Homer 1a uncouples metabotropic glutamatereceptor 5 from postsynaptic effectorsPaul J. Kammermeier*† and Paul F. Worley‡

*Department of Pharmacology and Physiology, University of Rochester Medical Center, 601 Elmwood Avenue, Box 711, Rochester, NY 14642; and‡Department of Neuroscience, Johns Hopkins School of Medicine, 905 Huntarian Building, 725 North Wolfe Street, Baltimore, MD 21205

Edited by Roger A. Nicoll, University of California, San Francisco, CA, and approved February 12, 2007 (received for review October 11, 2006)

Metabotropic glutamate receptors (mGluRs) and Homer proteinsplay critical roles in neuronal functions including plasticity, noci-ception, epilepsy, and drug addiction. Furthermore, Homer pro-teins regulate mGluR1/5 function by acting as adapters and facil-itating coupling to effectors such as the inositol triphosphatereceptor. However, although Homer proteins and their interactionwith mGluRs have been the subject of intense study, direct mea-surements of Homer-induced changes in postsynaptic mGluR–effector coupling have not been reported. This question wasaddressed here by examining glutamatergic excitatory postsynap-tic currents (EPSCs) in rat autaptic hippocampal cultures. In mostneurons, the group I mGluR agonist (S)-3,5-dihydroxyphenylgly-cine strongly inhibited the EPSC acutely. This modulation occurredpostsynaptically, was mediated primarily by mGluR5, and wasinositol triphosphate receptor-dependent. Expression of the dom-inant negative, immediate early form of Homer, Homer 1a,strongly reduced EPSC modulation, but the W24A mutant of Homer1a, which cannot bind mGluRs, had no effect. (S)-3,5-dihydroxy-phenylglycine-mediated intracellular calcium responses in the pro-cesses of Homer 1a-expressing neurons were reduced comparedwith those in Homer 1a W24A-expressing cells. However, neitherthe distribution of mGluR5 nor the modulation of somatic calciumchannels was altered by Homer 1a expression. These data dem-onstrate that Homer 1a can reduce mGluR5 coupling to postsyn-aptic effectors without relying on large changes in the subcellulardistribution of the receptor. Thus, alteration of mGluR signaling bychanges in Homer protein expression may represent a viablemechanism for fine-tuning synaptic strength in neurons.

autapse � calcium channel � excitatory postsynaptic current � hippocampal

Metabotropic glutamate receptors (mGluRs) are class 3 Gprotein-coupled receptors expressed throughout the

brain. Group I mGluRs 1 and 5 couple to the phospholipaseC/calcium release cascade and to pathways regulating ion chan-nels and synaptic currents (1, 2). In the brain, group I mGluRsare often expressed postsynaptically, where they regulateNMDA and AMPA receptor function and mediate multipleforms of plasticity (3).

The Homer family of postsynaptic scaffolding proteins wasdiscovered by the regulated expression of the member subse-quently termed Homer 1a (4). Levels of Homer 1a and variantAnia-3 are increased after periods of activity or stress, injury, ornovel experience (4, 5). Other ‘‘long’’ Homer proteins (1b, 1c, 2,and 3) are expressed constitutively (6–8) and, unlike Homer 1a,form clusters mediated by their long C termini. Homer proteinsorganize postsynaptic proteins around the active site (7) bybinding several targets including group I mGluRs, inositoltriphosphate receptors (IP3Rs), Shank, and the TRPC1 cationchannel (9–12).

Homer proteins also influence the function of their bindingpartners. Binding to Homers can alter the function of TRPC1(12), and group I mGluRs couple more strongly to someeffectors (e.g., ion channels) when bound to Homer 1a (13) andmore strongly to others, such as IP3Rs (8), when bound to longHomer proteins. Thus, Homer proteins regulate localization of

binding partners (13–18) and assemble groups of molecules intoputative signaling microdomains (8, 13). However, no study todate has demonstrated the functional consequences of Homerexpression on receptor–effector coupling for mGluRs located atthe postsynaptic site in mammalian neurons.

In the present study, acute modulation of excitatory postsyn-aptic currents (EPSCs) by mGluR5 was examined in autaptichippocampal neurons in the absence and presence of Homer 1aexpression to determine its effect on mGluR signaling at thepostsynapse, where critical functions of mGluRs are most relevant.

