Network stability through homeostatic scaling of excitatory and inhibitory synapses following inactivity in CA3 of rat organotypic hippocampal slice cultures

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Mol. Cell. Neurosci. 31 (2006) 805 – 816

Network stability through homeostatic scaling of excitatory and

inhibitory synapses following inactivity in CA3 of rat organotypic

hippocampal slice cultures

Lucy E. Buckby,1 Thomas P. Jensen,1 Paul J.E. Smith, and Ruth M. Empson*

School of Biological Sciences, Royal Holloway University of London, Egham, Surrey TW20 0EX, UK

Received 4 October 2005; revised 6 December 2005; accepted 11 January 2006

Available online 24 February 2006

Homeostatic plasticity is a phenomenon whereby synaptic strength is

scaled in the context of the activity that the network receives. Here, we

have analysed excitatory and inhibitory synapses in a model of

homeostatic plasticity where rat organotypic hippocampal slice

cultures were deprived of excitatory synaptic input by the NMDA

and AMPA/KA glutamate receptor antagonists, AP5 and CNQX. We

show that chronic excitatory synapse deprivation generates an

excitable CA3 network where enhanced amplitude and frequency of

spontaneous excitatory post-synaptic potentials were associated with

increased glutamate receptor subunit expression and increased

number and size of synapsin 1 and VGLUT1 positive puncta. Intact

spontaneous inhibitory post-synaptic potentials coincided with persis-

tent expression of the GABA-A receptor alpha subunit and GAD65

and an enhancement of parvalbumin-positive puncta. In this model of

homeostatic plasticity, scaling up of synaptic excitation and mainte-

nance of fast synaptic inhibition promote an excitable, but stable, CA3

network.

D 2006 Elsevier Inc. All rights reserved.

Keywords: Hippocampus; Synapsin 1; Glutamate; VGLUT1; Synapsin 1;

GABA; GABA-A receptor; NR1; GluR1; Homeostatic plasticity; Homeo-

static scaling; EPSP; IPSP

Introduction

Over the past decade, there has been a transformation in our

understanding of activity-dependent synaptic plasticity especially

regarding the development and refinement of synaptic connections.

Throughout life, axonal arbours and synaptic connections are

continuously reshaped, refined and reconfigured by activity-

dependent mechanisms (Goodman and Shatz, 1993; Munno and

Syed, 2003). These activity-dependent mechanisms can be cate-

1044-7431/$ - see front matter D 2006 Elsevier Inc. All rights reserved.

doi:10.1016/j.mcn.2006.01.009

* Corresponding author. Fax: +44 1784 434326.

E-mail address: [email protected] (R.M. Empson).1 These authors contributed equally to this work.

Available online on ScienceDirect (www.sciencedirect.com).

gorised into Hebbian plasticity including long-term potentiation and

depression (LTP and LTD) and homeostatic plasticity (Turrigiano et

al., 1998; Davis and Bezprozvanny, 2001; Burrone and Murthy,

2003). Hebbian and homeostatic plasticity are complementary yet

contrasting.Whilst Hebbian plasticity can act to destabilise neuronal

networks in either a positive or negative direction, homeostatic

plasticity promotes network stabilisation by returning synaptic

strength and neuronal firing to within a set range (Turrigiano, 1999;

Turrigiano and Nelson, 2004; Burrone and Murthy, 2003).

The concept of physiological homeostasis was originally

championed by Cannon in 1932 (Cannon, 1939). However, the

idea that neuronal networks could undergo the same form of

adaptation was only recently suggested (Davis and Goodman,

1998; Turrigiano et al., 1998). This may be because up until

recently, the idea seemed contradictory, how can homeostatic

regulation of neuronal activity also permit the changes in synaptic

strength required by Hebbian plasticity and learning ? Rather, the

view is that instead of returning neuronal activity to a fixed level,

homeostatic plasticity acts like a Fnet_ ensuring that neuronal

networks do not reach the extremes of their activity. This is

particularly important for highly epileptogenic structures such as

the hippocampus and cortex.

The classical way to model homeostatic plasticity is to subject

neurones to a period of long-term inactivity or overactivity.

Previous studies examining homeostatic plasticity in a variety of

neuronal preparations have reported increases in neuronal excitation

entirely consistent with the network acting to correct the inactivity

by upregulating synaptic excitation. This has been measured as an

increase in the amplitude and frequency of spontaneous miniature

excitatory post-synaptic currents (mEPSCs) indicating direct

changes in the properties of the synapse, increases in glutamate

receptor expression (O’Brien et al., 1998; Turrigiano et al., 1998;

Watt et al., 2000; Bacci et al., 2001; Burrone et al., 2002; Kilman et

al., 2002; Galvan et al., 2003) and increases in synapse size,

including the pre-synaptic active zone and number of vesicles

(Murthy et al., 2001; De Gois et al., 2005).

Although homeostatic plasticity has been examined in dispersed

primary cortical and hippocampal cultures, there are fewer examples

L.E. Buckby et al. / Mol. Cell. Neurosci. 31 (2006) 805–816806

where the consequences of changes in synaptic excitation within

intact neuronal networks have been studied (Aptowicz et al., 2004;

Galvan et al., 2003; Royer and Pare, 2003). There are fewer studies

where the impact of changes in synaptic inhibition has been

examined. Intuitively, homeostatic plasticity in a neuronal network

deprived of excitatory synapse input should down-scale synaptic

inhibition, as it can be argued that it is not required. This is the case

in dispersed neocortical cultures where homeostatic plasticity

reduces the frequency of miniature inhibitory post-synaptic currents

(mIPSCs) (Kilman et al., 2002); however, this could have grave

consequences to additionally destabilise the network in the presence

of enhanced excitation.

In the present study, we therefore chose to examine the

phenomenon of homeostatic plasticity using organotypic hippo-

campal slice cultures where both synaptic excitation and inhibition

exist together to balance the output properties of the hippocampus

(Stoppini et al., 1991). To do this, we subjected developing

hippocampal slice cultures to a period of synapse inactivity by the

Fig. 1. Network excitability is increased in AP5/CNQX-treated organotypic

extracellular recordings evoked from the CA3 region of control and AP5/CN

stimulation (as stated). Recordings are the average of a minimum of three successi

control (n = 10) and AP5/CNQX-treated slice cultures (n = 12), values are means

indicating increased excitation, for a given stimulus input. (C) fEPSP duration stim

slice cultures (n = 12), values are means T SEM. The duration of the fEPSP

compared to control. #Denotes the stimulation artefact.

chronic application of NMDA and AMPA/Kainate glutamate

receptor antagonists, AP5 and CNQX. The result, on release from

the inactivity, was enhanced synaptic excitation via pre- and post-

synaptic mechanisms, but where sufficient fast synaptic inhibition

ensured the stability of this highly epileptogenic region.

