC recorded at –80 mV decayed to 5% of its

peak amplitude. When EPSCs were recorded at +40 mV the AMPA

component decayed completely by that time because the driving force

is significantly smaller at +40 compared with –80 mV. Therefore, small

voltage-dependent changes in the decay kinetics of the AMPA EPSC as

those previously reported (Cathala et al. 2005) do not affect our

measurement of the NMDA EPSC. In some neurons, the AMPA/NMDA

ratio was estimated by measuring the area under the AMPA and NMDA

EPSC waveforms, which represents the synaptic charge transfer.

The EPSC recorded at –80 mV was used to estimate the area of the

AMPA EPSC waveform. The NMDA EPSC area was estimated by

measuring the area of the EPSC recorded subsequently from the same

neuron at a holding potential of +40 mV and after the AMPA EPSC was

blocked by application of CNQX. Stimulus intensity, stimulus duration,

and the position of the stimulation electrode were kept constant before

and after CNQX application to stimulate the same presynaptic fibers.

The area under the curve was calculated between EPSC onset and the

time at which the EPSC decayed to 5% of its peak amplitude.

Analysis of the Kinetics of AMPA and NMDA EPSCsTo compare the decay kinetics of AMPA EPSCs between age groups,

EPSCs were recorded at a holding potential of –80 mV in the presence of

a GABAA-receptor antagonist (either bicuculline or gabazine, 20 lM). To

compare the decay kinetics of NMDA EPSCs, EPSCs were recorded at

a holding potential of +40 mV. For both AMPA and NMDA EPSCs, at least

20 consecutive sweeps were recorded and averaged, and curve fitting

was performed on the average trace. Single-exponential decay functions

were fit to the data using curve-fitting routines in Igor (Wavemetrics,

Lake Oswego, OR).

Rate of NMDA EPSC Block by MK801MK801 is an open-channel blocker that inhibits NMDA receptor--

activated currents in an essentially irreversible manner (Huettner and

Bean 1988). Therefore, the rate of NMDA EPSC block by MK801 at

a constant interstimulus interval is directly proportional to the proba-

bility that each stimulus evokes glutamate release (Pr), leading to a faster

rate of NMDA EPSC block by MK801 (Rosenmund et al. 1993). Assessing

the actual probability of release (Pr) requires knowledge of parameters

related to the time course of the glutamate concentration transient in

the synaptic cleft and the kinetics of NMDA channel opening (Rose-

nmund et al. 1993). Because such parameters were not available in the

present study, only the rate of NMDA EPSC block was used for

comparison of the Pr between age groups. NMDA EPSCs were isolated

by application of bicuculline or gabazine and CNQX, 20 lM each. NMDA

EPSCs typically had a stable peak amplitude of 50--150 pA when evoked

at a frequency of 0.1 Hz. In preliminary experiments (n = 5), the

stimulation was interrupted and resumed a few minutes later to verify

that pausing the stimulation did not affect the NMDA EPSC amplitude

(Fig. 6A). Before MK801 application, stimulation was paused, and the

cells were held at –80 mV to avoid NMDA channel opening. MK801

(25 lM) was applied for at least 15 min to allow equilibration of its

concentration in the extracellular space. Stimulation was then resumed

to evoke NMDA EPSCs at a holding potential of +40 mV in the

continuous presence of MK801 (Fig. 6A). In contrast to previously

published findings (Wasling et al. 2004), we found that the amplitude of

the first NMDA EPSC recorded after resuming the stimulation in the

presence of MK801 was essentially identical to that of the last EPSC

recorded right before interrupting the stimulation (Fig. 6A). For data

analysis, NMDA EPSC amplitude in each experiment was normalized

relative to that of the first NMDA EPSC recorded after resuming

stimulation in the presence of MK801 (Fig. 6B). Single- or double-

exponential decay functions were fit to the data using curve-fitting

routines in Igor (Wavemetrics, Lake Oswego, OR).

Morphological AnalysisAll layer 3 pyramidal cells in which dendritic spine density was

measured were located in dorsolateral PFC areas, specifically in the

dorsal portion of area 9 or in area 46, in the medial bank of the principal

sulcus. In slices from 15-month-old animals, 6 out of 11 neurons were

located in area 9, and 4 out of 11 cells were in area 46. In slices from 42-

month-old animals, 3 out of 12 neurons were located in area 9, and 6 out

of 12 neurons in area 46. For the remaining cells (1/11 in 15-month-old

and 3/12 in 42-month-old animals), the location in area 9 versus area 46

could not be determined reliably. Layer 3 pyramidal neurons were filled

with 0.5% biocytin during recording and after recording, the slices were

fixed in 4% paraformaldehyde and stored in an antifreeze solution (1:1,

glycerol:ethylene glycol in 0.1 M phosphate buffer) at –80 �C until

processed for visualization of the biocytin label. Slices were resectioned

at 60 lm, incubated with 1% H2O2, and immersed in blocking serum

containing 0.5% Triton X-100 for 2 h at room temperature. Slices were

then incubated with Avidin Biotinylated enzyme complex-peroxidase

and developed using the Nickel-enhanced diaminobenzidine chromo-

gen to visualize the biocytin-labeled pyramidal neurons.