ResultsEPSCs in Hippocampal Autapses. To examine group I mGluRsignaling to effectors at the synapse, hippocampal neurons weregrown as autapses (19–21) in which single neurons on a substrate‘‘microisland’’ grow and form synapses onto themselves. Fig. 1Ashows an autaptic hippocampal neuron. When autaptic cellswere patch-clamped in the whole-cell configuration, EPSCs weremeasured by stepping to �40 mV for 2 msec to trigger an actionpotential volley in the distal, unclamped processes, resulting insynchronized neurotransmitter release and a measurablepostsynaptic current (Fig. 1B, EPSC).

Although some apparent inhibitory neurons were observed(excluded from analysis), indicated by a slow inhibitory current(22), most exhibited EPSCs that were sensitive to the nonselec-tive ionotropic glutamate receptor (iGluR) inhibitor kynurenicacid (Fig. 1C), indicating that these neurons were glutamatergic(23). These EPSCs were inhibited 47 � 7% by 10 �M kynurenicacid (average � SEM; n � 6) (Fig. 1 C and D). EPSCs were rapid,reaching peak in �10 msec. The non-NMDA iGluR inhibitor6-cyano-7-nitroquinoxaline-2,3-dione (20 �M) reduced theEPSC by 67 � 16% (median inhibition was 81%; n � 10),suggesting that the EPSC was primarily non-NMDA, as expectedbecause currents were recorded at �80 mV, and with 1.2 mMMg2� in the bath to inhibit currents through NMDA channels at�80 mV. These data indicate that autaptic hippocampal neuronswere predominantly excitatory glutamatergic neurons.

EPSC Modulation by Group I mGluRs. Application of the group ImGluR-selective agonist (S)-3,5-dihydroxyphenylglycine(DHPG) to autapses for 30–50 sec produced a reversible inhi-

Author contributions: P.J.K. and P.F.W. designed research; P.J.K. performed research; P.J.K.and P.F.W. contributed new reagents/analytic tools; P.J.K. analyzed data; and P.J.K. andP.F.W. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Abbreviations: mGluR, metabotropic glutamate receptor; iGluR, ionotropic glutamatereceptor; IP3R, inositol triphosphate receptor; DHPG, (S)-3,5-dihydroxyphenylglycine; HEP,low-molecular-weight heparin; EPSC, excitatory postsynaptic current; IRES, internal ribo-some entry sequence.

†To whom correspondence should be addressed. E-mail: paul�kammermeier@urmc.rochester.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0608991104/DC1.

© 2007 by The National Academy of Sciences of the USA

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bition of the EPSC (Fig. 2A). This effect was variable, rangingfrom undetectable to near total inhibition. Overall, 50 �MDHPG produced a 51 � 6% inhibition (n � 23) of the EPSC. Adose–response curve (n � 7) demonstrated that the half-maximal DHPG dose was 3.9 �M, consistent with a group ImGluR effect (Fig. 2B). Because of variability and run-down ofthe current in some cells, apparent effects less than �15% werein fact negligible.

In addition to the DHPG effect, the mGluR5 selective agonist(RS)-2-chloro-5-hydroxyphenylglycine inhibited the EPSC sim-ilarly (54 � 8%, n � 7; 1 mM). Furthermore, the specificmGluR1 antagonist (S)-(�)-�-amino-4-carboxy-2-methylbenze-neacetic acid (LY367385) did not alter the DHPG effect. Incontrast, the specific mGluR5 antagonist 2-methyl-6-(phenyl-ethynyl) pyridine (MPEP) reduced the effect of DHPG [sup-porting information (SI) Fig. 9 A and B]. This experiment wasperformed by applying 50 �M DHPG, then DHPG in thepresence of 50 �M LY367385 or 30–300 nM MPEP (or bothseparately), followed by DHPG alone again (if the cells sur-vived). The first DHPG application resulted in a 70 � 7%inhibition (n � 10). In the presence of LY367385, DHPGinhibition was 61 � 14% (n � 7), and, with MPEP, DHPGproduced only a 25 � 9% inhibition (n � 8). The final applicationof DHPG alone inhibited the EPSC by 72 � 12% (n � 3) (SI Fig.9). These data demonstrate that the predominant group ImGluR in hippocampal autapses is mGluR5 (23, 24).

mGluR5 Acts Postsynaptically. To determine whether the EPSCmodulation by mGluR5 arose pre- or postsynaptically, the ratioof EPSC amplitudes using a paired-pulse protocol was examined.Evoking two EPSCs with a 50-msec interval produces pairedEPSCs (Fig. 3) with the second current ranging in relative sizefrom depression to facilitation. This ratio (P2/P1), influenced byfactors including extracellular Mg2� and Ca2�, and the size of thereadily releasable pool of transmitter (25, 26), is a function oftransmitter release. Therefore, only a presynaptic modulatorymechanism would normally alter the ratio.