Results

Evoked fEPSP excitability in CA3 is increased in

AP5/CNQX-treated organotypic hippocampal slice cultures

but there is no alteration in cell number or viability compared

with controls

Evoked fEPSPs, arising from the stimulation of mossy fibres, in

control organotypic hippocampal slice cultures and AP5/CNQX-

treated sister cultures revealed an increase in excitability in treated

slice cultures, see Fig. 1A. In response to a range of stimuli (0–40V)

hippocampal slice cultures compared with control. (A) Representative

QX-treated organotypic hippocampal slice cultures using 10 V or 40 V

ve events. (B) fEPSP peak amplitude stimulus response curves evoked from

T SEM. The curve is shifted to the left in AP5/CNQX-treated slice cultures

ulus response curves evoked from control (n = 10) and AP5/CNQX-treated

is increased at all stimulus strengths in AP5/CNQX-treated slice cultures

L.E. Buckby et al. / Mol. Cell. Neurosci. 31 (2006) 805–816 807

applied in CA3c/d, stimulus–response curves for fEPSP peak

amplitudes (Fig. 1B) and duration (Fig. 1C) from AP5/CNQX-

treated organotypic hippocampal slice cultures were shifted to the

left of those from control slice cultures, indicating an increase in the

excitability of the CA3 network, although as seen in Fig. 1B, a

graded response could still be observed from the more excitable,

treated slice cultures. The fEPSP peak amplitude stimulus–response

curve of AP5/CNQX-treated slice cultures (Fig. 1B) had a steeper

gradient between 0 and 10 V, 8.2 mV/V compared to 3.8 mV/V for

control slice cultures; a higher normalised fEPSP peak amplitude at

10 V, 81.5 T 3.9% of maximum (n = 14) compared to 38.1 T 4.8% for

control slice cultures (P < 0.001, t test, n = 10, Fig. 1B) and a lower

50% stimulation value (the stimulus strength required to evoke the

half-maximal fEPSP peak amplitude) 5.3 V, a third of the

stimulation required in control slice cultures, 15 V. The mean

duration of the evoked fEPSPs in AP5/CNQX-treated slice cultures

was also increased at all stimulus strengths (P < 0.001, t test, n =

10). As seen in Figs. 1A and C, the duration of fEPSPs evoked in

AP5/CNQX-treated slice cultures was increased compared with

control responses, from 27 T 4 ms and 42 T 10 ms (n = 10), at 10 V

and 40 V, respectively, in control slice cultures, to 178 T 31 ms and

178 T 21 ms (n = 14), respectively, in AP5/CNQX-treated slice

cultures. We also examined the fEPSPs in control slice cultures for

their mossy fibre component using the metabotropic glutamate

receptor type II agonist DCGIV (5 AM). Application of DCGIV

reduced the peak amplitude of evoked fEPSPs by 66.9 T 4.1% (P <

0.0001, n = 16, t test), indicating that the evoked CA3 fEPSPs were

approximately 65% mossy fibre-derived.

Despite the clear increase in the amplitude and duration of the

evoked fEPSP responses in the AP5/CNQX-treated slice cultures,

we never observed any spontaneous epileptiform activity. All

indications from these electrophysiology experiments indicated

that despite chronic incubation with glutamate receptor antago-

nists, the responses from the AP5/CNQX-treated slice cultures

were excitable but stable. Further parallel experiments showed

that there was no significant loss or gain of neurones or glia, or

any changes in slice culture thickness in the AP5/CNQX-treated

slice cultures compared with control. Estimations of the area of

living (calcein-stained) regions in both CA3 and CA1 revealed

no significant differences between control and AP5/CNQX-

treated slice cultures at 2, 4 and 6 DIV (P = 0.23, ANOVA, n = 4

slice cultures for each time point and condition), consistent with

no detectable changes in slice culture thickness (P = 0.1, ANOVA,

n = 3 slice cultures for each time point and condition). Neither

was there any change in the intensity or area of dead (propidium

iodide-stained) neurones at all ages (P = 0.99, P = 0.85,

respectively, ANOVA, n = 4 slice cultures for each time point

and condition) in slice cultures from AP5/CNQX-treated and

control conditions, nor in the expression of the glial cell marker

protein GFAP within the CA3 cell layer of control and AP5/

CNQX-treated slice cultures (P = 0.37, t test, n = 13). All these

results confirmed that the observed network excitability in the

AP5/CNQX-treated slice cultures was unlikely to be a result of

the loss or gain of neurones or glial cells. We also tested the

reversibility of the alterations in excitability within our chronic

AP5 and CNQX model. If we treated slice cultures for only 1–2

days with AP5 and CNQX (instead of 7 days), we observed a

similar and also reversible enhancement to the excitability, as

described above. In contrast, after 7 days treatment with AP5 and

CNQX, the alterations to the excitability were not easily

reversed.

The amplitude and frequency of spontaneous EPSPs were

increased in CA3 pyramidal neurones from AP5/CNQX-treated

organotypic hippocampal slice cultures compared with controls

Whole cell recordings of spontaneous excitatory post-synaptic

potentials (spEPSPs) from pyramidal cells held at hyperpolarised

levels (Fig. 2A, to similar levels across control and AP5/CNQX-

treated cells) revealed a large increase in the amplitude and

frequency of the spEPSPs from AP5/CNQX-treated cells (n = 7)

compared with control (n = 9), across all spEPSPs within the

recorded population, as shown in Fig. 2B (P < 0.0001, ANOVA,

n = 16). We also detected an alteration in the mean frequency of

spEPSPs, as shown in Fig. 2C, where mean values changed from

3.2 T 1.0 spEPSP per second in cells from control slice cultures to

11.8 T 1.9 spEPSPs per second in cells from AP5/CNQX-treated

slice cultures (P < 0.001, t test, n = 16). None of the cells from

either the control slice cultures or the AP5/CNQX-treated slice

cultures demonstrated epileptiform behaviour in the form of

spontaneous bursts of action potentials or synchronous and

spontaneous depolarising shifts, even when depolarised. However,

we did observe a slightly depolarised average resting membrane

potential in the cells from the AP5/CNQX-treated slice cultures.

The mean resting potential in cells from control slice cultures was

�66 T 1 mV compared with�62 T 1 mV in cells from AP5/CNQX-

treated slice cultures (P < 0.05, t test, n = 16). Added to this, the

cells from the AP5/CNQX-treated slice cultures also demonstrated

a slightly enhanced input resistance; mean values were 305 T 24

MV in control cells compared with 368 T 19 MV in cells from AP5/

CNQX-treated slice cultures (P < 0.05, t test, n = 16).

Our electrophysiological recordings revealed excitable but

stable cells and slice cultures following chronic AP5/CNQX

treatment. In order to further explain our results thus far, we

conducted parallel Western blotting experiments to identify any

changes in the expression of excitatory synaptic proteins at both

the pre- and post-synaptic level.

Enhanced expression of the excitatory post-synaptic receptors

GluR1 and NR1 in AP5/CNQX-treated organotypic hippocampal

slice cultures compared with controls in the absence of any change

in expression of post-synaptic protein markers PSD95 and SAP102

As shown in Fig. 3A, we identified an approximately 50%

increase in the expression of the AMPA and NMDA receptor

subunits GluR1 and NR1 in the AP5/CNQX-treated slice cultures.

Mean NR1 expression increased to 136.4 T 13.1% of the control

value (P < 0.05, t test, n = 3) and mean GluR1 expression increased

to 140 T 14.4% of the control value (P < 0.05, t test, n = 3).

Western blot analysis of the post-synaptic proteins SAP102 and

PSD95, however, showed no alteration in their expression in slice

cultures from control or AP5/CNQX-treated slice cultures, as

shown in Fig. 3B. Mean values of PSD95 and SAP102 expression

from control and AP5/CNQX-treated slice cultures remained

similar at 95 T 12% and 94 T 11% of control values, respectively

(P = 0.47, t test, n = 8 and P = 0.57, t test, n = 4, respectively).