Previous studies have shown that the changes in excitatory synapse

density during postnatal development occur throughout the entire

dendritic tree of layer 2/3 pyramidal neurons in the monkey DLPFC

(Anderson et al. 1995). In order to avoid any potential confounds

associated with proximity to the recording site at the soma, the analysis

of spine density was focused on the most distal portions of the apical

dendrite (apical tuft) in layers 1 and 2. Using the Neurolucida tracing

system (MicroBrightField, Williston, VT), a sampling frame (200 lmdeep by 400 lm wide), containing 50 sampling squares (40 lm side),

was superimposed on the image of the apical dendritic tuft. The top

border of the sampling frame was oriented parallel to and 30 lm below

the pial surface and was centered relative to the main trunk of the apical

dendrite. Each sampling frame had 42% of the squares randomly

selected for sampling. A different frame was used for sampling dendritic

spines in each neuron. The selected portions of the apical dendrite were

reconstructed using a 633 oil immersion objective with differential

interference contrast optics using Neurolucida (MicroBrightField,

Williston, VT) with the diameters of the dendritic shafts recorded.

The total number of spines counted per neuron was divided by the total

length of dendritic shaft sampled to obtain an estimation of dendritic

spine density per cell. The total number of spines counted per neuron

varied from 129 to 949 spines. Spine density was not correlated with the

total dendritic length sampled in each neuron (Pearson correlation

coefficient r = –0.04, P = 0.83). Mean spine density per neuron was then

compared across age groups.

The method used for counting spines may underestimate the true

spine density due to ‘‘hidden’’ spines, oriented perpendicular to the

plane of section. Therefore our spine counts are relative rather than

absolute. Most likely, however, this issue applies equally to all cells

examined and is independent of age group. For example, because the

number of hidden spines is related to dendritic diameter, age-related

differences in diameter could bias the obtained relative spine densities.

However, previous studies (e.g., Anderson et al. 1995) of animals in the

age range used herein did not detect age-related differences in diameter

of layer 3 pyramidal cell dendrites and no differences in mean dendrite

diameter were found in the present study. Furthermore, previous

studies using 3-dimensional reconstructions (see for example http://

synapse-web.org/) of pyramidal cell dendrites did not find a particular

bias in spine distribution around the dendritic shaft in developing or

mature pyramidal neurons (Fiala and Harris 1999; Bourne et al. 2007).

Thus, it seems unlikely that our relative spine density measures were

substantially biased by age-dependent differences in the fraction of

spines oriented perpendicular to the plane of section.

Previous studies demonstrated that changes in estrogen levels are

associated with dendritic spine density in rat hippocampal pyramidal

neurons and that estradiol administration increases spine density in

pyramidal neurons from the DLPFC of ovariectomized monkeys (Tang

et al. 2004; Hao et al. 2006). To determine if gonadal steroids levels could

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confound dendritic spine measures in this study, we measured the

serum levels of estradiol and progesterone on each day when brain

tissue was obtained from the 42- and 84-month-old animals. Spine

density did not differ (P = 0.47) between or within animals when slices

were prepared during the luteal or follicular phases of the menstrual

cycle.

Statistical AnalysisThe data are expressed as mean ± standard deviation. Statistical

significance of the difference between group means was determined

employing Student’s t-test or analysis of variance (ANOVA) followed by

comparisons, as indicated in each particular case. Differences were

considered significant when P < 0.05.

Results

The Density of Dendritic Spines in Layer 3 PyramidalNeurons Significantly Declines between 15 and 42Months of Age

During adolescence, spine density significantly decreases

throughout the dendritic tree of layer 3 pyramidal neurons in

themonkey DLPFC (Anderson et al. 1995) in a manner consistent

with the elimination of excitatory synapses in layers 1 to 4 (Rakic

et al. 1986; Bourgeois et al. 1994). In contrast to spine density, the

size of layer 3 pyramidal cell bodies or the length of their dendrites

are close to adult values at 3monthsof age and essentially identical

to adult values at 15 months of age (Anderson et al. 1995). The

absence of changes in layer 3 pyramidal cell size suggests that the

significant decrease in spine density during adolescence repre-

sents a reduction in the total numberof excitatory synaptic inputs.

To confirm that an adolescence-related decrease in spine density

was present in our neuron sample, we determined the spine

density in the distal (apical tuft) dendrites of layer 3 pyramidal

neurons that were filledwith biocytin during electrophysiological

recordings (Fig. 1A).

The spines in the distal dendrites of layer 3 neurons displayed

various morphologies (Fig. 1B). However, using light micros-

copy, the morphology of many individual spines could not be

reliably characterized. Therefore, spine density was measured

independently of morphological spine type (Fig. 1C). Mean

spine density was significantly lower (17%) in layer 3 pyramidal

neurons from 42-month-old compared with the 15-month-old

animals (Fig. 1D). It was previously shown that in this pyramidal

cell class, spine density decreases by 40--50% by the end of

adolescence when averaged across dendritic membrane com-

partments (Anderson et al. 1995). However, the actual magni-

tude of this decrease depends on the dendritic compartment

studied. For example, in the apical tuft dendrites, spine density

decreases by ~15% between 18 and 32 months of age (Anderson

et al. 1995). Therefore, the 17% decrease in spine density in the

distal apical dendrites between 15 and 42 months of age

reported here is consistent with the age-related changes

described in previous studies.