Paired EPSCs were examined before and during DHPGinhibition, and the P2/P1 ratio was examined. Fig. 3A showssample currents from such a cell. Although the EPSC wasstrongly reduced by DHPG, the P2/P1 ratio was unchanged. Onaverage, P2/P1 was 0.82 � 0.07 and 0.83 � 0.13 (n � 23) beforeand during DHPG inhibition, respectively (Fig. 3B). By contrast,EPSC inhibition by group III mGluRs, which act predominantlypresynaptically (2), with 300 �M L-AP4, increased P2/P1 from0.53 � 0.08 to 0.75 � 0.11 (n � 6). Finally, direct block ofpostsynaptic iGluRs with kynurenic acid left P2/P1 unchanged(0.60 � 0.12 in control and 0.61 � 0.10 in kynurenic acid)(Fig. 3B). These data demonstrate that DHPG-mediated inhi-bition was at least largely postsynaptic, consistent with previousreports (27).

Homer 1a Uncouples Postsynaptic mGluR5 from the EPSC. The longHomer proteins (Homer 1b, 1c, 2, and 3) are constitutivelyexpressed in hippocampal neurons (4). Homer 1a, a dominantnegative subtype, is expressed at high levels after neuronal insultor stress and less severe activity such as that during novelexperience (5). To assess its role in regulating mGluR5 couplingto effectors at the postsynapse, Homer 1a was expressed inautapses by using the Sindbis viral expression system (28–30).Fig. 4B illustrates a fluorescence image of an autaptic neuroninfected with Sindbis encoding GFP and, after an internalribosome entry sequence (IRES), Homer 1a.

Cells expressing Homer 1a (Homer 1a IRES GFP or GFPIRES Homer 1a) showed reduced EPSC inhibition by DHPG(Fig. 4A). Uninfected cells were inhibited 52 � 7% (n � 17,median � 52%) by 50 �M DHPG, whereas those expressingHomer 1a showed 8 � 9% inhibition (n � 8; median � 18%).By contrast, EPSC inhibition by presynaptic group III mGluRsusing the selective agonist L-AP4 was unaltered by Homer 1aexpression, as was EPSC modulation by the muscarinic acetyl-choline receptor agonist oxotremorine-M (SI Fig. 10). Thesedata demonstrate that Homer 1a expression selectively uncou-ples mGluR5 from effector targets in the postsynaptic density.

To ensure that the uncoupling observed with Homer 1a wasnot the result of protein overexpression or the indirect result ofSindbis virus infection, a W24A point mutant of Homer 1a,which cannot bind mGluRs (8, 31, 32), was expressed in hip-

Fig. 1. EPSCs in hippocampal autapses. (A) Phase contrast image of ahippocampal neuron grown in autaptic culture for 7 days. (B) Sample EPSCtrace illustrating the voltage protocol used to evoke an action potential volleyfrom a holding potential of �80 mV (above), the action current, and the EPSC(as indicated). (C) Sensitivity of the EPSC to 10 �M kynurenic acid. Samplecurrents before (Con), during (KYN), and after (Wash) application of KYN areshown. (D) Average � SEM inhibition by KYN and 20 �M CNQX (6-cyano-7-nitroquinoxaline-2,3-dione). The number of cells in each group is indicated inparentheses.

Fig. 2. The hippocampal autaptic EPSC is modulated by mGluR5. (A) Sensi-tivity of the EPSC to 50 �M DHPG. Sample currents before (Control), during (50�M DHPG), and after (Wash) application of DHPG are shown. (B) Dose–response relationship for DHPG inhibition of the EPSC. The estimated half-maximal effective concentration (EC50) was 3.9 �M.

Fig. 3. Modulation of the EPSC by DHPG occurs postsynaptically. (A) Samplepaired-pulse current traces illustrating the paired-pulse ratio in uninhibited(Con) and DHPG-inhibited currents. (B) Average � SEM P2/P1 ratio before(open bar) and during (filled bar) EPSC inhibition by the indicated drug.

