Phosphorylation of proteins involved in activity-dependent formsof synaptic plasticity is altered in hippocampal slices maintainedin vitro

Oanh H. Ho,* Jary Y. Delgado� and Thomas J. O’Dell*

*Department of Physiology and �Interdepartmental PhD Program for Neuroscience, David Geffen School of Medicine at UCLA, Los

Angeles, California, USA

Abstract

The acute hippocampal slice preparation has been widely

used to study the cellular mechanisms underlying activity-

dependent forms of synaptic plasticity such as long-term

potentiation (LTP) and long-term depression (LTD). Although

protein phosphorylation has a key role in LTP and LTD, little is

known about how protein phosphorylation might be altered in

hippocampal slices maintained in vitro. To begin to address

this issue, we examined the effects of slicing and in vitro

maintenance on phosphorylation of six proteins involved in

LTP and/or LTD. We found that AMPA receptor (AMPAR)

glutamate receptor 1 (GluR1) subunits are persistently

dephosphorylated in slices maintained in vitro for up to 8 h.

a calcium/calmodulin-dependent kinase II (aCamKII) was also

strongly dephosphorylated during the first 3 h in vitro but

thereafter recovered to near control levels. In contrast, phos-

phorylation of the extracellular signal-regulated kinase ERK2,

the ERK kinase MEK, proline-rich tyrosine kinase 2 (Pyk2),

and Src family kinases was significantly, but transiently,

increased. Electrophysiological experiments revealed that the

induction of LTD by low-frequency synaptic stimulation was

sensitive to time in vitro. These findings indicate that phos-

phorylation of proteins involved in N-methyl-D-aspartate

(NMDA) receptor-dependent forms of synaptic plasticity is

altered in hippocampal slices and suggest that some of these

changes can significantly influence the induction of LTD.

Keywords: brain slices, hippocampus, long-term depression,

long-term potentiation, protein phosphorylation.

J. Neurochem. (2004) 91, 1344–1357.

Since its development more than 50 years ago by HenryMcIlwain, the acute in vitro brain slice preparation has beenextensively used in cellular, biochemical, and molecularstudies of neuronal physiology (Collingridge 1995).Although brain slices provide a convenient and powerfulway to study the physiology of neurons and synapses in theCNS, the techniques used to prepare and maintain brainslices in vitro can have significant effects on cell physiology.For instance, soon after tissue slicing there is a precipitousdrop in cellular levels of glycogen, phosphocreatine, andATP (McIlwain et al. 1951; Whittingham et al. 1984; Feigand Lipton 1990), a near complete loss of dendriticmicrotubules (Burgoyne et al. 1982; Fiala et al. 2003), anda dramatic increase in cAMP and cGMP levels (Whittinghamet al. 1984). Although many of these changes recover withtime, slices also undergo more persistent alterations such as adecrease in the glutamate receptor 1 (GluR1) and GluR3subunits of AMPA-type glutamate receptors (Taubenfeldet al. 2002) and increases in mRNA levels for several

transcription factors, including c-fos and zif268 (Zhou et al.1995; Taubenfeld et al. 2002). Hippocampal slices also havemore synapses than perfusion-fixed tissue, suggesting that aconsiderable amount of structural reorganization occurs inbrain slices (Kirov et al. 1999).

Received May 25, 2004; revised manuscript received August 11, 2004;accepted August 17, 2004.Address correspondence and reprint requests to Dr Thomas J. O’Dell,

Department of Physiology, David Geffen School of Medicine at UCLA,53–231 Center for the Health Sciences, Box 951751, Los Angeles, CA90095, USA. E-mail: todell@mednet.ucla.eduAbbreviations used: ACSF, artificial cerebrospinal fluid; AMPAR,

AMPA receptor; aCamKII, a calcium/calmodulin-dependent kinase II;APV, 2-amino-5-phosphonovaleric acid; ERK, extracellular signal-regulated kinase; fEPSP, field excitatory post-synaptic potential; GluR,glutamate receptor; LFS, low-frequency stimulation; LTD, long-termdepression; LTP, long-term potentiation; MAPK, mitogen-activatedprotein kinase; MEK, mitogen-activated protein kinase kinase; NMDA,N-methyl-D-aspartate; Pyk2, proline-rich tyrosine kinase 2.

Journal of Neurochemistry, 2004, 91, 1344–1357 doi:10.1111/j.1471-4159.2004.02815.x

1344 � 2004 International Society for Neurochemistry, J. Neurochem. (2004) 91, 1344–1357

The biochemical and structural changes that occur in brainslices indicate that cellular physiology in slices can deviatesignificantly from that in vivo. Despite the importance of thisissue, few studies have attempted to identify how resultsobtained from experiments in brain slices might be influ-enced by biochemical perturbations produced by tissueslicing and maintenance in vitro. For example, the acutebrain slice preparation has been widely used to study themechanisms underlying activity-dependent forms of synapticplasticity such as long-term potentiation (LTP) and long-termdepression (LTD). Although LTP and LTD arise from acomplex network of protein kinases and protein phosphatases(Sheng and Kim 2002), the effects of tissue slicing andin vitro maintenance on phosphorylation of proteins in thesesignaling pathways are unknown.

In the experiments described here, we used a western blotanalysis with phospho-specific antibodies to examine howphosphorylation of proteins involved in N-methyl-D-aspartate(NMDA) receptor-dependent LTP and LTD might be alteredin hippocampal slices. We focused on five different proteinsthat are especially important for activity-dependent forms ofsynaptic plasticity: the GluR1 subunit of AMPA receptors(AMPARs) (Song and Huganir 2002), a calcium/calmodulin-dependent kinase II (aCamKII) (Lisman et al. 2002),extracellular signal-regulated kinase 2 (ERK2) (Thomasand Huganir 2004), proline-rich tyrosine kinase 2 (Pyk2:also known as CAKb) (Huang et al. 2001), and Src familytyrosine kinases (Salter and Kalia 2004). Remarkably, wefound that phosphorylation of all five proteins is strongly,and in some cases persistently, altered in hippocampal slices.In electrophysiological experiments, we found that theinduction of NMDA receptor-dependent LTD by low-frequency synaptic stimulation was also surprisingly sensi-tive to time in vitro. Together, these results demonstrate thatprotein phosphorylation is significantly altered in hippocam-pal slices and suggest that some of these changes can have astrong influence on synaptic plasticity.

Materials and methods

Slice preparation and electrophysiology

Standard techniques approved by the UCLA Institutional Animal

Care and Use Committee were used to prepare slices from the

hippocampus of 8–10-week-old male C57BL/6 mice. Animals were

anesthetized with halothane, killed by cervical dislocation, and the

brain was rapidly removed and placed in a beaker containing cold

(� 4�C) artificial cerebrospinal fluid (ACSF) consisting of 124 mM

NaCl, 4.4 mM KCl, 25 mM Na2HCO3, 1 mM NaH2PO4, 1.2 mM

MgSO4, 2 mM CaCl2, and 10 mM glucose that was oxygenated with

95% O2/5% CO2. Both hippocampi were dissected from the brain

and 400-lm thick slices were cut using a manual tissue chopper. For

some experiments, the dentate gyrus, CA3 region, and subiculum of

freshly cut slices were removed to provide ‘mini-slices’ containing

just the CA1 region (Opazo et al. 2003). Unless noted otherwise,

slices were placed into interface-type chambers (Fine Science Tools,

Foster City, CA, USA) and continuously perfused with warm (30�C)ACSF at a rate of 2–3 mL/min.