Postsynaptic Function Changes between 3 and 15Months of Age

In the developing rat cortex, immature synapses contain NMDA

receptors but possess few or no AMPA receptors and are

therefore weak or silent at the resting membrane potential

(Malinow and Malenka 2002). Glutamate synapse maturation

involves activity-dependent potentiation by AMPA receptor

insertion into the postsynaptic membrane (Liao et al. 2001;

Zhu and Malinow 2002; Lu and Constantine-Paton 2004; Mierau

et al. 2004), which gradually decreases the proportion of

synapses with low AMPA receptor content (Rumpel et al.

1998, 2004). To determine if the relative contribution of

AMPA receptors to the EPSCs increases in layer 3 pyramidal

neurons during maturation of the primate DLPFC, we first

determined the AMPA/NMDA ratio in EPSCs evoked by focal

extracellular stimulation (see Methods). Figure 2A shows

examples of EPSCs from neurons of the 3- and 15-month-old

age groups, recorded at somatic holding potentials of –80 and

+40 mV. As displayed in Figure 2B, the AMPA/NMDA ratio

increased significantly between 3 and 15 months of age, but did

not show further changes at 42 and 84 months of age. An

increase in the AMPA/NMDA EPSC ratio is consistent with

a significant decrease in the proportion of functionally imma-

ture synapses between 3 and 15 postnatal months.

In rat neocortex and hippocampus, the NMDA EPSC decay

accelerates during early postnatal development in coincidence

Figure 1. Analysis of dendritic spine density in layer 3 pyramidal neurons of monkeyDLPFC. (A) An example of the morphology of the dendritic tree of layer 3 pyramidalneurons filled with biocytin during electrophysiological recordings in slices frommonkey DLPFC. (B) High magnification micrograph of a distal apical dendritic segmentof a layer 3 pyramidal neuron. Note the presence of spines with heterogeneousmorphology. (C) Reconstruction of the segment of apical dendrite shown in Figure 1B.For quantification of spine density, dendritic protrusions identified as spines werecounted independently of their apparent morphology. (D) Bar graph illustrating that themean spine density significantly decreased by ~17% in neurons from 15- and 42-month-old animals. Statistical significance of the difference between means wasdetermined using a 1-tailed t-test, P \ 0.05. These results are similar to previousfindings showing that spine density in the distal portion of the apical dendrite of layer 3pyramidal neurons in monkey DLPFC decreases by ~15% between 18 and 32 monthsof age (Anderson et al. 1995).

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with synaptic circuit maturation (Flint et al. 1997). Therefore,

we tested whether the duration of pharmacologically isolated

NMDA EPSCs in layer 3 pyramidal neurons differs between age

groups. Figure 3A shows examples of NMDA EPSCs recorded

from pyramidal cells in slices of the 3- and 15-month-old age

groups, with exponential function fits superimposed on the

current traces. We found that the time constant of exponential

decay of the NMDA EPSCs decreased significantly between 3

and 15 months of age (Fig. 3B). After 15 months, the time

constant did not change further (Fig. 3B). These results are

consistent with a maturation of NMDA receptor function at

glutamatergic inputs onto layer 3 pyramidal cells, between 3

and 15 months of age.

The decrease in the duration of the NMDA EPSCs observed in

our studies (Fig. 3B) may contribute to the increase in the

AMPA/NMDA ratio when the NMDA contribution to this ratio is

measured at late phases in the decay of EPSCs recorded at

positive holding potentials (Fig. 2B). If so, then AMPA/NMDA

ratio and NMDA EPSC duration should be inversely correlated

when measured from the same neuron. To test this possibility,

we determined whether the AMPA/NMDA ratio and the NMDA

EPSC duration were correlated in a subsample of neurons (n =29) in which both parameters were measured sequentially from

the same cell. As shown in Figure 3C, there was no significant

correlation between AMPA/NMDA ratio and NMDA EPSC

duration (Pearson correlation coefficient r = –0.11, P = 0.57),

showing that the AMPA/NMDA ratio did not show an associa-

tion with the NMDA EPSC duration. To further test the idea that

the AMPA/NMDA ratio increases independently of the decrease

in NMDA EPSC duration, in a subsample of neurons (3 months,

n = 11; 15 months n = 12; 42 months, n = 5; 84 months, n = 9),

we determined the AMPA/NMDA ratio by recording from each

neuron isolated AMPA and NMDA EPSCs and by estimating the

area under the AMPA and NMDA EPSC waveforms separately.

