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ACUTE AND CHRONIC COCAINE DIFFERENTIALLY ALTER THESUBCELLULAR DISTRIBUTION OF AMPA GluR1 SUBUNITS INREGION-SPECIFIC NEURONS WITHIN THE MOUSE VENTRALTEGMENTAL AREA

D. A. LANE,a* A. JAFERI,a M. J. KREEKb ANDV. M. PICKELa,b

aDepartment of Neurology and Neuroscience, Weill Medical College of

Cornell University, New York, NY 10065, USA

bLaboratory of the Biology of Addictive Diseases, The Rockefeller

University, New York, NY 10065, USA

AbstractCocaine administration increases AMPA GluR1 ex-pression and receptor-mediated activation of the ventral teg-

mental area (VTA). Functionality is determined, however, by

surface availability of these receptors in transmitter- and

VTA-region-specific neurons, which may also be affected by

cocaine. To test this hypothesis, we used electron micro-

scopic immunolabeling of AMPA GluR1 subunits and ty-

rosine hydroxylase (TH), the enzyme needed for dopamine

synthesis, in the cortical-associated parabrachial (PB) and in

the limbic-associated paranigral (PN) VTA of adult male

C57BL/6 mice receiving either a single injection (acute) or

repeated escalating-doses for 14 days (chronic) of cocaine.

Acute cocaine resulted in opposing VTA-region-specific

changes in TH-containing dopaminergic dendrites. TH-la-

beled dendrites within the PB VTA showed increased cyto-

plasmic GluR1 immunogold particle density consistent withdecreased AMPA receptor-mediated glutamatergic transmis-

sion. Conversely, TH-labeled dendrites within the PN VTA

showed greater surface expression of GluR1 with in-

creases in both synaptic and plasmalemmal GluR1 immu-

nogold density after a single injection of cocaine. These

changes diminished in both VTA subregions after chronic

cocaine administration. In contrast, non-TH-containing, pre-

sumably GABAergic dendrites showed VTA-region-specific

changes only after repeated cocaine administration such that

synaptic GluR1 decreased in the PB, but increased in the PN

VTA. Taken together, these findings provide ultrastructural

evidence suggesting that chronic cocaine not only reverses

the respective depression and facilitation of mesocortical

(PB) and mesolimbic (PN) dopaminergic neurons elicited by

acute cocaine, but also differentially affects synaptic avail-ability of these receptors in non-dopaminergic neurons of

each region. These adaptations may contribute to increased

cocaine seeking/relapse and decreased reward that is re-

ported with chronic cocaine use. 2010 IBRO. Published by

Elsevier Ltd. All rights reserved.

Key words: synaptic plasticity, dopamine, tyrosine hydroxy-lase, ultrastructure, reward.

Dopaminergic neurons originating in the ventral tegmentalarea (VTA) play a critical role in the rewarding effects ofcocaine and other drugs of abuse (Koob and Bloom, 1988;Le Moal and Simon, 1991). Changes in alpha-amino-3-

hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) recep-tor-mediated glutamatergic activation of dopaminergicneurons are thought to contribute to the development ofaddiction (Kalivas and Stewart, 1991; Wolf, 2003; Kesselsand Malinow, 2009). Stimuli that induce long term poten-tiation (LTP) increase the number of functional AMPA re-ceptors at the synapse (Liao et al., 1995; Malinow andMalenka, 2002; Park et al., 2004). Additionally, LTP can beelicited from neurons within the VTA (Bonci and Malenka,1999) suggesting that AMPA receptor-mediated plasticityin the VTA may be important for the development of ad-diction to cocaine. This is supported by findings that bothacute and chronic systemic cocaine administration in-crease AMPA-mediated glutamatergic activation of dopa-

minergic neurons in the VTA (Ungless et al., 2001; Saal etal., 2003; Borgland et al., 2004; Bellone and Luscher,2006; Chen et al., 2008; but see Chen et al., 2008). More-over, chronic cocaine administration increases both AMPAGluR1 protein expression (Fitzgerald et al., 1996) andresponsiveness to local AMPA infusion (White et al., 1995;Zhang et al., 1997) demonstrating changes in AMPA re-ceptor-mediated transmission in VTA neurons after long-term cocaine administration.

Little is known, however, regarding the effects of co-caine administration on GluR1-containing AMPA receptorsin the diverse neuronal subtypes and/or subregions of theVTA (Saal et al., 2003). Functional differences in the VTA

are highly dependent on anatomical connectivity and thetransmitter phenotype of neurons, the majority of which aredopaminergic or GABAergic (Johnson and North, 1992).Changes in glutamate activation have been shown to af-fect dopamine release in both the medial prefrontal cortex(mPFC) and nucleus accumbens (NAc) (Kalivas et al.,1989; Taber and Fibiger, 1995; Karreman et al., 1996;Meltzer et al., 1997; Kretschmer, 1999), the respectiveprimary targets of parabrachial (PB) and paranigral (PN)projection neurons within the VTA (Carr and Sesack, 2000;Lane et al., 2008). Dopaminergic neurons of the PB VTAreceive monosynaptic glutamatergic inputs from themPFC, whereas these glutamatergic efferents target

*Corresponding author. Tel: 1-646-962-8257.E-mail address: [email protected] (D. A. Lane).

Abbreviations: ABC, avidin-biotin complex; BSA, bovine serum albu-min; LTP, long term potentiation; mPFC, medial prefrontal cortex; NAc,nucleus accumbens; PB, parabrachial; PB VTA, parabrachial ventraltegmental area; PN, paranigral; PN VTA, paranigral ventral tegmentalarea; TBS, tris buffered saline; TH, tyrosine hydroxylase; VTA, ventraltegmental area.