A bipolar, nichrome wire stimulating electrode placed in stratum

radiatum of the CA1 region of the slice was used to antidromically

activate commissural/associational fiber synapses onto CA3

pyramidal cells and field excitatory post-synaptic potentials

(fEPSPs) were recorded using ACSF-filled glass microelectrodes

placed in stratum radiatum of the CA3 region (electrode

resistance ¼ 5–10 MW). Pre-synaptic fiber stimulation pulses were

delivered once every 50 s using a stimulation strength that evoked

fEPSPs that were approximately 25% of the maximal fEPSP

amplitude that could be elicited by strong intensity stimulation.

LTD was induced using a low-frequency stimulation (LFS) train of

pre-synaptic stimulation (900 pulses delivered at 1 Hz). For each

experiment, fEPSPs were normalized to the averaged pre-LFS

baseline, and the average of fEPSP slopes recorded between 50

and 55 min after the start of LFS was used for statistical

comparisons (paired and unpaired t-tests).

Western immunoblotting

In all experiments, slices obtained from the same animal were placed

in up to four separate interface chambers (typically 3–6 slices/

chamber) and allowed to recover for various amounts of time. At the

appropriate time, slices were rapidly transferred into pre-chilled

microcentrifuge tubes and snap frozen by plunging the microcen-

trifuge tube into a bed of crushed dry ice. Slices were then stored at

)80�C for up to 2 weeks prior to homogenization in an ice-cold

buffer consisting of 50 mM Tris-HCl, 50 mM NaF, 10 mM EGTA,

10 mM EDTA, 80 lM sodium molybdate, 5 mM sodium pyrophos-

phate, 1 mM sodium orthovanadate, 0.01% Triton X-100, and 4 mM

para-nitrophenylphosphate. The homogenization buffer also con-

tained cocktails of protease inhibitors (Protease Inhibitors Complete;

Roche Molecular Biochemicals, Indianapolis, IN, USA) and protein

phosphatase inhibitors (Protein Phosphatase Inhibitor Cocktail I and

II; Sigma-Aldrich, St. Louis, MO, USA). For some experiments, we

used a previously described protocol to prepare synaptoneurosomes

from hippocampal slices (Johnson et al. 1997). In these experi-

ments, slices were homogenized in an ice-cold buffer consisting of

140 mM NaCl, 5 mM KCl, 5 mM NaHCO3, 1.2 mM NaH2PO4,

1.0 mM MgSO4, 1.8 mM CaCl2, 10 mM glucose, and 20 mM HEPES

(pH ¼ 7.4) that contained cocktails of protease and protein

phosphatase inhibitors. The homogenate was sequentially filtered

through 100-lm and 5-lm pore membranes and the filtrate was

centrifuged at 3000 g for 20 min at 4�C. The pellet containing

synaptoneurosomes was resuspended in homogenization buffer and

stored at )80�C.Protein concentrations were determined using the Bradford

method and equal amounts of protein (typically 10–20 lg/lane)were loaded onto 12% sodium dodecyl sulfate–polyacrylamide gels

(4% stacking gel). After electrophoresis, proteins were transferred

onto nitrocellulose membranes and the blots were then blocked for

30–60 min in Tris-buffered saline containing 0.025% Tween-20

(TBST) and 4% non-fat dry milk. Blots were incubated overnight at

4�C in TBST containing 1–4% non-fat dry milk with primary

antibodies specific for phosphorylated forms of GluR1, aCamKII,

mitogen-activated protein kinase kinase 1/2 (MEK1/2), ERK1/2,

Pyk2, or Src family kinases. Total levels of these proteins in the

Protein phosphorylation in hippocampal slices 1345

� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 91, 1344–1357

same samples were determined using antibodies that recognize both

phosphorylated and unphosphorylated forms of each protein.

Following incubation in primary antibody, blots were washed three

times with TBST and then incubated for 1–2 h at room temperature

(22–23�C) in TBST plus 1–4% non-fat dry milk containing the

appropriate horseradish peroxidase-conjugated secondary antibody

(1 : 1000 or 1 : 2000). Immunoreactive bands were visualized using

enhanced chemiluminescence (Immun-Star HRP detection kit, Bio-

Rad, Hercules, CA, USA) and images of the blots were acquired

using a cooled CCD camera-based image acquisition and analysis

system (Chemi-Doc and Quantity One software package from Bio-

Rad). To control for potential variations in loading, blots were re-

probed (without stripping) with primary antibodies against tubulin

or b-actin, and the density values measured for each protein of

interest were normalized to the density values obtain for one of these

loading controls. The normalized values were then expressed as a

percentage of levels seen in samples from control slices. All results

are reported as the mean ± SEM. In general, one-way ANOVAs or

Kruskal–Wallis one-way ANOVAs on ranks were used to assess

statistical significance. Repeated measures versions of these tests

were used where appropriate. Student–Newman–Keuls tests were

used to examine significance of multiple pair-wise comparisons and

Dunnett’s tests were used for repeated statistical comparisons to

controls.

Primary antibodies

Primary antibodies to AMPAR GluR1 subunits phosphorylated at

serine 845 (S845, 1 : 1000), GluR1 phosphorylated at serine 831

(S831, 1 : 1000), total GluR1 (1 : 2000), aCamKII phosphorylated

at theronine 286 (T286, clone 22B1, 1 : 1000), total aCamKII

(clone 6G9, 1 : 2000), total Pyk2 (clone 74, 1 : 500), and a

neuronal-specific isoform (bIII) of tubulin (clone 2G10, 1 : 10 000)

were obtained from Upstate Biotechnology (Lake Placid, NY, USA).

Antibodies against ERK1/2 dually phosphorylated at threonine 202

and tyrosine 204 (T203/Y204, clone E10, 1 : 1000), total ERK1/2

(1 : 1000), MEK1/2 dually phosphorylated at serine 217 and serine

221 (S217/S221, 1 : 1000), total MEK1/2 (1 : 1000), Pyk2 phos-

phorylated at tyrosine 402 (Y402, 1 : 2000) and Src phosphorylated

at tyrosine 416 (Y416, 1 : 500) were obtained from Cell Signaling

Technology (Beverly, MA, USA). An antibody against b-actin(clone AC15, 1 : 2000–4000) was obtained from Sigma.

The polyclonal antibody we used to measure Pyk2 phosphory-

lation at Y402 reacted with three high molecular weight proteins

(approximately 112, 130, and 190 kDa) in samples obtained from

hippocampal slices. All three proteins show qualitatively similar

changes in slices maintained in vitro for different amounts of time,

and detection of all three bands was blocked when the peptide used

to generate the phospho-Pyk2 antibody was present during the

primary antibody incubation step (peptide concentration ¼ 0.1 lg/mL). Because the lowest molecular weight protein co-migrated with

total Pyk2 (data not shown) this band was used to measure phospho-

Pyk2 levels. The sequence surrounding Y416 in Src (Y423 in

mouse) is highly conserved in Src family kinases and thus the

phospho-Y416 specific antibody used in our experiments may cross-

react with other Src family kinases phosphorylated at an equivalent

site. We therefore used changes in phosphorylation detected with

this antibody as a general measure of phosphorylation of Src family

kinases rather than a specific measure of phospho-Src levels.