Excitatory synapse pre-synaptic marker protein expression was

also enhanced in AP5/CNQX-treated organotypic hippocampal

slice cultures compared with controls

Homeostatic scaling of synaptic strength in a variety of

models involves pre- and/or post-synaptic changes at excitatory

Fig. 2. The amplitude and frequency of spontaneous EPSPs is increased in pyramidal neurones from AP5/CNQX-treated organotypic hippocampal slice

cultures. Panel A shows representative traces from a whole cell recording from a control neurone (upper trace) and a neurone from an AP5/CNQX-treated slice

culture (lower trace) both held slight hyperpolarised from their resting membrane potential by the injection of a small amount of hyperpolarising current.

Spontaneous EPSPs (spEPSPs) are seen as small depolarising deflections (that were sensitive to the AMPA/KA receptor antagonist CNQX and the NMDA

receptor antagonist, AP5, data not shown) and are less frequent and smaller in the cell from the control slice culture. In panel B, the distribution of the number

and amplitude of spEPSPs collected over similar time epochs from several control (white bars, n = 9) and AP5/CNQX-treated (black bars, n = 7) cells shows an

enhancement of the number of both small and large events in the AP5/CNQX-treated cells. This is confirmed in panel C where the average number of EPSPs

detected per second during the same epochs used to generate the data in panel B is increased in AP5/CNQX-treated cells (black bars), compared with the

controls (white bars). **Denotes significance at P < 0.001.

L.E. Buckby et al. / Mol. Cell. Neurosci. 31 (2006) 805–816808

synapses. Although our Western blot experiments identified an

enhancement in post-synaptic glutamate receptor expression,

the enhanced excitability seen with our electrophysiological

recordings could also be a result of enhanced neurotransmitter

release. We therefore chose to analyse the levels of a protein

expressed exclusively at excitatory synapse terminals, the

vesicular glutamate transporter protein VGLUT1 (Takamori et

al., 2000) and synapsin1, a vesicular protein present in all

pre-synapses. As shown in Fig. 4A, we detected an elevation

of VGLUT1 in AP5/CNQX-treated slice cultures compared

with control, where the mean expression increased to 133 T7.6% of the control value (P = 0.01, t test, n = 5). We could

also detect a similar level of enhancement of VGLUT1

expression in slice cultures (n = 4) treated with AP5/CNQX

for 48 h. To compliment this finding, we also detected an

increase in the number of VGLUT1 expressing puncta in

stratum radiatum of CA3 in AP5/CNQX-treated slice cultures,

as shown in Fig. 4B. The mean number of VGLUT1-labelled

puncta increased from 621 T 12.1 in control slice cultures to

673 T 16.2 (P < 0.05, t test, n = 5). Consistent with the

upregulation of VGLUT1 puncta, we also identified an

increase in the number of synapsin1 expressing puncta in

the CA3 cell body layer of AP5/CNQX-treated slice cultures.

Synapsin1 labelling appeared as larger puncta than seen with

VGLUT1 staining and although we detected fewer particles in

each field of view, the detected changes were greater. Mean

values for the number of synapsin1 puncta detected increased

from 141 T 8 in the control slice cultures to 273 T 12

particles in AP5/CNQX-treated slice cultures (P < 0.0001, t

test, n = 12). We also noted a significant change in the size

distribution of synapsin1-labelled particles in AP5/CNQX-

treated slice cultures where we detected more, larger synap-

Fig. 3. AP5/CNQX treatment of organotypic hippocampal slice cultures increases the expression of post-synaptic NMDA and AMPA glutamate receptor

subunits without changing the expression of post-synaptic density marker proteins. Panels A and B show Western blot-based comparison of the expression of

post-synaptic marker proteins in AP5/CNQX-treated and control organotypic hippocampal slice cultures. Slice cultures were probed with primary antibodies

raised against NR1, an NMDA receptor subunit (¨120 kDa), n = 4, GluR1 an AMPA receptor subunit (¨90 kDa), n = 3, in panel A and to SAP102 (102

kDa), n = 4, and PSD95 (95 kDa), n = 4, shown in panel B. All lanes were equally loaded with 15 Ag protein and the position of the standard protein markers

is shown, 116 and 66 kDa. Values representing protein expression for the AP5/CNQX-treated slice cultures (black bars) are obtained from the optical density

of the band compared as a percentage to the 100% control level (white bars) on the same blot. Bars represent mean expression T SEM for the different

proteins. *Denotes significance at P < 0.05. Representative examples of the protein expression from the same Western blots are shown below each bar graph.

L.E. Buckby et al. / Mol. Cell. Neurosci. 31 (2006) 805–816 809

sin1-stained puncta, compared with control, data not shown (P <

0.05, ANOVA, n = 12).

The frequency of spontaneous IPSPs was increased in CA3

pyramidal neurones from AP5/CNQX-treated organotypic

hippocampal slice cultures compared with controls

Simultaneous with the analysis of spEPSPs, we also recorded

spontaneous synaptic events in cells that were slightly depolarised

so as to better reveal them as negative-going events. We were

careful to ensure that the level of depolarisation in both control and

AP5/CNQX-treated cells was similar. These events represent

spontaneous inhibitory post-synaptic potentials (spIPSPs) driven

by the action of GABA at GABA-A receptors, as evidenced by

their sensitivity to bicuculline (data not shown). We noted that

although cells from control slice cultures showed robust inhibition,

as expected, shown in Fig. 5A, in cells from AP5/CNQX-treated

slice cultures, there was an increase in the mean frequency of

spIPSPs, Fig. 5C. Mean values changed from 2.7 T 0.6 spIPSPs persecond to 5.2 T 0.8 spIPSPs per second (P < 0.05, t test, n = 5) in

control versus AP5/CNQX-treated cells. However, there was no

significant change in the amplitude distribution of the spIPSPs

recorded from control and AP5/CNQX-treated cells, Fig. 5B (P =

0.98, ANOVA, n = 5), although more, larger spIPSP events can be

Fig. 4. AP5/CNQX treatment of organotypic hippocampal slice cultures increases the expression of the pre-synaptic vesicular glutamate transporter protein

VGLUT1. Panel A shows a Western blot-based comparison of the expression of VGLUT1 in AP5/CNQX-treated and control organotypic hippocampal

slice cultures. All lanes were equally loaded with 15 Ag protein and the position of the standard protein markers is shown, 66 and 45 kDa. Values

representing VGLUT1 expression for the AP5/CNQX-treated slice cultures (black bars) are obtained from the optical density of the band compared as a

percentage of the 100% control level (white bars) on the same blot. In panel A, bars represent mean expression T SEM for VGLUT1 under the two

different conditions. *Denotes significance at P < 0.05. A representative example of VGLUT1 expression from the same Western blot is shown below the

bar graph. In panel B, fluorescent immunohistochemical detection of VGLUT1 shows small punctate particles in stratum radiatum of hippocamal CA3 in

images obtained from control (left panel) and AP5/CNQX-treated slice cultures (right panel). Using thresholding techniques, we quantified the number of

VGLUT1-stained particles in each field of view (as described in Experimental methods) and found the number of VGLUT1 particles to be significantly

enhanced in AP5/CNQX-treated slice cultures (black bars) compared with control (white bars). *Denotes significance at P < 0.05.

L.E. Buckby et al. / Mol. Cell. Neurosci. 31 (2006) 805–816810

seen. Under the stimulation and recording conditions used here,

stimulus-evoked responses were always positive-going EPSPs,

with the evoked IPSPs masked by strong excitation in both control

and AP5/CNQX-treated slice cultures. For this reason, evoked

EPSPs and IPSPs were not analysed further.