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pocampal autapses by using the Sindbis system (Fig. 4 C and D).Homer 1a W24A had no effect on DHPG-mediated EPSCmodulation. The EPSC was inhibited 49 � 12% (n � 6;median � 43%) and 58 � 8% (n � 8; median � 57%) inuninfected and Homer 1a W24A-expressing cells, respectively.These data demonstrate that Homer 1a dramatically reducescoupling of mGluR5 to postsynaptic targets.

Modulation of the EPSC by mGluR5 Requires IP3R Activation. Theprecise mechanism of this EPSC inhibition by mGluR5 is notknown. Acute iGluR modulation via group I mGluRs maydepend on second messengers in the Gq/phospholipase C path-way (33–35). To better understand the Homer-dependent effectsdescribed above, it was important to determine whether themechanism of modulation required activation of the IP3R,because Homer proteins bind the IP3R and regulate mGluRcoupling to it (36). Thus, sensitivity of EPSC modulation byDHPG to low-molecular-weight heparin (HEP), an IP3R inhib-itor, was examined. Fig. 5A shows EPSC traces from a controlautaptic neuron (Fig. 5A Left) and one in which 100 �g/ml HEPwas included in the patch pipette (Fig. 5A Right). EPSC modu-lation by DHPG was inhibited by HEP, reducing the inhibitionfrom 44 � 10% (n � 7; median 48%) to 15 � 7% (n � 7; median10%) in neurons with intracellular HEP. In each cell, DHPG wasapplied at least 2 min after achieving whole-cell mode to allowfor HEP dialysis into cells. However, inclusion of 3 �M bisin-dolylmaleimide, a protein kinase C inhibitor, in the pipette didnot alter DHPG-mediated EPSC modulation (Fig. 5B). Thesedata suggest that mGluR5-mediated EPSC inhibition in hip-pocampal autapses requires activation of the IP3R, but not

activation of PKC. Therefore, overexpression of Homer 1a,which can disrupt mGluR5 association with the IP3R, uncouplespostsynaptic mGluR5 from EPSC modulation, perhaps by pre-venting efficient signaling through IP3Rs (37).

Homer 1a Expression Reduces the mGluR5-Mediated Rise in [Ca2�]i.The data above demonstrate that Homer 1a expression reducesIP3R-dependent EPSC modulation by postsynaptic mGluR5. Alikely mechanism for this uncoupling is disruption of efficientsignaling between mGluR5 and the IP3R (7), both of which bindthe EVH1 domain of Homer proteins and indirectly interact inthe presence of long Homer proteins. This model predicts that

Fig. 4. Expression of Homer 1a uncouples mGluR5 from EPSC modulation.(A) Scatter plot illustrating the maximal inhibition by DHPG of the EPSC in eachuninfected (Con; filled circles) and Homer 1a-expressing (H1a; open circles;neurons expressing either Homer 1a IRES GFP and GFP IRES Homer 1 wereused) autaptic neuron. The mean (heavy black line) and median (gray line)inhibition for each group are also shown. The Inset shows DHPG inhibition ina sample Homer 1a-expressing neuron. (B) Epifluorescence image showingGFP expression in one GFP IRES Homer 1a-expressing neuron. (C) Scatter plotillustrating the maximal inhibition by DHPG of the EPSC in each uninfected(Con; filled squares) and Homer 1a W24A-expressing (W24A; open squares)autaptic neuron. The mean (black lines) and median (gray lines) inhibition foreach group are also shown. (D) Epifluorescence image showing GFP expressionin one Homer 1a W24A IRES GFP-expressing neuron.

Fig. 5. Modulation of the EPSC by DHPG requires the IP3R but not PKC. (A)Sample current traces showing DHPG modulation of the autaptic EPSC incontrol cells (Left) and cells recorded in the presence of 100 �g/ml intracellularlow-molecular-weight heparin (Right). (B) Scatter plot illustrating the maxi-mal EPSC inhibition in each cell by 50 �M DHPG in control cells (open circles),cells recorded with intracellular heparin (filled circles), or 3 �M intracellularbisindolylmaleimide (filled squares). The mean (black lines) and median (graylines) inhibition for each group are also shown.