Measurement of the NMDA EPSC area provides an estimation of

the total NMDA current contribution to the synaptic charge

transfer, including the contribution of both peak current and

decay, independent of differences in decay time between age

groups. The mean AMPA/NMDA ratio calculated based on EPSC

areas increased by about 81% between 3 and 15 months and

remained higher than 3 months in the older age groups,

although the differences between group means did not reach

statistical significance (3 months: 0.22 ± 0.16, n = 11; 15 months:

0.40 ± 0.24, n = 12; 42 months: 0.30 ± 0.13, n = 5; 84 months: 0.37

± 0.23, n = 9; F3,28 = 2.50, P = 0.08, Single-factor ANOVA). The

increase in the AMPA/NMDA area ratio observed between 3 and

15 months was similar to the statistically significant increase

observed for the ratio of currents (about 60% increase between

3 and 15 months, Fig. 2B). Moreover, the AMPA/NMDA area

ratio in the 3-month-old group was significantly different from

that of the other age groups after they were pooled (P = 0.03).

Figure 2. The ratio between AMPA and NMDA receptor contribution to EPSCs issmaller at 3 months of age. (A) Average traces representing EPSCs recorded whileholding cells at negative (�80 mV) and positive (þ40 mV) membrane potentials.Traces are the average of at least 20 sweeps recorded at each membrane potential.Note the differences, between 3 and 15 months, in the ratio between the peak inwardcurrent at�80 mV and the amplitude of the outward current recorded atþ40 mV. (B)For EPSCs recorded at �80 and þ40 mV from each neuron, the ratio between AMPAand NMDA receptor contribution was estimated as described in the Methods section.Bars with different letters are significantly different. (Single-factor ANOVA, (F3,59 55.26, P 5 0.003, followed by Tukey’s tests).

Figure 3. The duration of NMDA EPSCs changes between 3 and 15 months of age.(A) Examples of average NMDA EPSCs recorded from 3- and 15-month-old neuronsscaled to the same peak amplitude and showing, superimposed, the exponentialfunctions fit to the traces. Note the apparent differences in NMDA EPSC duration. (B)The decay kinetics of NMDA EPSCs is significantly longer in EPSCs recorded fromneurons of 3-month-old animals. Bars with different letters are significantly different(Single-factor ANOVA, followed by Tukey’s tests, F3,103 5 9.39, P5 0.0002). (C) TheAMPA/NMDA ratio and the duration of NMDA EPSCs were compared for individualneurons, irrespective of age group. The absence of significant correlation suggests noassociation between changes in NMDA EPSC duration and the AMPA/NMDA ratio(Pearson’s correlation coefficient r: �0.110, P 5 0.57).

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Together, the results shown in Figure 3 suggest that in addition

to an acceleration of the NMDA EPSC decay there is an actual

increase in the relative contribution of AMPA receptors

between 3 and 15 months of age.

Multiple factors may contribute to the acceleration of the

NMDA EPSC decay during development. For instance, develop-

mentally regulated modifications in synaptic structure (Cathala

et al. 2005) may shorten the duration of the glutamate

concentration transient in the synaptic cleft by changing the

kinetics of glutamate release or the speed of transmitter

clearance (Diamond 2005; Koike-Tani et al. 2005; Takahashi

2005). Changes in the cleft glutamate concentration transient

may not alter synaptic NMDA receptor activation (Lester et al.

1990). However, NMDA EPSCs are mediated in part by extra-

synaptic NMDA receptors (Lozovaya et al. 2004; Thomas et al.

2006), the activation of which may depend on the duration of

the glutamate concentration transient (Lozovaya et al. 1999;

Arnth-Jensen et al. 2002). If the acceleration of NMDA EPSC

decay is in part due to changes in the time course of the

synaptic glutamate concentration transient, then the decay of

AMPA EPSCs should also be affected (Cathala et al. 2005; Koike-

Tani et al. 2005). To test this possibility, we examined the decay

kinetics of AMPA EPSCs recorded from monkey DLPFC layer 3

pyramidal neurons (Fig. 4A). We found that the decay time

constant of AMPA EPSCs did not differ significantly between age

groups (Fig. 4B) when assessed in neurons that displayed

significant acceleration of NMDA EPSC decay between 3 and

15 months. These results indicate that the acceleration of EPSC

decay is specific for NMDA EPSCs.

An important factor regulating NMDA EPSC duration during

development in rat hippocampus and neocortex is a switch in

the subunit composition of the NMDA receptor complex. Early

in development, NMDA receptor subunit B (NR2B) subunits are

prevalent and determine a longer NMDA EPSC duration.

Conversely, NR2A subunits are more abundant in the mature

cortex (Carmignoto and Vicini 1992; Erisir and Harris 2003; Liu

et al. 2004) and produce shorter NMDA EPSCs (Monyer et al.

1994; Sheng et al. 1994; Flint et al. 1997; Fu et al. 2005). To test

whether the decrease in NMDA EPSC duration with age

observed here is associated with a decrease in the contribution

of NR2B subunits, we determined the effects of ifenprodil,

a selective antagonist of NR2B subunit--containing NMDA

receptors (Dingledine et al. 1999). Application of ifenprodil

significantly reduced the amplitude of NMDA EPSCs recorded

from neurons of 3-, 15-, or 42-month-old animals (Fig. 5A).

However, the depression of NMDA EPSC amplitude by ifenpro-

dil was significantly strong in neurons from 3-month-old animals

(Fig. 5B). In addition, ifenprodil decreased the duration of

NMDA EPSCs, but this effect was statistically significant only in

neurons from 3-month-old animals (Fig. 5C). Our findings thus

suggest that the decrease in NMDA EPSC duration during

postnatal development of monkey DLPFC is at least in part

due to a decreased contribution of NR2B receptor subunits.