Results

AMPA receptor glutamate receptor 1 subunits undergo

dephosphorylation in hippocampal slices

Changes in AMPAR phosphorylation, particularly at S831and S845 in GluR1 subunits, have a crucial role in both LTPand LTD (Song and Huganir 2002). Thus, to begin toexamine whether phosphorylation of proteins involved inNMDA receptor-dependent forms of synaptic plasticity isaltered in hippocampal slices, we compared levels of totalS845 phosphorylated and S831 phosphorylated GluR1 inslices maintained in vitro for different amounts of time.Although total GluR1 levels did not change in slicesmaintained in vitro for up to 3 h, phosphorylation of GluR1at both S845 and S831 was strongly affected by time in vitro(Fig. 1a). Compared to levels in control slices collected after30 min in vitro, GluR1 phosphorylation at S845 was reducedto 74 ± 5% of control after 60 min in vitro (n ¼ 9, p < 0.05)and declined to 52 ± 5% of control after 120 min in vitro(n ¼ 9, p < 0.05). In slices maintained in vitro for 180 min,GluR1 phosphorylation at S845 was reduced more thantwofold to 40 ± 4% of control (p < 0.05, n ¼ 9). In contrast,phosphorylation of GluR1 at S831 was not significantlyaltered during the first 2 h in vitro but did show a smalldecrease in slices maintained in vitro for 180 min (levelswere 67 ± 8% of control, n ¼ 9, p < 0.05) (Fig. 1a). Thus,although GluR1 phosphorylation at S831 appears to be morestable than phosphorylation at S845, S831 also undergoessignificant dephosphorylation in hippocampal slices main-tained in vitro.

Although the results shown in Fig. 1(a) indicate thatGluR1 phosphorylation is surprisingly labile in hippocampalslices, the mechanisms responsible for GluR1 dephosphory-lation are not clear from these results alone. One possibility isthat slicing transiently activates protein kinases that phos-phorylate GluR1. If so, then the decrease in GluR1phosphorylation that occurs with time in vitro might reflectthe restoration of basal levels of GluR1 phosphorylation dueto the activity of protein phosphatases. Alternatively, slicingby itself could have no effect on GluR1 phosphorylation, andthe decay in phosphorylation that occurs over time in vitromight be due to an alteration in the basal protein kinase and/or protein phosphatase activity that favors GluR1 dephosph-orylation. To address this issue, we did four separateexperiments where a small sample of hippocampus wascollected immediately after dissection and slices preparedfrom the remaining tissue were maintained in vitro for30 min before being collected for western analysis. Asshown in Fig. 1(b), there was no difference in either totalGluR1 levels or GluR1 phosphorylation at S845 in slicesmaintained in vitro for 30 min compared to unsliced controltissue. This suggests that an alteration in protein phosphataseand/or protein kinase activity is responsible for the

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� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 91, 1344–1357

dephosphorylation of S845 that occurs in slices maintainedin vitro for longer times. Although the results shown inFig. 1(a) suggested that GluR1 phosphorylation at S831 wasmore stable than phosphorylation at S845, levels of S831phosphorylated GluR1 in slices maintained in vitro for30 min were reduced to 52 ± 15% of those in unslicedcontrol tissue (p < 0.05). Phosphorylation of GluR1 subunitsat S831 thus undergoes a rapid initial phase of dephosph-orylation either during or shortly after slicing, followed by aslower and less robust phase of dephosphorylation as slicesare maintained in vitro for longer periods of time. In contrast,GluR1 phosphorylation at S845 is not altered by slicing and ashort period of recovery in vitro but is instead stronglydephosphorylated over longer times in vitro. The combinedeffects of slicing and in vitro maintenance on phosphoryla-tion at both sites appear similar; after 180 min in vitro, levelsof phosphorylation at both S845 and S831 are reduced morethan twofold compared to levels in unsliced hippocampaltissue.

a Calcium/calmodulin-dependent kinase II is

dephosphorylated in hippocampal slices

The changes in GluR1 phosphorylation that occur in hippo-campal slices suggest that there may be equally dramaticchanges in protein phosphorylation within the variousupstreamsignalingpathways that regulateAMPARphosphory-lation in LTP. The calcium/calmodulin-dependent proteinkinase CamKII phosphorylates GluR1 at S831 (Barria et al.1997; Mammen et al. 1997) and has a crucial role in LTP(Lisman et al. 2002).Autophosphorylation ofCamKII at T286(in the a isoform) converts CamKII into a persistently activeform that is thought to be required for LTP induction (Lismanet al. 2002). Thus, to determine whether phosphorylation ofproteins in signaling pathways upstream ofAMPARs is alteredin hippocampal slices we measured phosphorylation ofaCamKII at T286 in hippocampal slices maintained in vitrofor different amounts of time. As shown in Fig. 2(a), slicesmaintained in vitro for times ranging from 30 min to 3 hhad similar levels of both total and phospho-T286 aCamKII(n ¼ 7). Although this suggests that aCamKII phosphoryla-tion at T286 is not altered in slices, we wondered whetheraCamKII phosphorylation at T286 might be sensitive to theeffects of slicing and a short recovery period. To examine this,we compared levels of total and T286 phosphorylatedaCamKII in control samples prepared from unsliced hippo-campal tissue to that in slices obtained from the samehippocampi that had been maintained in vitro for 30 min.Total aCamKII levels were unchanged in slices maintainedin vitro for 30 min, whereas phosphorylation of aCamKII atT286 was reduced to 51 ± 14% of control (n ¼ 4, p < 0.05)(Fig. 2b). Thus, much like the S831 phosphorylation site inGluR1, slicing and a short period of time in vitro induces arobust and long-lasting dephosphorylation of aCamKII atT286.

(a)

(b)

Fig. 1 AMPA receptor glutamate receptor 1 (GluR1) subunits undergo

dephosphorylation in hippocampal slices maintained in vitro. (a)

Hippocampal slices from the same animal were maintained in

interface type slice chambers for 30, 60, 120, or 180 min post-slicing.

Each point represents the mean (± SEM) from 9 separate experi-

ments. Compared to levels in slices maintained in vitro for 30 min,

GluR1 phosphorylation at S845 (pS845, circles) was significantly re-

duced at all time points measured. Phosphorylation at S831 (pS831,

triangles) was relatively constant over the first 2 h in vitro but was

significantly reduced after 180 min in vitro. *p < 0.05 compared to

levels in slices maintained in vitro for 30 min. Total GluR1 levels

(diamonds) did not change with time in vitro. The inset shows immu-

noblots from one experiment demonstrating the effect of time in vitro

on levels of GluR1 phosphorylated at S831, S845, and total GluR1. (b)

The histogram shows levels of total and phosphorylated GluR1 in

slices maintained in vitro for 30 min normalized to those in unsliced

tissue samples from the same hippocampi. Note that S831 is strongly

dephosphorylated in hippocampal slices maintained in vitro for

30 min, whereas GluR1 phosphorylation at S845 and total GluR1

levels are unchanged (n ¼ 4, *p < 0.05 compared to levels in unsliced

control samples). The inset shows immunoblots from one experiment

where samples from unsliced hippocampus (0 min in vitro) and slices

maintained in vitro for 30 min were probed with antibodies specific for

S831 phosphorylated GluR1, S845 phosphorylated GluR1, or total

GluR1.