Pharmacological removal of GABA-A-ergic network inhibition

revealed enhanced network excitability in AP5/CNQX-treated slice

cultures

The importance of GABA-A receptor-mediated inhibition for

the stability of the CA3 network under both control and AP5/

CNQX conditions was revealed by the application of the GABA-A

receptor antagonists bicuculline (10 AM, n = 4) and picrotoxin (10

AM, n = 2). Under these conditions, evoked fEPSPs were enhanced

in amplitude and duration and demonstrated an enhanced number of

afterdischarges, see Fig. 5D, downward pointing triangles. In the

AP5/CNQX-treated slice cultures, the number of afterdischarges

was significantly enhanced compared with control slice cultures.

Mean values for the number of afterdischarges evoked by similar

stimulus intensities (1–2 V) under control conditions (picrotoxin or

bicuculline alone) were 3.4 T 1.2 compared with 10 T 2.5 from AP5/

CNQX-treated slice cultures (P < 0.05, t test, n = 6). However, we

did not observe any difference in the frequency of the spontaneous

epileptiform events between control and AP5/CNQX-treated slice

cultures. Mean values were unchanged from 5.2 T 0.8 events per

minute in control slice cultures to 4.8 T 0.6 events per minute in

AP5/CNQX-treated slice cultures (P > 0.05, t test, n = 3).

These results indicated clearly enhanced excitability within the

CA3 network of the AP5/CNQX-treated slice cultures once fast

GABA-A receptor-mediated synaptic inhibition was removed.

Expression of GABA-ergic synaptic marker proteins in

AP5/CNQX-treated organotypic hippocampal slice cultures

In order to confirm the presence of fast synaptic inhibition in

AP5/CNQX-treated slice cultures, we also analysed the expression

of proteins exclusively expressed at GABA-ergic synapses. As

shown in Fig. 6A, there was little change in the presumably post-

synaptic expression levels of the alpha 1 subunit of the GABA-A

receptor or in the pre-synaptic expression of the GABA synthetic

enzyme glutamic acid decarboxylase, GAD65, present in GABA-

ergic terminals, in slice cultures from control or AP5/CNQX-treated

slice cultures. Mean values for GABA-A alpha 1 receptor expression

were unchanged at 93.5 T 4.7% of the 100 T 11.7% of the control

values (P = 0.55, t test, n = 4), whilst mean values for GAD65

expression were not significantly changed at 77 T 9.5% of the 100 T14.7% control values (P = 0.24, t test, n = 7), although there was a

tendency towards a decrease as can be seen in Fig. 6A. In light of the

tendency towards a change in the expression of GAD65, we

Fig. 5. Intact synaptic inhibition in AP5/CNQX-treated CA3, seen as frequent spontaneous IPSPs in pyramidal neurones helps stabilise the excitability of

the network. Panel A shows representative traces from a whole cell recording from a control neurone (upper trace) and a neurone from an AP5/CNQX-

treated slice culture (lower trace) both held slightly depolarised from their resting membrane potential by the injection of a small amount of depolarising

current. Spontaneous IPSPs (spIPSPs) are seen as small hyperpolarising deflections (that were sensitive to bicuculline, data not shown) and are less

frequent, though of a similar size, in the cell from the control slice culture. Panel B shows the distribution of the number and amplitude of spIPSPs

collected over similar time epochs from several control (white bars) and AP5/CNQX-treated (black bars) cells where a larger number of larger events can be

seen in the AP5/CNQX-treated cells compared with control. In panel C, we show that the average number of spIPSPs detected per second during the same

epochs used to generate the data in panel B is increased in AP5/CNQX-treated cells (black bars), compared with the controls (white bars). In panel D, the

effects of abolishing fast GABA-A receptor-mediated events are shown by the population fEPSPs recorded from the cell body layer of CA3 in response to

minimal (1–2 V) electrical stimulation to the mossy fibres. In the example from a control slice culture, when fast synaptic GABA-A receptor-mediated

inhibition is abolished, the fEPSP is lengthened and displays 4 clear afterdischarges, as denoted by small downward triangles. In contrast, in the example

from an AP5/CNQX-treated slice culture, the number of afterdischarges is increased to 10, lower trace, indicating the enhanced recurrent excitation within

these slice cultures.

L.E. Buckby et al. / Mol. Cell. Neurosci. 31 (2006) 805–816 811

conducted immunohistochemistry but could not detect any alter-

ations in the number or distribution of GAD65-stained puncta, in the

CA3 cell body layer, between control and AP5/CNQX-treated slice

cultures. Mean values were 263 T 21 in control slice cultures

compared with 248 T 27 in AP5/CNQX-treated slice cultures (P =

0.67, t test, n = 6). Nor could we detect any change in the amplitude

distribution of GAD65 positive puncta (P = 0.95, ANOVA, n = 6). In

contrast, immunohistochemistry for parvalbumin (PV), a classical

marker protein of a subtype of inhibitory terminals (Freund and

Buzsaki, 1996), showed an enhancement of the number of PV

positive puncta, see Fig. 6B. The mean number of PV puncta in a

field of view increased from 94 T 9 in control slice cultures to 136 T14 in AP5/CNQX-treated slice cultures (P < 0.05, t test, n = 9).

Discussion

The present study set out to examine the consequences of

homeostatic plasticity for both synaptic excitation and inhibition

following a period of chronic inactivity within hippocampal

CA3.

Our key findings are that inactivity scales up excitatory synapse

strength and number at both pre- and post-synaptic sites. In

addition, the strength of fast GABA-A receptor-mediated synaptic

inhibition stabilised the excitable, hippocampal CA3 network. Our

findings highlight the intrinsic ability of this network to rebalance

excitation and inhibition during alterations in excitatory synapse

strength.

Fig. 6. Changes in pre- and post-synaptic markers for GABA-ergic inhibitory synapses in AP5/CNQX-treated organotypic hippocampal slice cultures

compared with control. AWestern blot-based comparison of the expression of two markers for GABA-ergic inhibitory synapses shows little difference in their

expression in AP5/CNQX-treated and control organotypic hippocampal slice cultures. Slice cultures were probed with primary antibodies raised against the

GABA-A receptor alpha 1 subunit and to GAD65. All lanes were equally loaded with 15 Ag protein and the position of the standard protein markers is shown,

66 and 45 kDa. Values representing protein expression for the AP5/CNQX-treated slice cultures (black bars) are compared to the 100% control level (white

bars) on the same blot. Bars represent mean expression T SEM for the different proteins. Representative examples of the protein expression from the same

Western blots are shown below each graph. Panel B shows fluorescent immunohistochemical detection of parvalbumin (PV)-positive terminals within the cell

body layer of CA3 in control (left panel) and AP5/CNQX-treated slice cultures (right panel). Using thresholding techniques, we quantified the number of PV-

stained particles in each field of view (as described in Experimental methods) and found the number of PV particles to be significantly enhanced in AP5/

CNQX-treated slice cultures (black bars) compared with control (white bars). *Denotes significance at P < 0.05.