EPSCs Display Immature Presynaptic Properties at 3Months of Age

Our results suggest that excitatory inputs onto neurons from 3-

month-old animals have a significantly larger proportion of

synapses with relatively low AMPA receptor content (Figs. 2 and

3). Immature synapses with low AMPA receptor content may

have low failure rate (Montgomery et al. 2001) and higher Pr

than AMPA receptor-rich synapses (Yanagisawa et al. 2004).

Maturation of presynaptic properties is usually associated with

a decrease in the Pr (Bolshakov and Siegelbaum 1995; Kumar

and Huguenard 2001; Wasling et al. 2004; Zhang 2004). Thus,

we determined if the maturation of postsynaptic properties

observed in this study between 3 and 15 months of age was

paralleled by changes in Pr. To compare the Pr between age

groups, we determined the rate of block of NMDA EPSCs by

MK801, an irreversible open-channel blocker (Huettner and

Bean 1988) that blocks NMDA EPSCs with faster kinetics the

higher Pr is (Rosenmund et al. 1993). As in previous studies, the

amplitude of NMDA EPSCs recorded from layer 3 pyramidal cells

in monkey DLPFC was reduced progressively by stimulation in

the continuous presence of 25 lM MK801 (Fig. 6A). Consistent

with the presence of synaptic subpopulations with heteroge-

neous Pr, in each age group double-exponential decay functions

(Fig. 6B) fit the data better than single-exponential decay

functions. At later stages of the experimental protocol, the

time course of NMDA EPSC block largely overlapped between

age groups (Fig. 6B). Conversely, the initial rate of NMDA EPSC

block was faster in neurons from 3-month-old animals (Fig. 6B),

as revealed by an analysis of the parameters of exponential

curve fitting. Tau F, the time constant of faster exponential

decay, was significantly shorter in neurons from 3-month-old

animals than in neurons from the other age groups (Fig. 6C).

Figure 4. The decay time course of AMPA EPSCs does not differ between agegroups. (A) Examples of average AMPA EPSCs recorded from 3- and 42-month-oldneurons, scaled to the same peak amplitude and showing, superimposed, theexponential functions fit to the traces. Note the apparent lack of differences in AMPAEPSC duration between ages. (B) The exponential decay time constant of AMPAEPSCs did not differ significantly between age groups (Single-factor ANOVA, F3,30 50.40, P 5 0.75).

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Comparisons of the remaining fitting parameters showed no

statistically significant differences (Table 1). These data suggest

that neurons from 3-month-old animals have a subpopulation of

synapses in which Pr is higher than in synapses of any of the

other age groups. Therefore, the analysis of the rate of NMDA

EPSC blockade by MK801 suggest that the contribution of

synaptic inputs with immature presynaptic properties de-

creases significantly prior to adolescence, sometime between

3 and 15 months of age.

If the fraction of NMDA channels blocked by MK801 differs

between EPSCs recorded from neurons of the different age

groups, then the rate of NMDA EPSC blockade may be de-

pendent not only on the Pr but also on the efficacy of MK801 to

block the NMDA channels once glutamate is released (Hessler

et al. 1993; Rosenmund et al. 1993). Because we employed

a saturating concentration of MK801 (25 lM), age-related

differences in binding affinity are unlikely to influence the

fraction of NMDA channels blocked by MK801. To rule out the

possibility that a different MK801-binding affinity or other

factors may determine a different fractional block of NMDA

channels, we determined the effects of MK801 on the NMDA

EPSC decay. Acceleration of the NMDA EPSC decay by MK801 is

an indicator of the fraction of channels blocked once glutamate

is released (Hessler et al. 1993; Rosenmund et al. 1993) because

MK801 blocks the NMDA channels not only at the EPSC peak

but also during deactivation (Chen et al. 1999). To determine if

the efficacy of NMDA channel block differed between age

groups, we tested the effect of MK801 on the NMDA EPSC

decay time constant in neurons from 3-, 15-, and 42-month-old

animals. We found that MK801 (25 lM) significantly accelerated

the decay time of NMDA EPSCs. For each cell, the decay time

was computed for the average of the last 5 NMDA EPSCs

recorded prior to MK801 application and also for the first 5

NMDA EPSCs recorded after resuming stimulation in the

presence of MK801 (3 months, control decay time: 84 ± 30 ms,

MK801 decay time: 47 ± 24 ms, n = 10 cells; 15 months, con-

trol decay time: 49 ± 9 ms, MK801 decay time: 30 ± 15 ms, n = 10cells; 42 months, control decay time: 63 ± 12 ms, MK801 decay

time: 48 ± 30 ms, n = 9 cells). Two-factor ANOVA indicated

a significant effect of MK801 (F = 18.6, P = 0.00007), a significanteffect of age (F = 7.27, P = 0.00165), but an absence of significant

interaction between MK801 and age effects (F = 1.03, P =0.36171). These results indicate that the efficacy of MK801 for

blocking NMDA channels did not differ substantially between

age groups, thus suggesting that differences in Pr are the most

likely determinant of the faster kinetics of NMDA EPSC block in

neurons from 3-month-old animals.