Neuroscience 169 (2010) 559573

0306-4522/10 $ - see front matter 2010 IBRO. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.neuroscience.2010.05.056

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mainly GABAergic neurons in the PN VTA (Carr andSesack, 2000; Sesack and Carr, 2002; Sesack et al., 2003;Geisler and Zahm, 2005). These differences in circuitryhave opposing effects on dopamine release when AMPAreceptor antagonist are administered into the VTA (Taka-hata and Moghaddam, 2000) and result in opposing be-

havioral output as measured by locomotor activity(Tzschentke and Schmidt, 2000a,b). Consequently, co-caine administration may produce cell type- and VTA re-gion-specific changes in the availability and functional as-sembly of GluR1-containing AMPA receptors at the syn-apse. Moreover, these changes may differ depending onthe duration of cocaine exposure. To test this hypothesis,we used electron microscopic immunogold labeling of the

AMPA receptor GluR1 subunit in the PB and PN VTAregions of mice receiving either acute (single injection) orchronic (14 days of intermittent escalating-dose) systemiccocaine administration. Dopaminergic neurons were iden-tified by immunoperoxidase labeling of tyrosine hydroxy-

lase (TH), a mandatory enzyme for dopamine synthesisnot present in other neurons of the VTA, the majority ofwhich are GABAergic (Johnson and North, 1992). Theresults provide evidence that acute cocaine administrationproduces region-specific subcellular relocation of AMPAGluR1 subunits consistent with decreased AMPA-medi-ated activation of mesocortical- but increased activation ofmesolimbic-projecting dopamine neurons, an effect that dis-appears with chronic cocaine administration. In contrast,

AMPA GluR1 distributions within presumably GABAergicdendrites show little change following acute cocaine, but aremore profoundly influenced by chronic cocaine administra-tion.

EXPERIMENTAL PROCEDURES

Subjects

Twenty-one naive adult male C57/BL6 mice (Jackson Laborato-ries, Bar Harbor, ME, USA), weighing 1925 g (starting weight)were individually housed and maintained on a 12-h light cycle(lights out at 6:00 PM). Food and water were available ad libitum.Experimental protocols involving animals followed NIH guidelinesto the care and use of research animals and were approved by theInstitutional Animal Care and Use Committee (IACUC) at WeillCornell Medicall College.

Cocaine administration paradigm

Mice were randomly assigned to one of five treatment groups:

Acute cocaine (n5), chronic cocaine (n5), single (n3) orrepeated (n5) saline, and non-injected (n3) controls. Acutecocaine mice received a single injection of cocaine (15 mg/kg i.p.).Chronic cocaine treated mice received three injections per day (at10:00 AM, 4:00 PM, and 10:00 PM) for 14 consecutive days of anescalating dose paradigm of cocaine (ranging from 7.5 mg/kg to60 mg/kg i.p. cumulative dose per day). This paradigm mimics theincreasing drug intake over time as witnessed with humans ad-dicted to cocaine (Zhang et al., 2002). Control mice were injectedwith saline in the same volume and intervals as mice receivingacute (one i.p. injection) or chronic cocaine (three i.p. injectionsper day for 14 consecutive days). In addition, to assess anychanges in subcellular GluR1 related to the stress of injections,tissue from non-injected (normal) control mice were also includedin the study.

Tissue preparation

All mice were deeply anesthetized with sodium pentobarbital (120mg/kg i.p.; Sigma, St. Louis, MO, USA). For all injected animals,pentobarbital was administered 30 min after either the single(acute) or last of the chronic injections of cocaine or saline. Afterfully anesthetized, mice were perfused through the right ventricle

with 20 ml heparin (1000 U/ml) in saline (American Pharmaceuti-cal Partners, Schaumburg, IL, USA) followed by 50 ml of 3.75%acrolein (Polysciences, Warrington, PA, USA) in 2% paraformal-dehyde in 0.1 M phosphate buffer and finally 100 ml of 2%paraformaldehyde in 0.1 M phosphate buffer (Sigma, St. Louis,MO, USA). The brains were removed from the cranium and cutinto 34 mm coronal blocks that were postfixed in 2% parafor-maldehyde for 30 min, then sliced to 40 m sections on a LeicaVibratome VT1000 (Leica Instruments, Nussloch, Germany) inchilled 0.1 M phosphate buffer. The collected vibratome sectionswere placed in a storage solution (30% sucrose, 30% ethyleneglycol in 0.05 M phosphate buffer, pH 7.4) at 20 C until used forimmunolabeling of GluR1 and/or TH.

Antisera

GluR1 immunolabeling was achieved using an affinity purifiedrabbit polyclonal antibody raised against a 13 amino acid peptidesequence corresponding to the C-terminus of rat GluR1 AMPAsubunit (Chemicon, Temecula, CA, USA). The GluR1 antiserum iswell characterized by immunolabeling and Western blot analysis,which shows that this antiserum recognizes a single band of 110kDa corresponding to the GluR1 subunit with no cross-reactionwith GluR2-4 subunits (Siegel et al., 1995; Aicher et al., 2002;Glass et al., 2005). To the best of our knowledge, it is unknownwhether this antiserum recognizes heteromer and/or homomers ofthe GluR1 subunit. However, both receptor types are most likelydetected by this antibody because it was derived from a small 13amino acid peptide sequence of the C-terminus of the AMPAreceptor GluR1 subunit in a portion of the tail that should not beblocked by potential conformational change associated with

dimerization. The recognition of both heteromeric and homomericforms of GluR1-containing AMPA receptors is further supportedby the observed subcellular distribution of GluR1 immunogoldparticles in association with cytoplasmic organelles as well assynaptic and extrasynaptic portions of the plasma membrane(Lane et al., 2008), neuronal regions with proposed differentAMPA receptor subunit compositions (Bellone and Luscher, 2006;Mameli et al., 2007; Argilli et al., 2008).