Fig. 6. Homer 1a reduces mGluR5-mediated rise in intracellular calcium. (A)Homer 1a expression reduces the mGluR5-induced intracellular calcium signalin the processes of hippocampal neurons. Ratio of Fura 2 fluorescence(340:380 nm excitation) time course (average � SEM) for hippocampal neu-rons expressing either Homer 1a W24A (gray) or Homer 1a (black). Data werebaseline-subtracted from the average of all points 10 sec prior to DHPGapplication. The data include measurements from 61 points on the processesof six infected neurons and 59 points on the processes of five infected neuronsin Homer 1a W24A and Homer 1a-expressing cells, respectively. (B) Peakcalcium responses minus baseline in response to 50 �M DHPG and 10 �Moxo-M, as indicated in dSRed-transfected neurons (filled bars) and neuronstransfected with dSRed plus Homer 1a (open bars). Calcium measurementswere obtained as in A.

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expression of Homer 1a will weaken the mGluR5-dependent risein intracellular calcium in hippocampal neurons, which expresslong Homer proteins constitutively. To measure this directly,intracellular calcium levels in the processes of cultured hip-pocampal neurons expressing either Homer 1a IRES GFP orHomer 1a W24A IRES GFP were monitored with Fura2 duringDHPG application (Fig. 6). Fig. 6A shows baseline-subtractedcalcium levels from several regions of interest on resolvedprocesses of infected neurons. The peak of the calcium responsewas smaller in neurons expressing Homer 1a than in thoseexpressing the W24A point mutant. Neurons expressing theW24A point mutant showed an �50% greater peak response toDHPG than those expressing wild-type Homer 1a, and theenhanced response persisted for several seconds after peak.Slowing of the peak of the calcium response was not detected.

Similar experiments were also carried out by using neuronstransfected with Homer 1a using electroporation and coexpress-ing the fluorescent protein dSRed, because GFP can interferewith detection of changes in Fura fluorescence. These data (Fig.6B) show a similar impairment of the DHPG-induced calciumsignal, consistent with the Sindbis expression data. Furthermore,Homer 1a expression in these same cells failed to reduce calciumsignaling in response to oxotremorine-M, a muscarinic acetyl-choline receptor agonist, confirming that the reduction in sig-naling observed with DHPG was not due to a general reductionin IP3R activity. Together, these data are consistent with dataobtained from cerebellar Purkinje neurons expressing eitherHomer 1a or Homer 1b in a previous study (8) and demonstratethat Homer 1a expression selectively disrupts efficient couplingbetween mGluR5 and IP3Rs in the processes of cultured hip-pocampal neurons.

Effect of Homer 1a on mGluR5 Distribution. Expression of short vs.long Homer proteins may alter the subcellular distribution ofmGluR5 in central neurons (14, 31, 37, 38), which may explainthe data observed here. Expression of Homer 1a may lead tolocalization of mGluR5 at the soma or other regions away fromthe postsynaptic site, thus uncoupling the receptor from EPSC

modulation. To determine whether such a change in mGluR5distribution occurred here, the mGluR5 was examined by usingimmunofluorescence.

Fig. 7 illustrates a field of mGluR5-labeled (Alexa Fluor 555,in red) uninfected neurons (Fig. 7A) and a neuron exposed to theSindbis GFP IRES Homer 1a construct (Fig. 7B). Similarcultures exposed to the fluorescently conjugated secondaryantibody without prior exposure to the primary anti-mGluRantibody showed no detectable red fluorescence (data notshown). Most, but not all, neurons appear to be mGluR5-positive, consistent with the observation that most neuronsresponded to DHPG in patch-clamp experiments (Figs. 2–5).Second, mGluR5 staining was apparent on the soma and pro-cesses of both uninfected and Homer 1a-expressing neurons(Fig. 7B and SI Fig. 11). Indeed, no qualitative change indistribution was detectable between these two groups. Thus,although mGluR5 may be selectively excluded from associationwith the postsynaptic density in Homer 1a-expressing neurons,the data in Fig. 7 do not support a mechanism in whichuncoupling of mGluR5 from postsynaptic targets results fromexclusion from the dendritic compartment.