Discussion

We determined the functional properties of excitatory synapses

onto layer 3 pyramidal neurons during postnatal development of

Figure 5. NMDA EPSCs recorded from 3-month-old neurons are more sensitive to the NR2B subunit antagonist ifenprodil. (A) In an experiment performed with a 3-month-oldneuron, application of ifenprodil (10 lM) reduced the amplitude of NMDA EPSCs. Plotted is the peak amplitude of the NMDA EPSC normalized relative to the average amplitudemeasured 5 min prior to ifenprodil application. Average NMDA EPSCs obtained by averaging 5 consecutive traces right before the indicated time points (1 and 2) are shown to theright of the plot. The scaled traces in the inset show that the decrease in NMDA EPSC amplitude by ifenprodil was accompanied by an acceleration of its decay. (B) Summary plotshowing the effects of 10 lM ifenprodil on NMDA EPSCs recorded from neurons of the 3- and 42-month-old groups. Although at both age groups ifenprodil inhibited the NMDAEPSCs, the effect was significantly stronger at 3 months of age. Bars with different letters are significantly different (Single-factor ANOVA F2,26 5 5.84, P 5 0.008, followed byTukey’s test) (C) Summary graph of the effects of ifenprodil on the NMDA EPSC duration, as assessed by the time constant of monoexponential decay. Ifenprodil applicationproduced a statistically significant decrease in the duration of EPSCs recorded from 3-month-old neurons but not on the duration of those recorded from 42-month-old cells. Barswith different letters are significantly different (Single-factor ANOVA F2,30 5 4.35, P 5 0.02, followed by Tukey’s test).

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monkey DLPFC. We found that between 3 and 15 months of age,

when synaptic density is constant, the contribution of synapses

with immature functional properties appears to decrease

significantly, as suggested by the following findings: 1) the

relative contribution of AMPA receptors to the EPSCs increased;

2) the speed of NMDA EPSC decay increased; 3) the NMDA

EPSCs became less sensitive to the NR2B subunit--selective

antagonist ifenprodil; and 4) the Pr, as assessed by the rate of

NMDA EPSC block by MK801, decreased. In contrast, the

properties of EPSCs at 15 months were indistinguishable from

those of EPSCs recorded from adult neurons at 42 or 84 months

of age. Thus, our data suggest that functional maturation of

excitatory synaptic inputs onto layer 3 pyramidal cells of

monkey DLPFC occurs prior to the adolescence-related re-

duction in excitatory synaptic input described in previous

studies.

Changes in Synaptic Function during PostnatalDevelopment through Adolescence

An increase in the AMPA/NMDA ratio such as that found here is

commonly associated with postsynaptic maturation (Durand

et al. 1996; Wu et al. 1996; Isaac et al. 1997; Zhu and Malinow

2002; Franks and Isaacson 2005). Although consistent with an

increase in the AMPA receptor contribution, the AMPA/NMDA

ratio may increase via a decrease in the NMDA contribution. In

the developing frog optic tectum (Wu et al. 1996) and during

activity-dependent maturation of thalamocortical synapses

(Isaac et al. 1997), the AMPA/NMDA ratio increases via an

increased AMPA but constant NMDA component. In contrast, in

rat olfactory cortex the AMPA/NMDA ratio increases, in part by

an increase in AMPA contribution but mostly from NMDA

downregulation (Franks and Isaacson 2005). The relative

contribution of AMPA increase versus NMDA downregulation

to the age-dependent increase in ratio observed in this study

remains to be determined.

In the neonatal rat hippocampus, nascent synapses are rapidly

silenced by removal of AMPA receptors from the postsynaptic

membrane resulting from presynaptic action potential firing

(Groc et al. 2006). We did not directly address the contribution

of activity-dependent silencing of AMPA-containing nascent

synapses to the lower AMPA/NMDA ratio observed in neurons

from 3-month-old animals. However, the AMPA EPSC amplitude

was typically stable during stimulation at 0.1 Hz and showed no

evidence of silencing mechanisms during our recordings.

Changes in the AMPA/NMDA ratio may be also due to an

Figure 6. The rate of NMDA EPSC blockade by MK801 indicates that the contributionof synapses with high Pr is more significant in neurons from 3-month-old animals. (A)EPSC amplitude as a function of event number. The horizontal line above the graphshows the holding potential. The traces displayed above the graph were obtained afteraveraging 5 consecutive events right before the time points indicated by the numbers1--5. The red arrows indicate the time points at which stimulation was interrupted andthe black arrows indicate the time at which stimulation was resumed. (B) Theamplitude of NMDA EPSCs was normalized relative to that of the first responserecorded after resuming stimulation in the presence of MK801. The lines representdouble-exponential functions fit to the data by nonlinear regression. (C) Summarygraphs showing that tau F, the faster time constant in the double-exponential decayfunctions fit to the data, is significantly smaller in neurons from 3-month-old animals.Bars with different letters are significantly different (Single-factor ANOVA, F3,57 5

3.838, P5 0.0019). For additional information on the parameters of exponential curvefitting, see Table 1.