A mouse monoclonal antibody raised against the rate-limitingenzyme, TH, was commercially obtained from Immunostar (Hud-son, WI, USA). The TH immunogen was purified to homogeneityfrom PC12 cells of rat origin. Western blot analysis shows that thisantiserum identifies a single 60 kDa band exclusively in TH trans-fected, but not non-transfected, HEK293 cells (Immunostar; Les-sard et al., 2010). Moreover, in brainstem catecholaminergic cellgroups the TH-immunoreactivity seen using this antibody is com-

parable to the distribution of TH mRNA as seen by in situ hybrid-ization (Rusnak and Gainer, 2005).

Immunocytochemistry

Coronal sections of tissue containing the VTA of cocaine andsaline injected animals were co-processed using a dual immuno-gold-silver method for the detection of the antiserum againstGluR1 subunit of the AMPA glutamate receptor and immunoper-oxidase for detection of TH antibody. Co-processing eliminatedpotential variability in labeling between experimental groups. Thepre-embedding dual-labeling protocol used in the present studywas adapted from that of Chan and colleagues (Chan et al., 1990).

In preparation for immunolabeling, sections of tissue contain-ing the VTA were placed in 1% sodium borohydride in 0.1 M

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phosphate buffer to remove excess aldehydes, then freeze-thawed to enhance penetration of immunoreagents. For this, thetissue was incubated in a cryoprotectant solution (25% sucroseand 2.5% glycol in 0.05 M phosphate buffer), then immersedsuccessively in liquid Freon, liquid nitrogen, and room tempera-ture 0.1 M phosphate buffer. Tissue sections were rinsed (0.1 Mphosphate buffer) followed by 0.1 M Trisbuffered saline (TBS,pH 7.4) and incubated in 0.5% bovine serum albumin (BSA) inTBS to minimize non-specific labeling. The tissue was then incu-bated for 42 h (24 h at room temperature and 18 h at 4 C) in aprimary antibody solution containing mouse anti-TH (1:50,000)and rabbit anti-GluR1 (1:150) antisera in 0.1% BSA in TBS. Forimmunoperoxidase detection of TH, the avidin-biotin complex(ABC) method was used (Hsu, 1990). Sections were incubated for30 min in biotinylated horse anti-mouse IgG (1:400) and then a1:200 ABC solution (Vector Laboratories, Burlingame, CA, USA).The peroxidase reaction product was visualized with 0.022% 3,3=-diaminobenzidine (Aldrich, St. Louis, MO, USA) and 0.003% H

2O2

in TBS for 6 min. For immunogold-silver visualization of GluR1immunoreactivity, the tissue sections were incubated for 2 h ingoat anti-rabbit colloidal gold (1 nm) IgG (1:50; Amersham Phar-macia, Piscataway, NJ, USA). The gold particles were further

adhered to the tissue by gluteraldehyde (2%) and enhanced witha silver solution (IntenS-EM kit, Amersham, Arlington Heights, IL,USA) for 7 min.

The tissue was post-fixed with 2% osmium tetroxide (ElectronMicroscopy Supply, Hatfield, PA, USA) in 0.2 M phosphate bufferfor 1 h and then dehydrated through a series of ethanols (30, 50,70, 95, 100%), which was followed by 100% propelyne oxide, andan overnight incubation in a 50/50 mixture of propylene oxide andEMbed 812 Epon substitute (Electron Microscopy Supply, Hat-field, PA, USA). The following day, the sections were incubated inthe Epon susbstitute for 2 h, then embedded between two sheetsof Aclar plastic and placed in an oven (60 C) for 24 h.

Selection and sectioning of VTA tissue

The PB and PN subdivisions of the VTA were separately isolated

from the flat-embedded coronal sections of tissue (see trapezoidin Fig. 1A) by comparing the distributions of TH-immunoreactivityin reference to landmark structures identified in a mouse brainatlas (Hof et al., 2000). A medial/caudal region of the VTA (rangingfrom 3.2 to 3.5 mm from Bregma) was selected based on thegreatest density of TH-immunoreactive cells and the prevalence ofboth VTA regions within the same coronal plane. Differences inthe ratio of dopaminergic and GABAergic neurons exist through-out the rostral to caudal aspects of VTA (Olson and Nestler, 2007),which may affect changes in GluR1 localization. However, theVTA level selected for the present study shows abundant distri-butions of both TH- and non-TH labeled cells allowing for sufficientinvestigation of both cell populations.

Ultrathin sections (70 nm) were cut from the isolated tissueusing an ultratome (Leica Instruments, Nussloch, Germany) and adiamond knife (Diatome, Fort Washington, PA, USA). The thin

sections were obtained from the surface (12 m) of the flat-embedded tissue, where there is optimal penetration of immu-noreagents. The sections were collected on 400-mesh coppergrids (Electron Microscopy Science, Fort Washington, PA, USA),counterstained with uranyl acetate and lead citrate (Reynolds,1963) and examined with a Phillips CM-10 electron microscope(FEI, Hillsboro, OR, USA). The microscopic images were capturedwith an AMT Advantage HR/HR-B CCD Camera System (Ad-vanced Microscopy Techniques, Danvers, MA, USA).