Homer 1a Does Not Affect Coupling to Somatic Calcium Channels.Recent work has shown clear differences in mGluR5 distributionwhen various Homer proteins are expressed (14). Data in Fig. 7suggest that, under the conditions used here, a dramatic rear-rangement of mGluR5 is not observed. Coupling of somaticmGluR5 to voltage-dependent calcium channels was thereforeexamined in 7- to 9-day-old hippocampal cultures. To avoid thetechnical (space clamp) problems of voltage clamping neuronswith processes, current measurements were made from outside-out patches pulled from the soma of hippocampal neurons.Barium (25 mM) was used as the charge carrier to enhancecurrents through high-voltage activated calcium channels.

Barium currents recorded in patches from uninfected cellswere reversibly inhibited 73 � 7% (n � 4) by 50 �M DHPG (Fig.8), confirming that mGluR5 resides on the cell soma in theabsence of Homer 1a expression. In contrast to mGluR5-mediated modulation of EPSCs, calcium channel modulation bysomatic mGluR5 was unaffected by Homer 1a expression. Inpatches from neurons expressing Homer 1a, barium currentswere inhibited 68 � 16% (n � 5), similar to control, suggesting

Fig. 7. Expression of Homer 1a in hippocampal neurons does not detectablyalter the distribution of mGluR5. (A) GFP-fluorescence image of a GFP-infectedhippocampal neuron (Left) and AF555 fluorescence indicating mGluR5 ex-pression (Right) from the same field. (B) GFP-fluorescence image of a Homer1a IRES GFP-infected hippocampal neuron (Left) and AF555 fluorescenceindicating mGluR5 expression (Right) from the same field. Both images illus-trate mGluR5 expression in the cell body and processes of these representativecells. (C) Control fluorescence images illustrating the lack of signal in the GFPchannel in an uninfected, mGluR5-positive cell (Left) and the lack of AF555fluorescence in a GFP-transfected, mGluR5-negative neuron (Right), confirm-ing the lack of bleed-over across the two channels.

Fig. 8. Expression of Homer 1a does not alter DHPG-mediated modulationof currents through somatic calcium channels. (A) Sample current traces fromthe indicated voltage protocol (Inset) illustrating DHPG modulation of thebarium currents from somatic outside-out patches in uninfected (Upper) andHomer 1a-expressing (Lower) hippocampal neurons. (B) Average � SEM inhi-bition of the somatic, outside-out patch barium current in uninfected andHomer 1a-expressing neurons, as indicated. Inhibitions were calculated frompeak currents during a voltage ramp from �80 to �80 mV (see Methods). Thenumber of cells in each group is indicated in parentheses.

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that a large-scale redistribution of mGluR5 does not occur afterHomer 1a expression, at least not during the time scale andwithin the parameters of this study. Thus, Homer 1a expressioncan alter the coupling of mGluR5 to certain effectors, such asthose at the postsynaptic density, by disrupting efficient signalingdomains that are constitutively assembled when long Homerproteins are predominant. This change in coupling need not relyon a large-scale redistribution of mGluR5.

DiscussionThis study provides the first demonstration of Homer-dependentregulation of postsynaptic mGluR5–effector coupling in mam-malian neurons. EPSC modulation in hippocampal autapses wasreduced in cells expressing Homer 1a, but not Homer 1a W24A,a mutant that cannot bind mGluRs. Furthermore, the effect ofHomer 1a did not appear because of dramatic changes insubcellular distribution of mGluR5. These data suggest thatHomer 1a induces a subtle molecular rearrangement to reducecoupling to effectors at the postsynapse, such as the IP3R, but notthose at the soma, such as voltage-dependent channels.

Ango et al. (14) examined the effect of Homer expression onmGluR5 distribution in neurons. In that study, mGluR5 was seenonly in the soma when expressed alone. Coexpression of Homer1b/c induced dendritic expression of mGluR5 whereas Homer 1awas associated with global mGluR5 distribution. Thus, effects inthe present study may be due to relocation of mGluR5 away fromthe postsynapse. However, a recent study showed that overex-pression of Shank 1B enhanced mGluR coupling to BK channelsin hippocampal neurons, although expression of Homer 1b andShank did not alter dendritic mGluR5 localization (38). Thosedata, obtained from culture conditions similar to this study,suggest that changes in mGluR5 coupling more likely result fromrearrangement of the postsynaptic environment than frommovement of mGluR5 from the synaptic site. This may occur inpart because of inclusion or exclusion of IP3Rs from the synapse(38). Data in the present study show that Homer 1a can alsoinfluence mGluR coupling to postsynaptic iGluRs. The strengthof this pathway may also depend on proximity of IP3Rs and theendoplasmic reticulum, because an IP3R-dependent mechanismis likely (33–35, 39), and EPSC modulation in hippocampalautapses appears to require IP3R activation (Fig. 5), althoughiGluR modulation via group I mGluRs by an apparent direct Gprotein mechanism has been observed in cortical neurons (40).