L.E. Buckby et al. / Mol. Cell. Neurosci. 31 (2006) 805–816812

Chronic inactivity during AP5 and CNQX application enhances

CA3 excitability

Long-term treatment with AP5 and CNQX during develop-

ment of organotypic hippocampal slice cultures led to a scaling

or adjustment of CA3 excitability towards a hyperexcitable state

and is predicted from earlier work defining homeostatic plasticity

(Turrigiano and Nelson, 2004). fEPSPs recorded from CA3 of

the AP5/CNQX-treated slice cultures in response to mossy fibre

stimulation were longer in duration and revealed enhanced input

output properties compared with control, likewise our whole cell

recordings revealed enhanced amplitude and frequency of

spontaneous EPSPs in AP5/CNQX-treated cells. However, at

no time did the CA3 network in the excitable AP5/CNQX-

treated slice cultures generate synchronised spontaneous epilep-

tiform behaviour. This enhanced excitability of the CA3 region,

upon release from the period of chronic inactivity, is consistent

with a number of previous reports examining homeostatic

plasticity in a variety of preparations including dispersed cultures

of neocortex, visual cortex, spinal cord and hippocampus and

more recently in vivo and in hippocampal slice cultures

(Furshpan and Potter, 1989; Ramakers et al., 1990; van den

Pol et al., 1996; Turrigiano et al., 1998; Desai et al., 1999;

Burrone et al., 2002; Kilman et al., 2002; Galvan et al., 2003;

Aptowicz et al., 2004). Moreover, our findings indicated that

despite the chronic nature of the inactivity in this model, there

was no sign of damage or changes to the numbers of neuronal

and glial cells at any stage. However, we cannot exclude that

glia may have an enhanced role in this model to prevent

excessive glutamate spillover.

L.E. Buckby et al. / Mol. Cell. Neurosci. 31 (2006) 805–816 813

Increased expression of excitatory pre- and post-synaptic proteins

underlies the enhanced excitability in CA3

Consistent with our electrophysiological recordings, we

detected enhancements specifically to excitatory synaptic trans-

mission at both post-synaptic and pre-synaptic sites within the

AP5/CNQX-treated slice cultures.

Firstly, as also shown by others (Watt et al., 2000; Galvan et al.,

2003), we observed an increased expression of GluR1 and NR1

receptor subunits in the AP5/CNQX-treated slice cultures. This

suggests that homeostatic plasticity requires stable parallel

increases in both GluR1 and NR1 expression at the post-synaptic

site and is consistent with the results shown by others (O’Brien et

al., 1998; Watt et al., 2000; Galvan et al., 2003), bar one (Rao and

Craig, 1997). More recently, enhanced expressions of Ca2+

permeable GluR1 homomers (Thiagarajan et al., 2005), and GluR2

subunits (Wierenga et al., 2005), as well as NR1, and also

selectively the NR2B subunit (Galvan et al., 2003; Slutsky et al.,

2004) have been suggested as candidates to drive homeostatic

plasticity. Despite the upregulation of glutamate receptor expres-

sion in this model, we did not detect any changes in the expression

of two post-synaptic density (PSD) proteins, PSD95 and SAP102,

in the AP5/CNQX-treated slice cultures (Okabe et al., 1999; Qin et

al., 2001). This may suggest that the PSD remains the same size

during synapse scaling but contains a higher density of glutamate

receptors. Alternatively, our results do not exclude the possibility

of insertion of new extrasynaptic glutamate receptors (Kullmann

and Asztely, 1998).

Secondly, we detected an increase in the expression of two pre-

synaptic marker proteins. Synapsin1 expressing puncta were

increased both in number and size in AP5/CNQX-treated slice

cultures and this result points to an increased number of vesicles

and a larger readily releasable pool (Murthy et al., 2001;

Thiagarajan et al., 2005) as well as the appearance of new release

sites containing any transmitter, whether excitatory or inhibitory.

Consistent with our findings in the hippocampus, earlier work also

detected increased synaptophysin expression and distribution in

hippocampal neurones both in vivo (Galvan et al., 2003) and in

dispersed hippocampal cultures (Murthy et al., 2001) whereas in

the neocortex synapsin1 staining is unchanged (Turrigiano et al.,

1998; Kilman et al., 2002). We therefore chose to refine our

approach by analysing the expression and distribution of

VGLUT1, one of a family of glutamate transporter proteins

expressed exclusively on glutamate-containing vesicles within

excitatory synaptic terminals (Takamori et al., 2000). Here, in

AP5/CNQX-treated slice cultures, VGLUT1 levels were enhanced

compared with control, a finding also confirmed by a significant

increase in the number of VGLUT1-stained particles specifically in

stratum radiatum of CA3 where VGLUT1 dominates (Wojcik et

al., 2004). Recently, overexpression of VGLUT1 has revealed an

enhanced amplitude of hippocampal mEPSCs with good evidence

for enhanced glutamate release per vesicle (Wilson et al., 2005). In

light of our findings here in hippocampal CA3, the increased

VGLUT1 expression and distribution are consistent with our

observed increased amplitude and number of spEPSPs and mossy

fibre-stimulated excitability. VGLUT1 expression has also recently

been shown to change in line with bidirectional scaling of

excitatory synapses in both hippocampus and neocortex (Wilson

et al., 2005; De Gois et al., 2005) suggesting it has a pivotal role

for adapting synapse strength at the pre-synapse. Interestingly in

this model, the elevation in VGLUT1 expression (approximately

30% increase) was far less than the increase in synapsin 1

expression (approximately doubled). It is possible that since

synapsin 1 can be detected at all pre-synapses, whereas VGLUT1

detects only a subset of excitatory pre-synapses, other subsets of

excitatory pre-synapses are also enhanced.

Fast GABA-A receptor-mediated synaptic inhibition persists in the

presence of enhanced excitability and stabilises the CA3 network

Given our accumulated evidence for significantly enhanced

excitatory drive in the AP5/CNQX-treated slice cultures, we were

surprised that the highly interconnected CA3 hippocampal region

did not express synchronised epileptiform activity. In light of the

importance of the balance between excitation and inhibition for the

prevention of epileptogenesis in CA3 (Miles and Wong, 1987), we

looked more closely at fast GABA-A receptor-mediated synaptic

inhibition in AP5/CNQX-treated slice cultures. Intuitively, in a

neuronal network deprived of excitatory synaptic connectivity, a

decrease in synaptic inhibition would be predicted, since synaptic

inhibition is less likely to be required.

However, in this model of homeostatic plasticity in the

hippocampus, we observed more frequent spontaneous IPSPs in

AP5/CNQX-treated cells, with also a greater incidence of larger

events, supporting the idea that fast synaptic inhibition was

certainly intact. To help confirm this, we determined the expression

and distribution of three markers of inhibitory synapses within this

model. The presumed post-synaptic expression of the GABA-A

receptor alpha 1 subunit and the pre-synaptic expression of the

GABA synthetic enzyme GAD65, present in interneurone termi-

nals, were both unchanged in our model. However, we did detect

an increase in the number of parvalbumin-stained presumed pre-

synaptic inhibitory interneurone terminals, suggesting an enhanced

number of pre-synaptic GABA-ergic release sites, at least of this

type of inhibitory interneurone. Currently, we cannot exclude the

contribution from other types of inhibitory interneurone and their

potential for diversity under activity-deprived conditions. Never-

theless, all the changes we detected are consistent with the

observed enhanced frequency of spIPSPs in the absence of any

changes in their amplitude. Also consistent with enhanced spIPSPs

would be an enhanced excitation of inhibitory interneurones,

although we have no direct evidence for this, bar the enhanced

number of pre-synaptic excitatory terminals. In terms of the CA3

network, the synaptic GABA-A receptor-mediated inhibition was

clearly important since when we removed it the full force of the

enhanced excitatory synaptic strength and recurrent excitation

within CA3 led to enhanced epileptogenesis in AP5/CNQX-treated

slice cultures.