We chose pre-embedded goldsilver immunocytochemistryto analyze the subcellular distribution of GluR1 synaptic labelingbecause it preserves the fine structural detail necessary for validquantification of receptor proteins on extrasynaptic membranesthat can be lost during the process of plastic embedding (Adamset al., 2002). The pre-embedding method for immunogold also

avoids spurious attachment of gold particles to the plastic, whichoften results in significant background labeling with post-embed-

Fig. 1. (A) Light micrograph of plastic embedded coronal section oftissue (3.40 mm Bregma) showing the area used for electron micro-scopic analysis (trapezoid). The sampled area contains both the PB andPN VTA. (B) A representative electron micrograph of the VTA showingGluR1 immunogold labeling in both dopaminergic (THGluR1 den) andnon-dopaminergic (Non-THGluR1 den) dendrites. TH is identified bydense immunoperoxidase reaction product. Within these dendrites,GluR1 immunogold particles are most evident in the cytoplasm (grayarrows), butare also seen on theplasma membrane (whitearrow). GluR1

labeling is highly selective for individual dendritic segments seen in coro-nal sections and absent from other similar profiles within the same neu-ropil (ul den). An axon terminal containing GluR1 immunogold (blackarrows, Glu ter) is also seen in the electron micrograph. Both the electronmicrograph and quantitative analysis of labeled profiles shown in the bargraph (C) demonstrate the predominantly post-synaptic (dendritic) distri-bution of GluR1 labeling. Quantitative analysis indicates that only a fewsoma, axons, or glia contain detectable GluR1 and/or TH labeling. PBVTA, parabrachial ventral tegmental area; PN VTA, paranigral ventraltegmental area; SNC, substantia nigra compacta; SN, substantia nigra;ML, medial lemniscus; IP, intrapeduncular nucleus; IF, intrafascicularnucleus; Glu te, GluR1-labeled axon terminal; ul te, unlabeled axonterminal; em, endomembrane; scale bar500 m(A) and 500 nm (B).

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ding immunogold labeling (Leranth and Pickel, 1989). Despitethese advantages, the pre-embedding method may underesti-mate receptor proteins within postsynaptic membrane specializa-tions, where there is a known impediment to penetration (Adamset al., 2002). This limitation, however, would be expected to applyequally to both saline- and cocaine-treated tissue making com-parisons of synaptic labeling appropriate.

Data analysis

A total tissue area of 40,237 m2 (1459 micrographs at 19,000 34,000magnification) was examined from the VTA of 21 mice infive treatment groups. Micrographs of the neuropil were collectedthroughout each region of the VTA to eliminate the possibility ofbiasing the data. In individual profiles, GluR1 immunogold labelingwas evident as small punctuate electron-dense particles and werecharacterized by their location within each profile (see below).Peroxidase immunoreactive profiles were defined as having anelectron density considerably greater than that seen in compara-ble structures in the surrounding neuropil. Classification of all thelabeled profiles was determined by criteria from Peters and col-leagues (Peters et al., 1991).

GluR1 immunogold labeling was relatively sparse, thus pro-files containing one or more gold particles were considered to beimmunogold labeled. To ensure specificity of this labeling crite-rion, the percentage of gold particles overlying myelin, which is notknown to express AMPA GluR1 subunits thus serving as a controlfor spurious goldsilver deposits, was examined. Less than 0.01%of observed gold particles were seen on myelin. On the contrary,GluR1 immunogold particles displayed a high degree of specific-ity, with 94% of all immunogold particles found in dendritic profileswithin the neuropil (Fig. 1).

The parameters used for between-group comparison include(A) profile size, (B) GluR1 immunogold distribution, (C) GluR1synaptic analysis, and (D) TH immunoreactivity. A power analysisof the significant parametric tests was completed to evaluateconfidence of the results and to ensure that adequate samplesizes were used. Significant findings had power values rangingfrom 0.634 to 0.995.

Profile size. The profile area, perimeter, average diameter,and form factor were measured for each profile [total of 4618profiles (4376 dendrites); 2222 in the parabrachial VTA and 2396in the paranigral VTA] using Microcomputer Image Device soft-ware (MCID; Imaging Research, St. Catherines, Ontario, Can-ada). A cluster analysis was performed to statistically sort theprofiles by size and Students t-tests were conducted to evaluatesize differences between the PB and PN VTA regions.

GluR1 immunogold labeling. GluR1 gold particles werecharacterized by their location within the profile, such as whetherthey were in contact with the plasma membrane, post-synapticdensity, or other cytoplasmic structures. These numbers werecombined with size measurements obtained from the MCID pro-gram to give the following measures of gold particle density: thenumber of GluR1 gold particles on the plasma membrane dividedby the perimeter of the profile (PM/perim), the cytoplasmic goldparticles divided by the area of the profile (cyt/area), and the totalnumber of gold particles divided by the area of the profile (total/area). The data for each neuronal population (either TH or non-THlabeled dendrites) were analyzed separately using a two factor(drug treatment groupdendritic size) ANOVA followed by posthoc analysis using a Tukey test.

GluR1 synaptic analysis. Synaptic labeling was examinedby quantifying the ratio of GluR1-labeled synapses out of the totalnumber of synapses per experimental group. In addition, thenumber of GluR1 gold particles at post-synaptic densities wascounted. The ratio of synaptic labeling was evaluated using Chi-

Squared analyses and differences in the number of synapticGluR1 gold particles were analyzed with a single factor ANOVA.