Table 1Time course of NMDA EPSC blockade by MK801 in layer 3 pyramidal neurons of different age groups

3 months(n 5 19)

15 months(n 5 17)

42 months(n 5 15)

84 months(n 5 10)

F P value

FB 0.45 ± 0.05 0.49 ± 0.07 0.37 ± 0.04 0.52 ± 0.14 0.450 0.717tau F (s) 16.4 ± 4.1 38.9 ± 11.1 37.6 ± 8.8 56.9 ± 19.8 3.838 \0.01SB 0.56 ± 0.04 0.48 ± 0.07 0.60 ± 0.05 0.46 ± 0.15 0.706 0.550tau S (s) 463 ± 81 1386 ± 1340 642 ± 158 609 ± 478 1.693 0.173

Note: For each age group, the time course of NMDA EPSC blockade by MK801 was fit by a double-exponential decay function of the form: E(t)5 FB3 exp(�t/tau F) þ SB3 exp(�t/tau S) (Fig. 3C). In

this equation, E is the amplitude of the NMDA EPSC; t is time after resuming stimulation in the presence of MK801 25 lM; FB and SB represent the fractions of the initial NMDA EPSC that are blocked

with faster and slower time course, respectively, by MK801; tau F and tau S are the time constants of faster and slower exponential decay, respectively. Data are shown as mean ± standard error.

Statistical significance of the differences in each parameter between age groups was determined using an F test and differences were considered significant if P\ 0.05. Pairwise comparisons showed

that tau F at 3 months was significantly different from tau F at 15 months (P 5 0.034), from tau F at 42 months (P 5 0.049) and from tau F at 84 months (P 5 0.008). In contrast, no significant

differences were found between the tau F values of age groups older than 3 months.

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increase in the Pr at presynaptically silent synapses, rather than

to postsynaptic mechanisms (Voronin and Cherubini 2004).

Our data, however, suggest that the Pr decreases between 3 and

15 months of age. We therefore conclude that the differences in

AMPA receptor contribution reported here are more likely due

to postsynaptic mechanisms.

We found that EPSCs recorded from neurons of 3-month-old

animals had both lower AMPA/NMDA EPSC ratios and longer

NMDA EPSC decay time due to a more prominent contribution

of NR2B subunits. These results are consistent with recent data

indicating that immature synapses with low AMPA receptor

content contain only NR2B-subtype NMDA receptors and that

activity-dependent maturation produces synaptic insertion of

AMPA receptors and of NMDA receptors containing ifenprodil-

insensitive NR2A subunits (Nakayama et al. 2005). Expression of

NR2B-containing NMDA receptors favors the induction of

synaptic plasticity bidirectionally (i.e., either potentiation or

depression), via specific properties of the NR2B-mediated

intracellular Ca2+transients or by the efficient association of

NR2B-containing receptors with Ca2+/Calmodulin protein ki-

nase II (Barria and Malinow 2005). Thus, the apparent decrease

in the contribution of NR2B subunits with maturation would

imply a relative decrease in the plasticity of synapses in the

cortex of adult primates. Nevertheless, our findings are consis-

tent with in situ mRNA hybridization studies indicating that

NR2B subunits expression is still significant in the adult primate

neocortex (Scherzer et al. 1998).

The peak open-channel probability for NR2B subunit--con-

taining NMDA receptors is 5 times lower than that of NR2A-

containing receptors (Chen et al. 1999). Thus, MK801 could be

less effective in inhibiting NMDA EPSCs in neurons from 3-

month-old animals. A less efficient block of NR2B-mediated

responses by MK801 would lead to slower EPSC block in-

dependent of the Pr, thus leading to underestimation of the

differences in Pr between the 3-month-old and other age

groups. However, previous studies showed that MK801 similarly

inhibits the amplitude of NR2B- and NR2A-mediated currents

(Chen et al. 1999), suggesting that the on-rate of MK801 block

does not differ between NMDA receptor subtypes. Consistent

with this idea are previous data showing that blockade of NR2B-

containing NMDA receptors with ifenprodil does not affect the

rate of NMDA EPSC amplitude block by MK801 (Yanagisawa

et al. 2004). Furthermore, we found that MK801 similarly

accelerated the decay of NMDA EPSCs recorded from layer 3

pyramidal neurons of 3-, 15-, and 42-month-old animals (see

results). Our data suggest that the efficacy of NMDA channel

block by MK801 does not differ between age groups, supporting

the conclusion that the faster rate of NMDA EPSC block in

neurons from 3-month-old animals is due to synapses with

higher probability of glutamate release.

Implications for Models of Synapse Elimination duringAdolescence

During early brain development, synapses are stabilized via

activity-dependent strengthening, leading to elimination of

immature synapses that remain weak (Le Be and Markram

2006). In mouse neocortex, a significant proportion of mor-

phologically immature synapses are eliminated during adoles-

cence (Zuo, Lin, et al. 2005; Zuo, Yang, et al. 2005), suggesting

that, in rodents, similar mechanisms could underlie synapse

elimination during early development and during adolescence.

Adolescence represents approximately a similar proportion of

the total lifespan in mammals (Clancy et al. 2001); however,

lifespan is substantially longer in primates than in rodents.