Our combined evidence shows an increased number of GABA

release sites, an enhanced frequency of spIPSPs and likely an

enhanced excitation of inhibition, in this model of homeostatic

plasticity, all of which will strengthen recurrent and feed forward

synaptic inhibition. Previous findings in other models, where

synaptic inhibition has been analysed following synapse inactivity,

have often produced conflicting and mixed results. In the

neocortex, clear reductions in GABA-A receptor (but not

GAD65) (Kilman et al., 2002) and vesicular GABA transporter

VIATT expression (De Gois et al., 2005) occur during homeostatic

plasticity, consistent with the reduced mIPSC frequency (Kilman et

al., 2002), although in the latter study, the mean GABA-A receptor

single channel conductance was unchanged. Similarly, GAD65

levels are unchanged in deprived ocular dominance columns in

L.E. Buckby et al. / Mol. Cell. Neurosci. 31 (2006) 805–816814

visual cortex (Silver and Stryker, 2000). More recently, in two

further studies in the hippocampus, estimates of synaptic inhibition

reveal little change. In one study where dispersed cultures were

chronically treated with the ionotropic glutamate receptor antag-

onist kynurenate (Ivanova et al., 2003), evoked IPSCs and IPSC

reversal potentials were unchanged, although activity-dependent

depression of inhibition was enhanced. Likewise, in dispersed

cultured hippocampal neurones following tetanus toxin activity

blockade, GAD65 and GABA-A alpha 2 receptor expression was

unchanged (Harms and Craig, 2005). Some of the diversity in the

literature may arise from the direct and potentially confounding

action of CNQX to enhance synaptic inhibition (Brickley et al.,

2001), although more recent findings using TTX to block synaptic

transmission for only 48 h, show, as we show here, that synaptic

inhibition is also upregulated (Echegoyen et al., 2005).

Homeostatic plasticity uses a variety of mechanisms, both

synaptic and intrinsic (Turrigiano and Nelson, 2000), to keep the

safety net intact for neuronal networks, especially under patholog-

ical conditions. In this study, we have highlighted the importance

of robust synaptic inhibition during homeostatic plasticity. Al-

though chronic inactivity clearly scaled up the excitatory synapses

in CA3, it did not scale down the inhibitory synapses; instead, the

strength of the synaptic inhibition remained and effectively

stabilised the network to prevent epileptogenesis.

Experimental methods

Organotypic slice culture preparation

Organotypic hippocampal slice cultures were prepared and grown with

a slight modification to the interface culture method (Stoppini et al., 1991;

Buckby et al., 2004). All experiments were carried out in accordance with

the UK (Scientific Procedures) Act 1986. Horizontal, 250 Am thick,

limbic brain slices were taken from P6-8 Wistar rats and sectioned in

preparation media (composition; 20 AM HEPES buffer in Minimal

Essential Media at 4–10-C) using a Vibroslice (Campden Instruments,

Loughborough, UK). Slices were transferred to membrane inserts

(Millicell-CM, Millipore), kept at 36-C in 5% CO2 and medium

exchanged daily with either control media or media containing 100 AMAP5 and 20 AM CNQX for a period of 7 days in vitro (DIV). At this

point, slice cultures were either snap frozen for SDS-PAGE and Western

blotting, fixed in 4% formaldehyde for immunohistochemistry or used for

electrophysiological recordings.

SDS-PAGE and Western blotting

Samples were prepared for SDS PAGE and Western blotting as

previously described (Buckby et al., 2004). Proteins were probed with

primary antibodies raised against VGLUT1 (1:500 Synaptic Systems),

PSD95 (1:1000 Cambridge Bioscience), SAP102 (1:500 Abcam), GAD,

glutamic acid decarboxylase, isoform 65 (GAD; 1:500 Chemicon), alpha 1

subunit of GABA-A receptor (1:2000) GluR1 (1:1000 Santa Cruz) and

NR1 (1:2000, Pharmingen). Secondary antibodies were conjugated to

horseradish peroxidase (Dako Ltd.) and visualised by the enhanced

chemiluminesence (ECL) method (Pierce). The film was scanned and the

grey levels of the different bands of interest were measured at

approximately 50% exposure using an image analysis program, Simple

PCI (Compix Inc., USA). In order to quantify and compare the density of

different bands, the grey levels were background subtracted and normalised

as a percentage of the control band grey level for each lane on the Western

blot. Blot grey levels were analysed blind as we routinely randomise the

order of gel loading. Whilst protein levels were always controlled prior to

loading, at 15 Ag per lane, additional post hoc analysis of Ponceau stains of

transfers and Coomassie-stained loading gels revealed little changes in

protein loading between lanes. Mean values T standard error of the mean

are stated for n samples, P values are quoted for Student’s t test and

ANOVA comparisons using Prism (Graphpad 3.03, USA).

Electrophysiology

Organotypic hippocampal slice cultures were transferred to a recording

chamber, where they were superfused at a rate of 2 ml/min with artificial

cerebrospinal fluid (aCSF), of composition (mM) NaCl 126, KCl 2.5,

NaH2PO4 1.2, MgCl2 1.3, CaCl2 2.4, NaHCO3 26, Glucose 10, saturated

with 95% 02/5% CO2 at 29–31-C. Extracellular DC field recordings were

made from the CA3 region of the hippocampus using 2–5 MV glass

pipettes filled with aCSF. The signal was amplified and filtered using a

Neurolog amplifier (Digitimer Systems, Herts UK) then filtered further

using a Humbug (Digitimer Systems, Herts, UK). For stimulation, a

monopolar stimulating electrode made from AgCl wire (outside diameter

0.125 mm; Goodfellow, UK) was placed beneath the granular cell layer of

the dentate gyrus and the axons of the granule cells and the mossy fibres

stimulated half-maximally using a constant voltage-isolated stimulator

(model DS2A-mkII, Digitimer Ltd., UK). The stimulating and recording

electrodes were positioned in the organotypic slice cultures under visual

control using the 10� objective of an upright light microscope (Eclipse

model E600FN, Nikon, Japan).

Whole cell recordings from CA3 pyramidal cells used patch electrodes

of resistance 6–10 MV filled with an internal solution of the following

composition in mM (NaCl, 5, KCl 10, K-gluconate 125, Mg-ATP 4, Na-

GTP 0.3, HEPES, 10, pH adjusted to 7.2 with KOH). Bridge mode

recordings obtained with an Axoclamp 2A were amplified (�20) and

filtered (dc/5k) using a Neurolog amplifier (Digitimer Systems, Herts UK)

then filtered further using a Humbug (Digitimer Systems, Herts, UK). Cells

with resting membrane potentials more negative than �55 mV and

exhibiting overshooting action potentials were accepted for recording.

Average resting membrane potential (junction potential corrected) and input

resistance (measured during short 30 ms duration 0.05 nA square pulses)

were recorded following a stabilisation period of at least 10 min following

seal breakthrough. Spontaneous synaptic events were then collected over a

period of 3–10 min. In order to record spontaneous EPSPs, the cell

membrane was held 5–8 mV negative to the resting membrane potential by

the injection of constant negative current whilst during the recording of

spontaneous IPSPs, the cell membrane was held between �52 and �55 mV

by injection of constant positive current.

Data acquisition, analysis and stimulus triggering were accomplished

using Signal software (Cambridge Electronic Design Ltd., Cambridge, UK)

on a PC connected to a 1401 plus (Cambridge Electronic Design Ltd.,

Cambridge, UK). The peak amplitude and duration of the field excitatory

post-synaptic potential (fEPSP) component of the waveform were measured

from the average of three successive events for input/output analysis and

from an average of 10 successive events after a 5 min drug wash-on period

for drug application. Spontaneous EPSPs and IPSPs recorded from

randomly selected 90–150 s of data during a recording period of 3–10

min were analysed off line using Mini Analysis software (Synaptosoft,

Decatur, USA). Data were expressed as mean T standard error of mean for n

samples and P values quoted for Student’s t test and ANOVA comparisons

using Prism.