Protein phosphorylation in hippocampal slices 1347

� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 91, 1344–1357

The extracellular signal-regulated kinase pathway is

transiently activated in hippocampal slices

The changes in GluR1 and aCamKII phosphorylationassociated with slicing and in vitro maintenance indicatethat key components of the core signaling pathway respon-sible for NMDA receptor-dependent forms of synapticplasticity are significantly altered in the hippocampal slices.In addition to aCamKII activation and GluR1 phosphoryla-tion, the induction of LTP depends on a number of distinctmodulatory pathways (Dineley et al. 2001; Sheng and Kim2002). For instance, the induction of LTP by some patterns ofsynaptic stimulation requires activation of mitogen-activated

protein kinases (MAPKs) such as ERK1 (p44 MAPK) andERK2 (p42 MAPK) (Thomas and Huganir 2004). Thus, toexamine whether some of these modulatory pathways arealso altered in hippocampal slices, we measured ERKactivation in slices maintained in vitro using a phospho-specific antibody that specifically recognizes the activatedforms of ERK1 and ERK2. Because ERK2 may bepreferentially involved in LTP (Selcher et al. 2001), wefocused on how ERK2 phosphorylation might change inslices. In four separate experiments, a small piece ofhippocampal tissue was collected immediately after dissect-ing both hippocampi from the brain and slices prepared fromthe remaining tissue were maintained in vitro for differentamounts of time. Compared to basal levels of ERK2phosphorylation in control samples from unsliced hippocam-pus, ERK2 phosphorylation in slices maintained in vitro for30 min was increased to 231 ± 23% of control (p < 0.05)(Fig. 3a). A smaller, but significant, increase in ERK2phosphorylation was still present in slices maintained in vitrofor 60 min (levels were 153 ± 10% of control; p < 0.05).Phosphorylation of ERK2 was not significantly differentfrom control, however, in slices maintained in vitro forlonger periods of time (Fig. 3a). There was no change inlevels of total ERK2 at any time point. Together, these resultsindicate that slicing and a short period of recovery in vitroare associated with a robust, but transient, activation of theERK pathway. Consistent with this, phosphorylation ofMEK, the upstream kinase responsible for ERK activation,was also transiently increased in hippocampal slices(Fig. 3b).

Proline-rich tyrosine kinase 2 and Src family kinases are

activated in hippocampal slices

Members of the Src family of tyrosine kinases, such as Srcand Fyn, can potently modulate NMDA receptor function(Chen and Leonard 1996; Kohr and Seeburg 1996; Yu et al.1997) and are thought have an important modulatory role inLTP induction (Grant et al. 1992; Lu et al. 1998). Althoughmultiple signals can activate Src family kinases, the focaladhesion kinase Pyk2 provides an essential link betweenNMDA receptors and activation of Src family kinases in LTP(Huang et al. 2001). Thus, to determine whether tissueslicing and in vitro maintenance are associated with changesin tyrosine kinase-dependent pathways involved in LTP weused antibodies specific for the phosphorylated, active formsof Pyk2 (phosphorylated at tyrosine 402) and Src familykinases (phosphorylated at Y416 in Src) to measure activa-tion of Pyk2 and Src family kinases.

In these experiments (n ¼ 4), we collected a small sampleof hippocampus prior to tissue slicing and then maintainedslices prepared from the remaining tissue in vitro for 30, 60,120, or 180 min. As shown in Fig. 4(a), phosphorylation ofPyk2 at Y402 was dramatically increased in slices main-tained in vitro for 30 min (levels were increased to

(a)

(b)

Fig. 2 a Calcium/calmodulin-dependent kinase II (aCamKII) is rapidly

dephosphorylated in hippocampal slices maintained in vitro. (a)

Compared to slices maintained in vitro for 30 min, levels of both T286

phosphorylated aCamKII (filled symbols) and total aCamKII (open

symbols) are unchanged in slices maintained in vitro for up to 180 min

(n ¼ 7). The inset shows an immunoblot from one experiment probed

with an antibody specific for T286 phosphorylated aCamKII (pT286)

and later re-probed with an antibody against b-actin (loading control).

(b) The histogram shows the average levels of total and T286 phos-

phorylated aCamKII (pT286) in slices maintained in vitro for 30 min

expressed as a percentage of those in samples of unsliced hippo-

campus (n ¼ 4, *p < 0.05 compared to levels in unsliced control

samples). The inset shows immunoblots from one experiment where

samples of unsliced hippocampus (0 min in vitro) and slices main-

tained in vitro for 30 min (from the same hippocampi) were probed

with antibodies specific for total aCamKII or T286 phosphorylated

aCamKII.

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� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 91, 1344–1357

741 ± 93% of control samples from unsliced hippocampus,p < 0.05 compared to unsliced control samples). AlthoughPyk2 phosphorylation declined with time in vitro, phospho-Pyk2 levels were significantly elevated at all time pointsexamined (levels were 545 ± 95% of control after 60 minin vitro, 461 ± 74% of control after 120 min in vitro, and

379 ± 28% of control after 180 min in vitro; p < 0.05 for alltime points compared to unsliced controls). Because Pyk2phosphorylation in samples from unsliced tissue was verylow relative to that in slices maintained in vitro, it was

(a)

(b)

Fig. 3 The extracellular signal-regulated kinase (ERK) pathway is

transiently activated in hippocampal slices. (a) Compared to levels in

samples of unsliced hippocampus (0 min in vitro), phospho-ERK2

levels (filled circles) were significantly elevated in slices maintained

in vitro for 30 and 60 min (*p < 0.05) but not at later time points (n ¼4). No change in total ERK2 levels was detected at any time point. The

inset shows immunoblots from one experiment that were probed with

antibodies against T202/Y204 phosphorylated ERK1/2 (pERK1/2) and

tubulin (loading control). (b) Phosphorylation of mitogen-activated

protein kinase kinase 1/2 (MEK1/2) (filled symbols) was also signifi-

cantly elevated in slices maintained in vitro for 30 and 60 min but not

in slices maintained in vitro for longer periods of time (n ¼ 4,

*p < 0.05). Time in vitro had no effect on total MEK1/2 levels (open

symbols). The inset shows immunoblots from one experiment that

were probed with antibodies against dually phosphorylated MEK1/2

(pMEK1/2) and total MEK1/2.

(a)

(b)

Fig. 4 Proline-rich tyrosine kinase 2 (Pyk2) and Src family kinases are

activated in hippocampal slices. (a) Compared to levels in control

samples from unsliced hippocampus (0 min in vitro), phosphorylation

of Pyk2 at Y402 was significantly elevated in slices maintained in vitro

for 30, 60, 120, and 180 min (filled symbols, n ¼ 4, *p < 0.05 com-

pared to control). Although total Pyk2 levels (open symbols) were not

significantly different from control in slices maintained in vitro for 30 or

60 min levels, total Pyk2 levels were significantly reduced in slices

maintained in vitro for longer times (*p < 0.05). The inset shows

immunoblots from one experiment that were probed with antibodies

against Y402 phosphorylated Pyk2 (pY402), total Pyk2, and tubulin

(loading control). (b) Phosphorylation of Src family kinases was also

significantly increased in slices maintained in vitro (n ¼ 4, *p < 0.05

compared to control). The inset shows immunoblots from one

experiment that were probed with antibodies against Y416 phos-

phorylated Src (pY416) and b-actin (loading control).