RESULTS

Visualization of GluR1 immunogold distribution

The distribution of GluR1 immunogold particles in themouse VTA of the present study was similar to that previ-ously reported in the rat VTA (Fig. 1; Lane et al., 2008). Inboth the PB and PN subdivisions of the normal mouseVTA, GluR1 immunogold labeling was most evident post-synaptically in somatodendritic profiles with or without THimmunoreactivity. GluR1 immunogold labeling was alsosometimes detected in axons and their terminals and inglia (Fig. 1B, C). The labeling was highly selective forindividual dendritic segments seen in coronal sections andabsent from other similar profiles within the same neuropil.

In both TH and non-TH labeled dendrites, GluR1 im-munogold particles were primarily found within the cyto-

plasm (75%; n4533) and associated with cytoplasmicendomembranes (Fig. 1B). The remaining GluR1 immuno-gold particles (25%; n1507) were in contact with thedendritic plasma membrane. Plasmalemmal GluR1 immu-nogold particles were roughly equally distributed betweenpost-synaptic densities (PSDs; 43%) as non-synaptic por-tions of the membrane (57%). All PSD-associated GluR1immunogold particles (n460) were located at asymmetricexcitatory-type synapses (Carlin et al., 1980; Rollenhagenand Lubke, 2006).

Dendritic size

The majority of GluR1-labeled dendrites, compiled of all

treatment groups, were small to medium sized as mea-sured by the average diameter of each dendrite. Smalldendrites (ranging from 0.1 to 0.6 m) composed 51%,whereas, medium dendrites (ranging from 0.6 to 1.0 m)composed 41% of the total number of GluR1 containingdendrites sampled. Only 8% of the dendrites were greaterthan 1.0 m in average diameter. These larger dendriteswere the most variable in size (ranging from 1.0 to 2.51m), and at least some of them may include portions oflabeled somata. Experimentally induced changes in GluR1immunogold labeling were seen only in small dendrites.These smaller dendrites are, most likely, more distal fromthe soma, where there are a higher proportion of excita-

tory-type inputs (Peters et al., 1991; Lane et al., 2008).

Effects of IP injections on subcellular GluR1

localization

There was a significant increase in both cytoplasmic(F(2225)13.44,p0.05) and plasmalemmal (F(2225)5.51,

p0.05) GluR1 immunogold density in small TH-labeleddendrites within the PB VTA of mice receiving saline injec-tions (either a single or multiple) compared to non-injectedcontrols. There were no significant changes in GluR1 im-munogold labeling between non-injected and saline in-

jected mice in TH-labeled dendrites of the PN VTA ornon-TH-labeled dendrites of either VTA region. Further,


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Fig. 2. Electron micrographs (AC) and quantitative analysis (bar graphs, DF) of GluR1 immunogold distributions in dendrites showing immuno-peroxidase labeling for TH in the mouse PB VTA. GluR1-gold particles in dopaminergic dendrites (THGluR1 den) have a predominantly cytoplasmicdistribution (gray arrows) with some plasmalemmal labeling (white arrows) in saline controls (A). Following a single injection of cocaine (B), GluR1immunogold particles are more readily seen in the cytoplasm (gray arrows) of small dendrites containing peroxidase labeling for TH. However, afterrepeated cocaine administration (C) GluR1 immunogold is once again located both in the cytoplasm (gray arrow) and on the plasma membrane (white

arrows). Bar graphs show a significant increase in cytoplasmic with non-significant decreases in plasmalemmal and synaptic GluR1 labeling afteracute cocaine, as compared to saline controls. These acute cocaine-induced changes in GluR1 distribution diminish with repeated cocaineadministration. Ul te, unlabeled axon terminal; curved black arrowsynapse; * p0.05; scale bar500 nm.


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Fig. 3. Electron micrographs (AD) and quantitative analysis (bar graphs, EG) showing GluR1 immunogold particles in dendrites containingimmunoperoxidase labeling for TH in the mouse PN VTA. Dopaminergic dendrites (THGluR1 den) of the PN VTA have a predominantly cytoplasmicdistribution of GluR1 immunogold particles in saline treated mice (A). After a single injection of cocaine, GluR1 immunogold particles are more oftenseen on the plasma membrane (white arrows, B) and near (gray arrow, C) or at (white triangle, C) asymmetric excitatory-type synapses. Followingrepeated cocaine administration (D), GluR1 gold particles are, once again, observed mostly within the cytoplasm (gray arrows) of dendrites containingperoxidase labeling for TH. The bar graphs show no significant differences between any of the cocaine treatment group and saline controls in meancytoplasmic GluR1 density (E). However, there is a significant increase in mean plasmalemmal GluR1 immunogold density (F) and a significantincrease in the proportion of GluR1-labeled synapses (G) after a single injection of cocaine. These changes are not seen with repeated cocaineadministration. MA, myelinated axon; ul te, unlabeled axon terminals; curved black arrowsynapse; white triangleGluR1 labeling at the post-synapticdensity; * p0.05; scale bar500 nm.


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there were no significant differences between a single(acute) or repeated (chronic) saline injections in either celltype or VTA region.