All electrophysiological experiments occurred in aCSF (unless stated)

thereby relieving AP5/CNQX-treated organotypic hippocampal slice

cultures of their NMDA and AMPA receptor blockade. When used, drugs

were applied in known concentrations into the superfusate and were 6-

cyano-7-nitroquinoxaline-2,3-dione (CNQX), d,l-2-amino-5-phosphono-

pentanoic acid (AP5) and (2S,2VR,3VR)-2-(2V,3V-Dicarboxycyclopropyl)glycine (DCGIV; all Tocris, UK).

Immunohistochemistry

Control organotypic hippocampal slice cultures and AP5/CNQX-treated

sister cultures were fixed in 4% formaldehyde in phosphate-buffered saline

L.E. Buckby et al. / Mol. Cell. Neurosci. 31 (2006) 805–816 815

(PBS) pH 7.4 for a minimum of 12 h and then rehydrated by washing in

PBS pH 7.4 of composition (mM) NaCl 137, KCl 2.7, KH2PO4 1.5,

Na2HPO4 8.1. For VGLUT1, GAD65, GABA-A receptor alpha 1 subunit

and glial fibrillary acidic protein (GFAP) staining, slice cultures were

incubated with 1% goat serum, 0.5% BSA (Sigma, UK) and 0.5% Triton-X

(Sigma) in PBS pH 7.4 for 4 h at 37-C to permeabilise slice cultures and

block non-specific binding. Following this, slice cultures were incubated

overnight at room temperature with the VGLUT1 (1:500), Parvalbumin

(1:250, Chemicon), Synapsin 1 (1:200), GAD65 (1:100), GABA-A

receptor alpha 1 subunit (1:100) and GFAP (1:100) antibodies diluted in

1% goat serum, 0.5% BSA in PBS pH 7.4. After this, slice cultures were

washed in PBS pH 7.4 and then incubated for 4 h in the dark with 1% goat

serum, 0.5% BSA in PBS pH 7.4 containing an Alexa-488 conjugate

(1:500; Molecular Probes). All slice cultures were then washed in PBS pH

7.4 and mounted using a 50% glycerol (Sigma) solution in PBS pH 7.4.

Confocal fluorescence microscopy and quantification

A laser scanning confocal microscope (Bio-rad Radiance 2100, Zeiss,

UK) attached to an upright light microscope (Eclipse model E600FN,

Nikon, Japan) fitted with a 60� oil immersion objective (Nikon, Japan) was

used to image VGLUT1, Parvalbumin, Synapsin 1, GAD65 and GFAP

distribution in control and AP5/CNQX-treated organotypic hippocampal

slice cultures. A thin section was obtained by using an optimised confocal

aperture with minimal laser power and gain from the cell body layer of the

CA3b/c region. Once these two parameters were optimised for a given

fluorophore, they remained the same across all analysed samples, and all

samples from different slice cultures within the data set were collected on

the same day. We used a 488-nm excitation line of an Argon laser to excite

the Alexa 488 fluorophore and fluorescence emission was collected at 515 T

30 nm by filtering with an HQ515/30 emission filter. The confocal

microscope was controlled by Lasersharp software (Zeiss, UK) and images

collected from 3 fields of view within each slice culture. These were then

analysed offline using Simple PCI where identical threshold values were

applied across a data set. For a given data set, identical threshold settings

were used although these varied across the different antibody and

fluorophore combinations used. Immunocytochemistry particulate analysis

was performed blind as we routinely randomise our slides. Values were

analysed using Prism to generate mean values T standard error of the mean

for n samples and P values quoted for Student’s t test and ANOVA

comparisons with Prism.

Semi-quantitative assay for neuronal viability and slice culture thickness

using confocal microscopy

Using a slight modification to the method of Monette et al., 1998), we

used propidium iodide (PI; Sigma) and calcein-AM (Molecular Probes) to

stain for neuronal damage (where PI stains the nucleic acid of neurones

with damaged membranes) and survival (where calcein-AM stains neurones

with intact membranes). Living organotypic slice cultures maintained on

the microscope stage at room temperature in a HEPES-buffered saline were

imaged following dye uptake using a 60� water immersion objective

(Nikon, Japan) on the confocal microscope as described above. By imaging

the stained slice cultures (with constant confocal settings between all slice

cultures), we collected 4 areas of interest in CA3 for each slice culture

under each condition. Images of PI and calcein-AM staining within the

hippocampal CA3 region were then analysed using a constant maximum

threshold of 10% within Simple PCI to generate an area of fluorescence in

Am2 for each dye under the different conditions. Slice cultures from control

and AP5/CNQX-treated slice cultures were examined at 2, 4 and 6 DIV

using PI and calcein-AM. The values for the area of fluorescence (Am2)

were then compared using Excel and Prism as mean values T standard error

of mean for n samples and P values quoted for ANOVA comparisons. To

gain an estimate of the thickness of each slice culture, we used the

transmitted light detector on the confocal with control of the z-axis in order

to measure each slice culture from top to bottom where the Millipore

membrane became visible.

Acknowledgments

This work was funded by the BBSRC grant number 111/

NEU15396, the Epilepsy Research Foundation, UK (to TPJ) and

Action Medical Research.

References

Aptowicz, C.O., Kunkler, P.E., Kraig, R.P., 2004. Homeostatic plasticity in

hippocampal slice cultures involves changes in voltage-gated Na+

channel expression. Brain Res. 998, 155–163.

Bacci, A., Coco, S., Pravettoni, E., Schenk, U., Armano, S., Frassoni, C.,

Verderio, C., De Camilli, P., Matteoli, M., 2001. Chronic blockade of

glutamate receptors enhances presynaptic release and downregulates the

interaction between synaptophysin–synaptobrevin–vesicle-associated

membrane protein 2. J. Neurosci. 21, 6588–6596.

Brickley, S.G., Farrant, M., Swanson, G.T., Cull-Candy, S.G., 2001.

CNQX increases GABA-mediated synaptic transmission in the

cerebellum by an AMPA/kainite receptor-independent mechanism.

Neuropharmacology 41, 730–736.

Buckby, L.E., Mummery, R., Crompton, M.R., Beesley, P.W., Empson,

R.M., 2004. Comparison of neuroplastin and synaptic marker protein

expression in acute and cultured organotypic hippocampal slices from

rat. Brain Res. Dev. Brain Res. 150, 1–7.

Burrone, J., Murthy, V.N., 2003. Synaptic gain control and homeostasis.

Curr. Opin. Neurobiol. 13, 560–567.

Burrone, J., O’Byrne, M., Murthy, V.N., 2002. Multiple forms of synaptic

plasticity triggered by selective suppression of activity in individual

neurons. Nature 420, 414–418.

Cannon, W., 1939. The Wisdom of the Body, 2nd ed. Norton Pubs, New

York.

Davis, G.W., Bezprozvanny, I., 2001. Maintaining the stability of neural

function: a homeostatic hypothesis. Annu. Rev. Physiol. 63, 847–869.

Davis, G.W., Goodman, C.S., 1998. Synapse-specific control of synaptic

efficacy at the terminals of a single neuron. Nature 392, 82–86.

De Gois, S., Schafer, M.K., Defamie, N., Chen, C., Ricci, A., Weihe, E.,

Varoqui, H., Erickson, J.D., 2005. Homeostatic scaling of vesicular

glutamate and GABA transporter expression in rat neocortical circuits.