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� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 91, 1344–1357

difficult to acquire an image where the chemiluminesentsignals for all five samples were in a linear range. Our analysisthus most likely overestimates the magnitude of the increasein Pyk2 phosphorylation in slices maintained in vitro. More-over, there was a significant decrease in total Pyk2 levels inslices maintained in vitro for 120 and 180 min (levels werereduced to 68 ± 15% and 63 ± 12% of control, respectively;p < 0.05 for both, Fig. 4a). Thus, quantification of the effectsof slicing and in vitro maintenance of Pyk2 phosphorylationwas also complicated by the fact that some of the decay inPyk2 phosphorylation seen in slices maintained in vitro forlonger periods of time is due to changes in total protein levels.Nonetheless, although it is difficult to determine accuratelythe true magnitude and time course of Pyk2 phosphorylation,these results clearly indicate that slicing and in vitro main-tenance are associated with a robust and long-lasting activa-tion of Pyk2 in hippocampal slices. Because Pyk2 is anupstream activator of Src, these results suggest that Src familykinases should also be strongly activated in hippocampalslices. Consistent with this, phosphorylation of Src familykinases was significantly increased in hippocampal slicesmaintained in vitro (Fig. 4b).

Phosphorylation of a calcium/calmodulin-dependent

kinase II is sensitive to slice maintenance conditions

In general, two different techniques are used to maintainbrain slices in vitro. In the interface-slice preparation, slicesare partially submerged in ACSF with the top surface of theslice exposed to a humidified atmosphere of 95% O2/5%CO2. As the name implies, in the submerged-slice prepar-ation, slices are completely submerged in ACSF. Given the

large changes in protein phosphorylation that occur inhippocampal slices maintained in interface-type chambers,we investigated whether similar changes also occur in slicesmaintained under submerged conditions. In four separateexperiments, both hippocampi from an animal were dissectedfrom the brain and a small portion of one hippocampus wascollected to provide a control sample of unsliced tissue.Slices prepared from the remaining tissue were then main-tained in vitro for either 30 min or 3 h in a commerciallyavailable, submerged-slice type chamber (Harvard Appar-atus, Holliston, MA, USA). Because slices are often kept atroom temperature when they are maintained in submergedchambers, we performed these experiments at 22–23�C.

As shown in Fig. 5, slices maintained in submergedconditions showed changes in GluR1, ERK, Pyk2, and Srcfamily kinase phosphorylation similar to those that occurin slices maintained in interface conditions. There was,

(a)

(b)

(c)

Fig. 5 Changes in glutamate receptor 1 (GluR1), extracellular signal-

regulated kinase 2 (ERK2), proline-rich tyrosine kinase 2 (Pyk2), and

Src phosphorylation in hippocampal slices maintained in submerged-

slice type chambers. (a) Phosphorylation of GluR1 subunits at S845

(circles) and S831 (triangles) is significantly reduced in slices main-

tained in submerged chambers (n ¼ 4, *p < 0.05). Total GluR1 levels

were unchanged. The inset shows immunoblots from one experiment

probed with antibodies against phospho-S845 and phospho-S831

GluR1. (b) The ERK pathway is transiently activated in slices main-

tained in submerged conditions. Phosphorylation of ERK2 (filled

symbols) was significantly increased in slices maintained in vitro for

30 min but not in slices maintained in vitro for 180 min (n ¼ 4,

*p < 0.05). Total ERK2 levels did not change with time in vitro. The

inset shows immunoblots from one experiment probed with antibodies

against dually phosphorylated ERK1/2 and total ERK1/2. The two

bands detected by the phospho-specific and total ERK1/2 antibodies

correspond to ERK1 and ERK2 (p44 and p42 mitogen-activated pro-

tein kinase, respectively). (c) Phosphorylation of Pyk2 (triangles) and

Src family kinases (circles) is significantly increased in slices main-

tained in vitro in submerged-slice chambers for 30 or 180 min (n ¼ 4,

*p < 0.05). The inset shows immunoblots from one experiment probed

with antibodies against Y402 phosphorylated Pyk2 and Y416 phos-

phorylated Src.

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� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 91, 1344–1357

however, a striking difference between submerged andinterface slices in how aCamKII phosphorylation wasaffected by time in vitro (Fig. 6). Phosphorylation ofaCaMKII at T286 was significantly increased in sub-merged slices maintained in vitro for 30 min (levels were198 ± 29% of controls, p < 0.05) but was not significantlydifferent from control in submerged slices maintainedin vitro for 3 h (levels were 107 ± 19% of controls). Thus,although aCamKII is dephosphorylated in slices main-tained in interface chambers, a robust, but transient,increase in phosphorylation at T286 occurs in slicesmaintained in submerged conditions. It is interesting thatin slices maintained in submerged conditions GluR1phosphorylation at S831 in significantly decreased after30 min in vitro, even though aCamKII phosphorylation isincreased. Since aCamKII is one of the kinases thatphosphorylate GluR1 at S831 (Mammen et al. 1997), this

finding suggests that the changes in GluR1 and aCamKIIphosphorylation associated with slicing and in vitro main-tenance may be occurring in distinct cellular compart-ments.

Protein phosphorylation is altered in CA1 mini-slices

Much of our current understanding regarding the role ofprotein phosphorylation in LTP and LTD has come fromstudies in the CA1 region of the hippocampus. Thus, toexamine how tissue slicing and in vitro maintenance mightalter phosphorylation of plasticity-related proteins in thehippocampal CA1 region, we prepared CA1 mini-slices fromfreshly cut slices and maintained these slices in interface-typechambers for different amounts of time. Similar to ourfindings in samples prepared from whole slices, phosphory-lation of GluR1 (at both S845 and S831) and aCamKII wassignificantly decreased in CA1 mini-slices (Figs 7a and b).Moreover, the increase in Pyk2 and Src phosphorylationassociated with slicing and in vitro maintenance was alsoevident in CA1 mini-slices (Fig. 7c). Surprisingly, althoughphospho-ERK2 levels are significantly elevated in samplesprepared from whole slices maintained in vitro for 30 min(Fig. 3), phosphorylation of ERK2 was not significantlyelevated in CA1 mini-slices maintained in vitro for the sameamount of time (Fig. 7d). Moreover, unlike whole slices, asignificant decrease in ERK2 phosphorylation was evident inCA1 mini-slices maintained in vitro for 3 h. This suggeststhat the transient increase in ERK2 phosphorylation observedin homogenates prepared from whole slices is most likelydue to ERK2 activation within the dentate gyrus and/orhippocampal CA3 region. Thus, most of the changes inprotein phosphorylation associated with tissue slicing andin vitro maintenance seen in samples prepared from wholeslices are also evident in samples from isolated CA1 regions.The effects of slicing and in vitro maintenance on ERKphosphorylation, however, appear to be region-specific.