Effects of cocaine administration on subcellular

GluR1 localization

TH-labeled dendrites. Cocaine administration re-sulted in opposing changes, dependent upon VTA region,in the subcellular localization of AMPA-receptor GluR1subunits in TH-labeled dendrites of putative dopaminergicneurons. Small TH-labeled dendrites within the PB VTAshowed an increase in cytoplasmic density of AMPA-re-ceptor GluR1 subunits, whereas, small TH-labeled den-drites within the PN VTA demonstrated an increase insurface expression (both synaptic and plasmalemmal den-sity) of GluR1 subunits after a single cocaine injection.Following chronic cocaine administration, however, thesubcellular distribution of AMPA-receptor GluR1 subunitsin these dendrites was not distinguishable from saline

controls in either region of the VTA.Parabrachial VTA. Dopaminergic dendrites of the PB

VTA showed a significant increase in cytoplasmic GluR1immunogold labeling (F (4,1285)3.22, p0.05) after asingle injection of cocaine (Fig. 2B, D). A small decrease inthe number of synapses labeled with GluR1 immunogoldparticles (2(4)2.63, p0.05; Fig. 2B, F) and plasmale-mmal GluR1 labeling (F (4,1285)1.82, p0.05; Fig. 2B,E) as well as an increase in total GluR1 density (F(4,1285)4.02, p0.05) were also observed followingacute cocaine administration. Taken together, the findingsdemonstrate that a single injection of cocaine results ininternalization and/or cytoplasmic retention of GluR1-con-

taining AMPA receptors.Following chronic cocaine administration, the subcel-lular distribution of immunolabeled GluR1 subunits re-verted to a level similar to that of saline control animals(Fig. 2C). Tukey post hoc tests confirm that the cytoplas-mic density of GluR1 immunogold particles (# of particles/dendritic area) significantly decreased compared to acutecocaine administration, while both plasmalemmal and syn-aptic GluR1 labeling slightly increased.

Paranigral VTA. TH-containing dopaminergic den-drites of the PN VTA showed a significant increase in thepercentage of synapses with GluR1 labeling (2(4)13.86,

p0.05, Fig. 3C, G) following a single injection of cocaine.In addition, there was a significant increase in plasmale-

mmal (F (4,1476)3.14, p0.05; Fig. 3B, F) and total (F(4,1476)2.77, p0.05) GluR1 immunogold particle den-sity in these dendrites. These data provide ultrastructuralevidence suggesting that the surface/synaptic availabilityof GluR1-containing AMPA receptors in TH-containingdendrites of the PN VTA is increased after a single injec-tion of cocaine.

Following chronic cocaine administration, the percent-age of synapses labeled with GluR1 immunogold particlesdecreased (2(4)13.86, p0.05; Fig. 3D, G) and plas-malemmal GluR1 distributions in TH-containing dendritesreturned to levels similar to saline controls (3DF). Thesechanges suggest a loss of cocaine effect on mesolimbic

dopaminergic neurons of the PN VTA with repeated co-caine administration.

Non-TH labeled dendrites. Acute cocaine producedno significant changes in AMPA GluR1 immunogold label-ing in dendrites without TH immunoreactivity in either PB

or PN VTA (see discussion). There was, however, a slightincrease in cytoplasmic GluR1 immunogold density inthese dendrites in both VTA subregions after a singleinjection of cocaine. The most pronounced changes inGluR1 immunogold labeling were seen following chroniccocaine administration when non-TH containing dendritesshowed opposing effects on synaptic labeling dependingupon VTA region.

Parabrachial VTA. In non-TH labeled dendrites of thePB VTA, acute cocaine administration resulted in a non-significant increase in cytoplasmic GluR1 labeling (F(4814)2.26, p0.05; Fig. 4B, D) and slight decrease inthe percentage of GluR1-labeled synapse (Fig. 4B, F).

Although, not statistically significant, this decrease in syn-

aptic GluR1 labeling was greater with repeated cocaineadministration (2(4)2.39, p0.05; Fig. 4C, F).

Paranigral VTA. In the PN VTA, the most pro-nounced changes in GluR1 immunogold labeling occurredwith repeated cocaine administration, which resulted inincreased numbers of synapses labeled with GluR1 immu-nogold particles (2(4)5.58, p0.05; Fig. 5C, D, G).There was also a small decrease in cytoplasmic GluR1immunogold density after repeated cocaine administration(n.s., F (4797)1.02, p0.05, Fig. 5CE). The findingssuggest that repeated cocaine administration may promotesynaptic translocation of GluR1-containing AMPA recep-tors in non-dopaminergic dendrites of the PN VTA.

DISCUSSION

Our results are summarized in Fig. 6, which schematicallyillustrates neuronal phenotype- and VTA region-specificchanges in response to cocaine administration. Specifi-cally, we show a significant increase in GluR1 immunogoldcytoplasmic density in TH-labeled, putatively dopaminesynthesizing, dendrites of the PB VTA after a single, butnot repeated, injection of cocaine. In contrast, TH-labeleddendrites of the PN VTA have increased surface expres-sion, both synaptic and plasmalemmal, of GluR1 immuno-gold particles after a single injection of cocaine, whichreverts to distributions similar to saline controls after

chronic cocaine. These results, together with evidence thatcytoplasmic GluR1 subunits are not available for surfaceactivation, suggest that acute cocaine administration pro-duces trafficking of GluR1-containing AMPA receptors al-lowing for decreased activation of mesocortical- but in-creased activation of mesolimbic-projecting dopaminergicneurons, an effect that disappears with chronic cocaineadministration. This suggests a homeostatic change in thefunction of glutamatergic transmission in the VTA thatcompensates for the ongoing presence of cocaine. Theschematic also illustrates our findings showing that non-TH, presumably GABAergic, dendrites have GluR1 distri-butions that are more profoundly influenced by chronic