J. Neurosci. 25, 7121–7133.

Desai, N.S., Nelson, S.B., Turrigiano, G.G., 1999. Activity-dependent

regulation of excitability in rat visual cortical neurons. Neurocomputing

26–27, 101–106.

Echegoyen, J.C., Graber, K.D., Soltesz, I., 2005. Homeostatic Plasticity in

the Adult Hippocampus in vivo. Abstract number 499.8. Society for

Neuroscience Abstract viewer, Washington, DC.

Freund, T.F., Buzsaki, G., 1996. Interneurons of the hippocampus.

Hippocampus 6, 347–470.

Furshpan, E.J., Potter, D.D., 1989. Seizure-like activity and cellular damage

in rat hippocampal neurons in cell culture. Neuron 3, 199–207.

Galvan, C.D., Wenzel, J.H., Dineley, K.T., Lam, T.T., Schwartzkroin, P.A.,

Sweatt, J.D., Swann, J.W., 2003. Postsynaptic contributions to

hippocampal network hyperexcitability induced by chronic activity

blockade in vivo. Eur. J. Neurosci. 18, 1861–1872.

Goodman, C.S., Shatz, C.J., 1993. Developmental mechanisms that

generate precise patterns of neuronal connectivity. Cell 72, 77–98.

Harms, K.J., Craig, A.M., 2005. Synapse composition and organization

following chronic activity blockade in cultured hippocampal neurons.

J. Comp. Neurol. 490, 72–84.

Ivanova, S.Y., Storozhuk, M.V., Melnick, I.V., Kostyuk, P.G., 2003.

Chronic treatment with ionotropic glutamate receptor antagonist

kynurenate affects GABAergic synaptic transmission in rat hippo-

campal cell cultures. Neurosci. Lett. 341, 61–64.

Kilman, V., van Rossum, M.C., Turrigiano, G.G., 2002. Activity depriva-

tion reduces miniature IPSC amplitude by decreasing the number of

postsynaptic GABA(A) receptors clustered at neocortical synapses.

J. Neurosci. 22, 1328–1337.

L.E. Buckby et al. / Mol. Cell. Neurosci. 31 (2006) 805–816816

Kullmann, D.M., Asztely, F., 1998. Extrasynaptic glutamate spillover in the

hippocampus: evidence and implications. Trends Neurosci. 21, 8–14.

Miles, R., Wong, R.K., 1987. Inhibitory control of local excitatory circuits

in the guinea-pig hippocampus. J. Physiol. 388, 611–629.

Monette, R., Small, D.L., Mealing, G., Morley, P., 1998. A fluorescence

confocal assay to assess neuronal viability in brain slices. Brain Res.

Brain Res. Protoc. 2, 99–108.

Munno, D.W., Syed, N.I., 2003. Synaptogenesis in the CNS: an odyssey

from wiring together to firing together. J. Physiol. 552, 1–11.

Murthy, V.N., Schikorski, T., Stevens, C.F., Zhu, Y., 2001. Inactivity

produces increases in neurotransmitter release and synapse size. Neuron

32, 673–682.

O’Brien, R.J., Kamboj, S., Ehlers, M.D., Rosen, K.R., Fischbach, G.D.,

Huganir, R.L., 1998. Activity-dependent modulation of synaptic AMPA

receptor accumulation. Neuron 21, 1067–1078.

Okabe, S., Kim, H.D., Miwa, A., Kuriu, T., Okado, H., 1999. Continual

remodeling of postsynaptic density and its regulation by synaptic

activity. Nat. Neurosci. 2, 804–811.

Qin, L., Marrs, G.S., McKim, R., Dailey, M.E., 2001. Hippocampal mossy

fibers induce assembly and clustering of PSD95-containing postsynap-

tic densities independent of glutamate receptor activation. J. Comp.

Neurol. 440, 284–298.

Ramakers, G.J., Corner, M.A., Habets, A.M., 1990. Development in the

absence of spontaneous bioelectric activity results in increased

stereotyped burst firing in cultures of dissociated cerebral cortex. Exp.

Brain Res. 79, 157–166.

Rao, A., Craig, A.M., 1997. Activity regulates the synaptic localization of

the NMDA receptor in hippocampal neurons. Neuron 19, 801–812.

Royer, S., Pare, D., 2003. Conservation of total synaptic weight through

balanced synaptic depression and potentiation. Nature 422, 518–522.

Silver, M.A., Stryker, M.P., 2000. Distributions of synaptic vesicle proteins

and GAD65 in deprived and nondeprived ocular dominance columns in

layer IV of kitten primary visual cortex are unaffected by monocular

deprivation. J. Comp. Neurol. 422, 652–664.

Slutsky, I., Sadeghpour, S., Li, B., Liu, G., 2004. Enhancement of synaptic

plasticity through chronically reduced Ca2+ flux during uncorrelated

activity. Neuron 44, 835–849.

Stoppini, L., Buchs, P.A., Muller, D., 1991. A simple method for

organotypic cultures of nervous tissue. J. Neurosci. Methods 37,

173–182.

Takamori, S., Rhee, J.S., Rosenmund, C., Jahn, R., 2000. Identification of a

vesicular glutamate transporter that defines a glutamatergic phenotype

in neurons. Nature 407, 189–194.

Thiagarajan, T.C., Lindskog, M., Tsien, R.W., 2005. Adaptation to synaptic

inactivity in hippocampal neurons. Neuron 47, 725–737.

Turrigiano, G.G., 1999. Homeostatic plasticity in neuronal networks: the

more things change, the more they stay the same. Trends Neurosci. 22,

221–227.

Turrigiano, G.G., Nelson, S.B., 2000. Hebb and homeostasis in neuronal

plasticity. Curr. Opin. Neurobiol. 10, 358–364.

Turrigiano, G.G., Nelson, S.B., 2004. Homeostatic plasticity in the

developing nervous system. Nat. Rev., Neurosci. 5, 97–107.

Turrigiano, G.G., Leslie, K.R., Desai, N.S., Rutherford, L.C., Nelson, S.B.,

1998. Activity-dependent scaling of quantal amplitude in neocortical

neurons. Nature 391, 892–896.

van den Pol, A.N., Obrietan, K., Belousov, A., 1996. Glutamate

hyperexcitability and seizure-like activity throughout the brain and

spinal cord upon relief from chronic glutamate receptor blockade in

culture. Neuroscience 74, 653–674.

Watt, A.J., van Rossum, M.C., MacLeod, K.M., Nelson, S.B., Turrigiano,

G.G., 2000. Activity coregulates quantal AMPA and NMDA currents at

neocortical synapses. Neuron 26, 659–670.

Wierenga, C.J., Ibata, K., Turrigiano, G.G., 2005. Postsynaptic expression

of homeostatic plasticity at neocortical synapses. J. Neurosci. 25,

2895–2905.

Wilson, N.R., Kang, J., Hueske, E.V., Leung, T., Varoqui, H., Murnick,

J.G., Erickson, J.D., Liu, G., 2005. Presynaptic regulation of quantal

size by the vesicular glutamate transporter VGLUT1. J. Neurosci. 25,

6221–6234.

Wojcik, S.M., Rhee, J.S., Herzog, E., Sigler, A., Jahn, R., Takamori,

S., Brose, N., Rosenmund, C., 2004. An essential role for vesicular

glutamate transporter 1 (VGLUT1) in postnatal development and

control of quantal size. Proc. Natl. Acad. Sci. U. S. A. 101,

7158–7163.