Alterations in protein phosphorylation associated with

tissue slicing and in vitro maintenance can be observed in

synaptoneurosomes

To determine whether the changes in protein phosphorylationwe observed in whole homogenates prepared from intactslices reflects changes in synaptic proteins, we comparedphosphorylation of GluR1, aCamKII, Pyk2, and Src familykinases in synaptoneurosomes prepared from freshly cuthippocampal slices and slices maintained in interface cham-bers for 3 h. (ERK2 phosphorylation was not measured inthese experiments because the transient activation of ERK2in slices fully recovers by 3 h in vitro, see Fig. 3.) Consistentwith the notion that phosphorylation of synaptic proteins isaltered in hippocampal slices maintained in vitro, all fourproteins showed changes in phosphorylation similar to thoseobserved in experiments using whole homogenates, i.e.GluR1 and aCamKII phosphorylation was significantly

Fig. 6 Phosphorylation of a calcium/calmodulin-dependent kinase II

(aCamKII) at T286 is transiently increased in slices maintained in

submerged chambers but persistently decreased in slices maintained

in interface chambers. In slices maintained in submerged conditions

(filled symbols), aCamKII phosphorylation at T286 was significantly

enhanced (*p < 0.05 compared to unsliced control tissue) after 30 min

in vitro but not after 180 min in vitro (n ¼ 4). For comparison, phos-

pho-T286 aCamKII levels in slices maintained in interfaces slices for

the same period of time are also shown (open symbols, n ¼ 5). In

slices maintained under interface conditions there is a persistent de-

crease in aCamKII phosphorylation at T286 (*p < 0.05 compared to

unsliced control tissue). At both 30 and 180 min, in vitro submerged

slices showed significantly higher levels of aCamKII phosphorylation

at T286 than interface slices (#p < 0.05). The inset shows immuno-

blots from a single experiment where samples obtained from unsliced

control tissue and submerged slices maintained in vitro for the indicate

times were probed with antibodies against T286 phosphorylation and

total aCamKII.

Protein phosphorylation in hippocampal slices 1351

� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 91, 1344–1357

decreased and phosphorylation of Pyk2 and Src familykinases was significantly increased in slices maintainedin vitro for 3 h (Fig. 8).

Changes in protein phosphorylation over an extended

period of time in vitro

Although the increase in ERK2 phosphorylation associatedwith slicing and in vitro maintenance recovers rapidly(within an hour or two), phosphorylation of GluR1, aCam-

KII, Pyk2, and Src family kinases remains significantlyaltered, even after 3 h in vitro. To determine how phos-phorylation of these proteins responds to even longer periodsof time in vitro, we examined phosphorylation of GluR1,aCamKII, Pyk2, and Src in whole homogenates prepared

Fig. 7 Changes in protein phosphorylation associated with slicing and

in vitro maintenance in CA1 mini-slices. (a) As in samples prepared

from intact slices, phosphorylation of AMPA receptor glutamate

receptor 1 (GluR1) subunits at S845 (pS845, circles) and S831

(pS831, triangles) is significantly reduced as a function of time in vitro

in isolated CA1 mini-slices (*p < 0.05 compared to control mini-slices

homogenized immediately after slicing, n ¼ 4). Total GluR1 levels

(diamonds) were unchanged. (b) Phosphorylation of a calcium/cal-

modulin-dependent kinase II (aCamKII) at T286 (pT286, filled sym-

bols) is significantly decreased in CA1 mini-slices maintained in vitro,

whereas total aCamKII levels (open symbols) were unchanged

(*p < 0.05 compared to control, n ¼ 4). (c) Phosphorylation of proline-

rich tyrosine kinase 2 (Pyk2) (circles) and Src family kinases (trian-

gles) is significantly increased in CA1 mini-slices (*p < 0.05 compared

to control, n ¼ 4). After 30 min in vitro, total Pyk2 levels (diamonds)

were not significantly different from control but were significantly

reduced by 180 min in vitro (*p < 0.05 compared to control, n ¼ 4).

(d) In contrast to samples prepared from intact slices, extracellular

signal-regulated kinase 2 (ERK2) phosphorylation (circles) was not

elevated after 30 min in vitro in isolated CA1 mini-slices and was

significantly reduced after 3 h in vitro (n ¼ 4, *p < 0.05 compared to

control). Total ERK2 levels (diamonds) were not significantly altered.

(a)

(b)

(c)

Fig. 8 Changes in protein phosphorylation associated with slicing and

in vitro maintenance are evident in synaptoneurosomes. Synapto-

neurosomes were prepared from intact slices collected immediately

after slicing (0 min in vitro controls) or from slices maintained in

interface slices for 180 min. (a) Glutamate receptor 1 (GluR1) phos-

phorylation at both S845 and S831 was significantly reduced in syn-

aptoneurosomes prepared from slices maintained in vitro for 180 min

(n ¼ 3). Total GluR1 levels were unchanged. The inset shows

immunoblots from one experiment where samples from control slices

and slices maintained in vitro for 180 min were probed with antibodies

against S845 phosphorylated GluR1, S831 phosphorylated GluR1,

and total GluR1. (b) Phosphorylation of a calcium/calmodulin-

dependent kinase II (aCamKII) at T286 is significantly reduced in

synaptoneurosomes prepared from slices maintained in vitro for

180 min (n ¼ 3). Total aCamKII levels were unchanged. The inset

shows immunoblots from one experiment that were probed with anti-

bodies against T286 phosphorylated and total aCamKII. (c) Phos-

phorylation of proline-rich tyrosine kinase 2 (Pyk2) and Src family

kinases is significantly increased in synaptoneurosomes prepared

from slices maintained in vitro for 180 min (n ¼ 3). Although total Pyk2

levels tended to be lower in synaptoneurosomes prepared from slices

maintained in vitro for 180 min, this difference was not statistically

significant (p ¼ 0.2). The inset shows immunoblots from one experi-

ment probed with antibodies against Pyk2 phosphorylated at Y402,

total Pyk2, and Src phosphorylated at Y416. *p < 0.05 paired t-test

compared to control.

1352 O. H. Ho et al.

� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 91, 1344–1357

from intact slices maintained in interface chambers for 2, 4,or 8 h. Compared to levels in unsliced tissue, GluR1phosphorylation at both S845 and S831 was stronglyreduced, even after 8 h in vitro (Fig. 9a). In contrast,although phosphorylation of aCamKII at T286 was signifi-cantly reduced in slices maintained in vitro for 2 h, levelsreturned to near control by 4 h in vitro (Fig. 9b). Likewise,the increase in phosphorylation of Pyk2 and Src familykinases associated with slicing and in vitro maintenance wasalso transient, and phosphorylation of both kinases returnedto near control levels after 8 h in vitro (Fig. 9c).