Therefore, if similar mechanisms underlie synapse elimination

during early development and adolescence in primates, then

such mechanisms must remain active for an exceptionally

prolonged time. For example, in macaque monkeys adolescence

begins around 2--3 years of age (Lewis 1997). Our data seem to

indicate that most synapses are mature prior to adolescence

and, therefore, that the mechanisms of synapse elimination

differ between early development and adolescence. It is

possible, however, that synapses eliminated during adolescence

are immature when considering molecular or ultrastructural

characteristics that are not reflected in the functional proper-

ties measured in this study.

Our experiments cannot directly determine if the changes

in functional properties observed between 3 and 15 months of

age are due to maturation of persistent synapses or synapse

replacement. Although no changes in synapse density occur in

layer 3 of the monkey DLPFC between 3 and 15 months of

age (Bourgeois et al. 1994; Anderson et al. 1995), functionally

mature synapses could replace immature ones maintaining total

synaptic density constant. During periods of constant spine

density in adulthood, most spines are long lasting and only

a small fraction is eliminated or added from pyramidal cell

dendrites in mouse neocortex (Grutzendler et al. 2002; Trach-

tenberg et al. 2002; Holtmaat et al. 2005; Zuo, Lin, et al. 2005;

Zuo, Yang, et al. 2005). However, newly added spines eventually

form synapses with axonal boutons, demonstrating that synap-

togenesis occurs in the neocortex of adult mice (Holtmaat et al.

2006; Knott et al. 2006). A recent study suggests that synapto-

genesis may be an actively ongoing process throughout life

also in the primate neocortex (Stettler et al. 2006). Therefore,

a higher rate of synapse elimination than synaptogenesis may

lead to the decrease in synapse number during primate

adolescence. Conversely, those rates would be balanced in

adult primate neocortex, when there is no net change in

synapse density, although the rate of synapse turnover is

surprisingly high, about 7% per week (Stettler et al. 2006).

Our experiments revealed that EPSCs had relatively immature

properties in neurons from 3-month-old animals, an age when

synaptogenesis still proceeds at a fast rate, and many newly

formed synapses are being added to layer 3 pyramidal cell

dendrites (Rakic et al. 1986; Bourgeois et al. 1994; Anderson

et al. 1995). Collectively, our findings and those of previous

anatomical studies suggest that around this age in primates,

functional maturation of single synapses and synaptic circuits is

coincident and perhaps causally related. For instance, formation

and plasticity of ocular dominance columns, thought to proceed

via synapse stabilization and elimination (Katz and Crowley

2002), in monkey visual cortex occurs during the first 12 weeks

after birth (LeVay et al. 1980; Horton and Hocking 1997). This

time window overlaps with the period of active synaptogenesis

in layer 4 of monkey visual cortex (Bourgeois and Rakic 1993).

Our data also suggest that global maturational changes in

excitatory synaptic function happen before adolescence and

that remodeling of excitatory circuits during adolescence

happens via mechanisms that seem to be independent of

significant maturational changes in synaptic strength.

An intriguing possibility is that refinement of excitatory

circuits during adolescence results from dynamic excitation--

inhibition interactions. For instance, administration of

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benzodiazepine modulators that potentiate GABAA receptor--

mediated transmission significantly alters the remodeling of

excitatory connections during critical periods of cortical de-

velopment (Hensch 2005). In contrast to excitatory synapses,

no significant changes in the density of inhibitory synapses are

observed in the neocortex during adolescence (Rakic et al.

1986; De Felipe et al. 1997). However, the efficacy of GABAergic

transmission may nevertheless change during adolescence

because during this developmental period substantial changes

occur in the levels of expression of GABAA receptors, GABA

transporters and parvalbumin, an interneuron-specific calcium-

binding protein (Lewis et al. 2004). Whether or not GABA-

mediated transmission can regulate excitatory synaptic pruning

during adolescence remains to be investigated.

Our results are relevant to current neurodevelopmental

models of schizophrenia based in the observations that the

expression of mRNAs for proteins that control the efficacy of

synaptic transmission is altered in the illness (Mirnics et al.

2001). According to this model, the exuberant number of

cortical synapses present prior to adolescence compensates

for this synaptic dysfunction, and the pruning of these synapses

during adolescence leads to the expression of the clinical

consequences of the synaptic dysfunction. Our findings indicate

that synaptic properties do not differ pre- and postpruning, at

least in the normal monkey DLPFC, suggesting that prior to

adolescence, functionally mature synapses might provide com-

pensation via an excess in number.

Funding

Young Investigator Award from NARSAD to GG-B; National

Institute of Mental Health (MH45156).

Notes

We thank Ms Olga Krimer and Ms Mary Brady for their help with

reconstruction of dendritic trees and spines. Conflict of Interest: None

declared.

Address correspondence to Dr Guillermo Gonzalez-Burgos, Trans-

lational Neuroscience Program, Department of Psychiatry, University of

Pittsburgh School of Medicine, W1651 Biomedical Science Tower, 200

Lothrop Street, Pittsburgh, PA 15261, USA. Email: gburgos@pitt.edu.

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