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Fig. 4. Electron micrographs (AC) and quantitative analysis (bar graphs, DF) showing GluR1 immunogold in dendrites without immunoperoxidaselabeling for TH in the mouse PB VTA. Non-TH dendrites (non-THGluR1 den) have a predominantly cytoplasmic (gray arrow) GluR1 immunogolddistribution following saline administration (A). However, GluR1 immunogold particles are also observed on the plasma membrane (white arrow) andnear (black arrow) or at a synapse (white triangle) on these dendrites (A). After a single injection of cocaine (B), GluR1 immunogold is more evidentin the cytoplasm (gray arrows), but still found near asymmetric, excitatory-type, post-synaptic densities (black arrow). This pattern in GluR1 labelingis not seen with repeated cocaine administration (C), where there are fewer synapses (black curved arrows) labeled with GluR1 immunogold particles.Quantitative analysis in the bar graphs (DF) show no significant differences between control and drug treatment groups in the cytoplasmic (E),plasmalemmal (F), or synaptic (G) density of GluR1 immunogold particles. However, there is a trend for increased cytoplasmic GluR1 labeling aftera single cocaine injection and decreased synaptic GluR1 labeling following chronic cocaine administration, as compared to saline controls. Ul te,unlabeled axon terminals; curved black arrowsynapse; white arrowGluR1 immunogold particle at the post-synaptic density; scale bar500 nm.


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Fig. 5. Electron micrographs (AD) and quantitative analysis (bar graphs, EG) showing GluR1 immunogold in small dendrites without immunoper-oxidase labeling for TH in the mouse PN VTA. Dendrites containing GluR1, but no peroxidase labeling of TH, (non-THGluR1 den) have apredominantly cytoplasmic (gray arrow) GluR1 immunogold distribution following saline administration (A). Immunogold particles are also seenoccasionally on the plasma membrane (white arrows). The prevalent cytoplasmic distribution of GluR1 immunogold particles is also seen in VTAsections from mice receiving acute cocaine administration (B), where GluR1 immunogold labeling is most evident in the cytoplasm (gray arrows) andmore rarely seen on the plasma membrane (white arrow). In contrast, after chronic cocaine administration (C, D), GluR1 immunogold particles aremore evident at synapses (white triangles in both C, D), although GluR1 immunogold particles are still seen in the cytoplasm (gray arrows) of thesedendrites. Quantitative analyses (bar graphs) show no significant cocaine-induced changes in cytoplasmic (E), plasmalemmal (F), or synaptic (G)GluR1 labeling as compared to saline controls. However, there is a small trend for increased cytoplasmic/decreased plasmalemmal GluR1 density innon-TH dendrites after acute cocaine administration. Further, there is a large, but non-significant, increase in the proportion of synapses labeled withGluR1 immunogold particles following chronic cocaine administration (G), as compared to animals receiving repeated injections of saline. Glu te,GluR1 labeled axon terminal; ul te, unlabeled axon terminal; gray arrowcytoplasmic GluR1 immunogold particle; curved black arrowsynapse; whitetriangleGluR1 immunogold labeling at the synapse; black block arrowpresynaptic GluR1 immunogold particle; scale bar500 nm.


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cocaine administration. In these dendrites, chronic cocaineincreased the proportion of synapses immunolabeled withGluR1 in the PN VTA, but decreased this labeling in the PBVTA. These results are discussed with regard to theirimplications for the increased locomotor activation anddecreasing reward associated with chronic cocaine use.

VTA region-dependent changes in subcellular GluR1

distributions within dopamine neurons after a single

cocaine injection

The significant increase in cytoplasmic GluR1 density, withdecreased plasmalemmal surface expression, in small TH-labeled dendrites of the PB VTA suggests that acute co-caine administration results in internalization of surface/synaptic GluR1 subunits into the cytoplasm of these den-drites. The decrease in GluR1 surface expression mayreflect enhanced glutamate release following acute co-caine administration (Kalivas, 1995) leading to decreasedactivation of dopaminergic neurons in the PB VTA. In

contrast, TH-containing dendrites within the PN VTA showan increase in synaptic and plasmalemmal GluR1 immu-nogold particles following acute cocaine administrationconsistent with increased AMPA receptor-mediated gluta-matergic transmission. This effect could facilitate burstingactivity of these dopaminergic neurons (Grace and Bun-

ney, 1984; Overton and Clark, 1992).In addition to the observed effects of acute cocaine on

GluR1 subcellular distribution, there was also a significantincreases in the total amount of GluR1 immunogold density indopaminergic dendrites of both VTA subregions following asingle injection of cocaine. No similar changes were seenafter chronic cocaine administration. Previous work showsthat blocking mRNA transcription prevents cocaine-inducedchanges in AMPA receptor GluR1-dependent activation ofdopaminergic neurons suggesting that new protein syn-thesis is required for this type of neuronal activation(Argilli et al., 2008). Similarly, blocking translational ac-tivity in the VTA prevents metabotropic glutamate recep-