Synaptic plasticity in hippocampal slices is sensitive to

time in vitro

The changes in protein phosphorylation that occur inhippocampal slices raise a number of questions. Onequestion that we considered to be especially important waswhether these changes might influence NMDA receptor-dependent forms of synaptic plasticity. Importantly, onceestablished, the decrease in GluR1 phosphorylation (at bothS845 and S831) was relatively constant over 8 h in vitro.Thus, although changes in basal levels of GluR1 phosphory-lation might strongly influence the induction of LTP and/orLTD, the effects of GluR1 dephosphorylation on synapticplasticity should be relatively constant over time in vitro. Incontrast to the stable dephosphorylation of GluR1 subunits,there is a strong, transient activation of Src family kinases inslices maintained in vitro. How might activation Src familykinases influence LTP and LTD induction? In general, LTP isinduced by patterns of synaptic stimulation that producestrong levels of NMDA receptor activation, whereas LTD isinduced by patterns of synaptic activity that result in lowerlevels of NMDA receptor activity (Winder and Sweatt 2001).Because activation of Src family kinases up-regulatesNMDA receptor activity (Salter and Kalia 2004), the strongactivation of these kinases in slices maintained in vitro forshort periods of time should facilitate the induction of LTPand oppose the induction of LTD by near threshold patternsof synaptic stimulation. Subsequently, as Src activitysubsides over longer time in vitro, the resulting down-regulation of NMDA receptor activity should favor theinduction of LTD and oppose the induction of LTP by nearthresholds patterns of synaptic activity. In addition, becauseelevated basal levels of constitutively active aCamKII (i.e.T286-phosphorylated) can facilitate the induction of LTD(Mayford et al. 1995), the decrease in aCamKII phosphory-lation at early time points in vitro may also suppress theinduction of LTD. Together, these time-dependent changes inprotein phosphorylation suggest that the induction of LTDmight grow stronger with time in vitro as Src activationsubsides and basal levels of constitutively active CamKIIreturn to normal. Thus, to begin to explore whether synapticplasticity is influenced by time in vitro, we examinedwhether the ability of low-frequency trains of synaptic

(a)

(b)

(c)

Fig. 9 Changes in glutamate receptor 1 (GluR1), a calcium/calmo-

dulin-dependent kinase II (aCamKII), proline-rich tyrosine kinase 2

(Pyk2), and Src phosphorylation in slices maintained for an extended

period of time in vitro. (a) Phosphorylation of AMPA receptor GluR1

subunits at S845 (circles) and S831 (triangles) is significantly

(* p < 0.01, n ¼ 4) reduced even in slices maintained in vitro for up

to 8 h. Total GluR1 levels (diamonds) are unchanged. The inset

shows immunoblots from one experiment that were probed with

antibodies against GluR1 phosphorylated at S845 (pS845), GluR1

phosphorylated at S831 (pS831), and total GluR1. (b) Dephospho-

rylation of aCamKII at T286 recovers after 4 h in vitro. Phospho-

T286 aCamKII levels were significantly reduced in slices maintained

in vitro for 2 h but not in slices maintained in vitro for longer times

(*p < 0.05 compared to control, n ¼ 4). Total aCamKII levels were

unchanged. (c) The increase in phosphorylation of Pyk2 (triangles)

and Src family kinases (circles) shows partial recovery in slices

maintained in vitro for up to 8 h (n ¼ 4, *p < 0.01 compared to

control. In contrast, the decrease in total Pyk2 levels associated with

in vitro maintenance was persistent (diamonds, n ¼ 4, #p < 0.05

compared to control).

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� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 91, 1344–1357

stimulation to induce NMDA receptor-dependent LTDchanges as a function of time in vitro. As shown inFig. 10(a), in 12 control experiments using slices maintainedin vitro for 2–8 h, LFS induced significant LTD at commis-sural/associational fiber synapses onto pyramidal cells in thehippocampal CA3 region (fEPSPs were depressed to

75 ± 4% baseline). Consistent with previous results showingthat the induction of LTD at these synapses is NMDAreceptor-dependent (Debanne et al. 1998), LFS-induced LTDwas blocked in slices bathed in the NMDA receptorantagonist 2-amino-5-phosphonovaleric acid (APV) (fEPSPswere 96 ± 7% of baseline after LFS in the presence of100 lM D,L-APV, n ¼ 8). To examine whether time in vitroaffects the induction of LTD, we used the 12 controlexperiments shown in Fig. 10(a) and compared the averageamount of LTD induced by LFS in five slices maintainedin vitro for the shortest amount of time (2–2.5 h) to theaverage LTD induced by LFS in five slices maintainedin vitro for the longest period of time (5–8 h). As shown inFig. 10(b), LFS induced small, but statistically significant,LTD in slices maintained in vitro for 2–2.5 h (fEPSPs weredepressed to 85 ± 4% of the baseline; p < 0.05 compared topre-LFS baseline). In contrast, LFS induced significantlylarger LTD in slices that were maintained in vitro for 5–8 h(fEPSPs were depressed to 65 ± 4% of baseline, p < 0.01compared to the LTD induced in slices maintained in vitrofor 2–2.5 h). As a second measure of how time in vitroaffects LTD induction, we performed a linear regressionanalysis of the relationship between time in vitro and LTDmagnitude across all 12 control experiments (Fig. 10c).There was a strong correlation between time in vitro and themagnitude of LFS-induced LTD (correlation coefficient, r ¼0.783; p < 0.01). Thus, the amount of time that slices aremaintained in vitro has a strong influence on the induction ofNMDA receptor-dependent LTD.

Discussion

The acute brain slice preparation has had a central role instudies of synaptic plasticity such as LTP and LTD. Given theprimary role of protein phosphorylation in these forms ofplasticity (Sheng and Kim 2002), it is surprising that so littleis known about how phosphorylation of proteins involved inLTP and LTD might be altered in brain slices. To begin toaddress this issue, we examined the phosphorylation state oftwo proteins that are part of the core mechanisms responsiblefor regulating synaptic strength in NMDA receptor-depend-ent forms of plasticity (aCamKII and the GluR1 subunitof AMPARs) as well as four proteins in two important modu-latory pathways (the ERK pathway and Pyk2/Src signaling).To our surprise, phosphorylation of every protein we exami-ned was altered.

Our results indicate that time in vitro is an importantvariable that should be carefully controlled in studies usingbrain slices. For instance, soon after tissue slicing there is arobust activation of the ERK pathway that requires one ormore hours to return to basal levels. The Pyk2/Src pathway isalso strongly activated in slices and recovers with a muchslower time course. Moreover, the GluR1 subunit ofAMPARs is strongly dephosphorylated in hippocampal

Fig. 10 Low-frequency stimulation (LFS)-induced long-term depres-

sion (LTD) is more robust in slices maintained in vitro for longer

periods of time. (a) A long-train of LFS (900 pulses at 1 Hz) induces

NMDA receptor–dependent LTD at associational/commissural fiber

synapses onto pyramidal cells in the hippocampal CA3 region. In

control experiments, LFS (indicated by the bar) induced significant

LTD (filled symbols, p < 0.05 compared to pre-LFS baseline, n ¼ 12).

In experiments where slices were continuously bathed in ACSF con-

taining the NMDA receptor antagonist D,L-APV (100 lM) LFS stimu-

lation had no significant lasting effect on synaptic transmission (open

symbols, n ¼ 8). (b) Of the 12 control experiments in (a), relatively

small LTD was induced in slices maintained in vitro for 2–2.5 h (cir-

cles, n ¼ 5), whereas significantly larger LTD was induced by LFS in

slices maintained in vitro for 5–8 h (triangles, n ¼ 5, p < 0.01 com-

pared to LTD in slices maintained in vitro for 2–2.5 h). (c) The

magnitude of LTD induced by LFS is strongly correlated with time

in vitro. Individual results for all 12 control experiments summarized in

panel (a) are plotted as a function of time in vitro. The line shows the

results of linear regression analysis (correlation coefficient ¼ 0.783,

p < 0.01).