Hippocampal neurons constitutively express long Homer pro-teins (4), which are associated with weakened coupling tomembrane calcium channels (13). So it was surprising thatHomer 1a did not enhance coupling of somatic mGluR5 tocalcium channels. It was also surprising that, in uninfected cells,mGluR5-mediated calcium channel modulation was strong (seeFig. 8). Group I mGluR agonists have been reported ineffectiveat modulating calcium currents in hippocampal neurons (27). Inour hands, acutely isolated hippocampal neurons exhibited nocalcium current inhibition by DHPG (data not shown), butmodulation was seen in neurons 7–9 days in culture (Fig. 8). Itis unclear why we saw strong coupling to calcium channels but

they did not (27). Nonetheless, in our hands, mGluR5/calciumchannel coupling suggests that, although long Homer proteinsare constitutively expressed, they may be restricted to thedendritic compartment and/or the postsynaptic region (37), suchthat somatic mGluRs may be unassociated with Homer proteins.Thus, overexpression of Homer 1a would not alter this coupling.

Homer proteins and mGluRs are implicated in processes suchas synaptic plasticity (41), addiction (42, 43), nociception (44),schizophrenia (45), and epilepsy (46, 47). In each case, regula-tion of synaptic strength via Homer expression appears to be acritical regulator. The data above show that regulation ofpostsynaptic mGluR signaling is an important aspect of Homer1a-dependent regulation of synaptic strength. Furthermore,Homer 1a expression alters mGluR signaling even when large-scale changes in mGluR distribution are not seen, suggesting thatthis mechanism may be used to fine-tune synaptic strength on ashort-term basis, rather than the more protracted time requiredto redistribute mGluRs between cellular compartments.

MethodsHippocampal Neuronal Cultures. Hippocampal autapses were gen-erated similar to that described previously (19). Detailed meth-ods on culturing and transfection are contained in SI Text.

Electrophysiology, Calcium Imaging, and Data Analysis. Patch-clampand calcium imaging experiments were performed by usingstandard methods (see ref. 13) and are described in detail in SIText.

The bath solution for EPSC recording had 140 mM NaCl, 5.4mM KCl, 10 mM 4-Hepes, 1.2 mM MgCl2, 1.2 mM CaCl2, and15 mM glucose. The bath solution for outside-out patches had140 mM Tris hydroxymethyl aminomethane, 20 mM Hepes, 10mM glucose, 25 mM BaCl2, and 0.0003 mM tetrodotoxin to pH7.4 with methanesulfonic acid. The pipette solution for EPSCrecording had 130 mM KCl, 1.1 mM EGTA, 10 mM Hepes, 0.1mM CaCl2, 4 mM MgATP, and 0.1 mM NaGTP (pH 7.2). Thepipette solution for barium currents had 120 mM N-methyl-D-glucamine, 20 mM TEA methanesulfonic acid, 11 mM EGTA, 10mM Hepes, 10 mM sucrose, 1 mM CaCl2, 4 mM MgATP, 0.3 mMNa2GTP, and 14 mM Tris creatine phosphate (pH 7.2).

Immunofluorescence. Neurons were fixed in 4% paraformalde-hyde/4% sucrose PBS, permeabilized in 0.05% Triton X-100,then exposed to rabbit polyclonal anti-mGluR5 (ChemiconInternational, Temecula, CA) antibody in PBS with 1% BSA for25 min, washed in PBS, exposed to an Alexa Fluor 555 goatanti-rabbit secondary antibody (Invitrogen, Carlsbad, CA) for 30min, and washed in PBS before imaging.

We thank D. I. Yule and L. E. Wagner (University of Rochester) for helpwith the calcium measurement experiments, S. Ikeda and H. Chen(National Institute on Alcohol Abuse and Alcoholism) for advice onhippocampal culture techniques, and T. B. Begenisich (University ofRochester) for providing HEP in a pinch. This work was supported bya grant from the Epilepsy Foundation of America (to P.J.K.).

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