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� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 91, 1344–1357

slices maintained in vitro and levels of GluR1 phosphoryla-tion at both S845 and S831 show little recovery, even inslices maintained in vitro for 8 h. Thus, for several hoursafter slicing, protein phosphorylation in hippocampal slices isin a highly altered and constantly changing state that couldinfluence cellular responses to various forms of stimulation.Indeed, we found that the induction of LTD by LFS wasstrongly dependent on time in vitro. Although the restorationof basal levels of T286-phosphorylated aCamKII and theslow decline in Pyk2/Src activation are likely candidates,additional experiments will be needed to identify the specifictime-dependent changes responsible for the enhancement ofLTD in slices maintained in vitro for longer periods of time.Indeed, some of the changes in protein phosphorylationassociated with slicing and in vitro maintenance, such as therobust decrease in phosphorylation of GluR1 AMPARsubunits at S845, might be expected to occlude LTD (Leeet al. 2000). The change in LTD induction as a function oftime in vitro is consistent, however, with the notion that thealterations in protein phosphorylation that occur in hippo-campal slices can have functionally significant effects onsynaptic plasticity.

Most of the changes in protein phosphorylation that occurin slices maintained in interface chambers at 30�C alsooccurred in slices maintained in submerged chambers at roomtemperature. This suggests that the changes in proteinphosphorylation that occur in slices are a general responseto tissue slicing and in vitro maintenance. It is interesting,however, that phosphorylation of aCamKII at T286 wastransiently enhanced in submerged slices but decreased ininterface slices. This suggests that phosphorylation of someproteins can be strongly influenced by the method and/ortemperature used to maintained slices in vitro. In addition,although total GluR1 levels did not change in any of ourexperiments, a previous study found that total GluR1 levelsare strongly reduced in hippocampal slices maintained insubmerged conditions at physiological temperatures (Tau-benfeld et al. 2002). Together with this finding, our resultsindicate that different methods commonly used to maintainslices in vitro can produce slices with different biochemicalproperties. Because NMDA receptor-dependent forms ofsynaptic plasticity arise from a highly interconnected networkof protein kinase and protein phosphatase signaling pathways,alterations in some components of this network produced byslicing and in vitro maintenance could influence the role ofother components of the network. For instance, the differentbasal levels of aCamKII phosphorylation in slices maintainedin submerged vs. interface conditions could influence the needfor activation of other kinases, such as PKA (Blitzer et al.1998; Makhinson et al. 1999) and ERK (Giovannini et al.2001), that can facilitate LTP induction by enhancing CamKIIactivation. Thus, our results indicate that the method of slicemaintenance is an important variable that might influence theapparent need for some signaling pathways in LTP.

In addition to the methodological implications discussedabove, our findings also have broader, more conceptualimplications for areas of research that use brain slices. Forexample, brain slices are often used in experiments wheregenetic approaches are used to study the molecular mech-anisms underlying LTP and LTD or to examine therelationship between LTP and behavioral learning. One issueraised by our findings is that changes in signaling pathwaysinduced by genetic mutations might interact in subtle, yetfunctionally significant ways with changes in protein phos-phorylation associated with tissue slicing and in vitromaintenance. Alterations in LTP and LTD produced bygenetic mutations thus might be enhanced, masked, or onlybecome apparent with time in vitro. Thus, the alterations inprotein phosphorylation that occur in hippocampal slicesneed to be carefully considered in genetic studies attemptingto relate in vitro plasticity to learning.

Our observations also have important implications forstudies that attempt to use brain slices to search for changesin neuronal and synaptic physiology that occurred in vivo.For example, previous studies have found that synaptictransmission is enhanced (McKernan and Shinnick-Gallagher1997) and LTP is reduced (Tsvetkov et al. 2002) in amygdalaslices obtained from animals that have undergone fearconditioning. The induction of LTP is also partially occludedin slices of piriform cortex obtained from animals trained inan olfactory discrimination task (Lebel et al. 2001). Simi-larly, monocular visual deprivation is associated with dep-hosphorylation of GluR1 subunits at S845 and a LTD-likedepression of synaptic responses in visual cortex thatpartially occludes LFS-induced LTD in vitro (Heynen et al.2003). Findings such as these are often interpreted asdemonstrating that experience induces changes in synapticfunction through mechanisms similar to those involved inLTP and LTD in vitro. The changes in protein phosphory-lation observed in our experiments are so large, however, thatit is difficult to imagine that experience-induced changes thatare regulated by protein phosphorylation could survive theeffects of tissue slicing and in vitro maintenance. Instead, ourresults are more consistent with the notion that other cellularchanges, such as alterations in NMDA receptor subunitcomposition (Zinebi et al. 2003; Quinlan et al. 2004), areresponsible for the apparent occlusion of in vitro plasticity byin vivo manipulations.

Unfortunately, our experiments do not provide muchinformation regarding the mechanisms responsible foraltered protein phosphorylation in hippocampal slices.Slices are typically maintained well below physiologicaltemperature and neurons in slices are deprived of many oftheir normal inputs. Thus, spontaneous neuronal activity inslices is likely to be far below that seen in vivo. If activity-dependent protein kinase activation maintains normal levelsof GluR1 and aCamKII phosphorylation, then the dep-hosphorylation of GluR1 and aCamKII in hippocampal

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� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 91, 1344–1357

slices might be due to abnormally low levels of proteinkinase activity. In the case of the ERK pathway it seemsclear that tissue slicing, either alone or in combination witha short time period of in vitro recovery, leads to ERKactivation. Because NMDA receptor stimulation can acti-vate the ERK2 pathway (Bading and Greenberg 1991;Kurino et al. 1995), glutamate released during braindissection and tissue slicing (Feig and Lipton 1990) mightbe responsible for ERK2 activation in slices. NMDAreceptor-dependent activation of protein phosphatases dur-ing slicing and in vitro maintenance could also promotedephosphorylation of GluR1 and aCamKII (Morioka et al.1995; Winder and Sweatt 2001). Thus, although multiplemechanisms are likely to be involved, it would beinformative to examine whether blocking NMDA receptorsduring slicing and in vitro maintenance prevents thechanges in protein phosphorylation observed in our experi-ments.

In summary, our results demonstrate that protein phos-phorylation is dramatically altered in hippocampal slices.These changes are large, protein-specific, and in some casespersistent. It is important to note that our findings do notindicate that the acute brain slice preparation is poorly suitedfor studies of phosphorylation-dependent physiological pro-cesses. This preparation was proven to be invaluable in manyareas of research and has provided crucial insights in cellularand molecular processes involved in brain development,synaptic transmission and plasticity, neurotransmitter actions,and intracellular signaling. Our results do indicate, however,that a more complete characterization of how proteinphosphorylation is altered in acute brain slices and a betterunderstanding of how these changes might influence cellularand synaptic physiology in slices is warranted.

Acknowledgements

This work was supported by a grant from the NIH (MH60919 to

TJO). JYD was supported by a MFP Fellowship in Neuroscience

from the American Psychological Association. We are grateful to

members of the UCLA Learning and Memory Project for helpful

discussion and to Pato Opazo and Joseph Watson for comments on

this manuscript.

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