Fig. 6. Schematic diagram summarizing changes in AMPA receptor GluR1 immunogold labeling in TH and non-TH containing dendrites of the mousePB and PN VTA following acute and chronic cocaine administration. TH-labeled dendrites of the PB VTA show a significant increase in GluR1immunogold cytoplasmic density (gold circles) after a single (acute), but not repeated (chronic), injection of cocaine. In contrast, TH-labeled dendritesof the PN VTA have increased surface expression, both synaptic (red circles) and plasmalemmal (green circles), of GluR1 immunogold particles aftera single injection of cocaine, which reverts to basal distributions (bottom left corner) similar to saline controls after chronic cocaine. Non-TH containingdendrites show little change in GluR1 immunogold labeling following acute cocaine administration and are more influenced by repeated cocaineadministration. Chronic cocaine decreases the proportion of synapses (darkened segments on the rim of the conical dendrites) immunolabeled withGluR1 in the PB VTA, but increases this labeling in the PN VTA. The drug dose paradigm used for each condition is illustrated in the graph at thebottom of the diagram. Doses for the chronic cocaine group are reported as the total dose per day (the sum of three daily injections). Three-dimensional conestransversely cut dendrites whose plasmalemmal and cytoplasmic components are defined by the dark brown rim and lightercenter; gold circlescytoplasmic GluR1 labeling; green circlesplasmalemmal GluR1 labeling; red circlessynaptic GluR1 labeling; tear dropcontaining small clear circlespresynaptic terminals; thickened dark portion of dendritic circlessynapses.


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tor-dependent long term depression, which plays a rolein cocaine-induced changes in AMPA receptor subunitcomposition (Mameli et al., 2007). Our finding of anoverall increase in GluR1 density supports the idea thatchanges in GluR1 immunogold labeling are not only aresult of AMPA receptor trafficking, but may also be ex-

plained by increased synthesis of AMPA receptor GluR1subunits after a single injection of cocaine.

AMPA receptors are composed of tetramers (twodimers) of four different types of subunits (GluR1GluR4).

All four subunits are found within the VTA, however, GluR1and GluR2 are more prevalent than GluR3 and GluR4(Sato et al., 1993; Paquet et al., 1997; Lu et al., 2001). Thestructure of AMPA receptors is important because theinclusion of the GluR2 subunit renders the receptor cal-cium impermeable, whereas AMPA receptors composedof GluR1 subunits allow for calcium permeability into thecell (Hollmann et al., 1991; Cull-Candy et al., 2006). Syn-aptic insertion of GluR1-containing AMPA receptors is a

necessary first step in the development of neuronalchanges leading to increased excitability of dopaminergicneurons (Liao et al., 2001; Argilli et al., 2008), a proposedmechanism of cocaine addiction (Bellone and Luscher,2006). This idea is supported by findings that show VTAGluR1 protein increases after administration of cocaine, aswell as other drugs of abuse (Fitzgerald et al., 1996), andincreases in VTA GluR1 are linked to the development ofbehavioral sensitization (Carlezon et al., 1997; Churchill etal., 1999; Grignaschi et al., 2004). As such, our finding ofopposing regional changes in subcellular GluR1 providesanatomical evidence suggestive of decreased activation ofmesocortical-projecting dopaminergic neurons of the PBVTA, but increased activation of mesolimbic-projecting do-paminergic neurons of the PN VTA after a single injectionof cocaine.

The observed regional differences of GluR1 traffickingin dopamine dendrites of the PB and PN VTA may be areflection of their anatomical connectivity and/or function.The PB VTA contains mesocortical dopaminergic neuronsthat primarily project to the mPFC (Carr and Sesack, 2000;Lane et al., 2008), a region important for drug seekingbehavior and reinstatement of cocaine self-administration(Goeders and Smith, 1983; Robbins, 1996; McFarland andKalivas, 2001; Park et al., 2002; Capriles et al., 2003; Sunet al., 2005). The PN VTA contains mesolimbic dopami-nergic neurons with principal projections to the NAc, a

brain region important for mediating reward (and aversion)salience (Jentsch and Taylor, 1999). Further, glutamater-gic inputs from the prefrontal cortex (PFC) to these twoVTA regions differ, with direct glutamatergic afferents ter-minating onto mesocortical dopaminergic neurons withinthe PB VTA and indirect input, via GABAergic neurons,terminating on mesolimbic dopaminergic neurons of thePN VTA (Carr and Sesack, 2000; Geisler et al., 2007). Thiscircuitry has been shown to have an opposing effect ondopamine release in the prefrontal cortex and nucleusaccumbens when an AMPA antagonist is applied into theVTA(Takahata and Moghaddam, 2000). Further, depletionof dopamine in the mPFC enhances dopaminergic release

in the NAc (Thompson and Moss, 1995), whereas in-creased mPFC dopamine dampens rewarding effects ofnatural stimuli (Mitchell and Gratton, 1992) supporting theidea that the mPFC acts as a negative feedback system forDA release in the NAc (reviewed in; Tzschentke andSchmidt, 2000b). Thus, the regional differences in the

subcellular GluR1 localization in TH-containing dendritesfollowing a single systemic injection of cocaine are func-tionally intuitive. They provide anatomical evidence fordecreased activation of mPFC-projecting dopaminergicneurons involved in drug seeking and reinstatement ofcocaine self-administration with a concurrent increase inglutamatergic activation of NAc-projecting dopaminergicneurons involved in reward.

Loss of cocaine effect on GluR1-containing AMPA

receptors in VTA dopaminergic dendrites after

repeated escalating doses of cocaine

Following 14 days of escalating doses of cocaine, dopa-minergic dendrites within both regions of the VTA havesimilar subcellular AMPA receptor GluR1-subunit distribu-tions as cocaine naive animals. The base-line distributionof AMPA receptor GluR1 subunits was seen despite thepresence of cocaine when administered 30 min prior tobrain perfusion and tissue processing, a time point atwhich there is a high systemic concentration of cocaine(Pan and Hedaya, 1998) and observed acute effects ofcocaine (see above). Conceivably, the comparable subcel-lular GluR1 distributions seen in both chronic cocaine-treated and saline control animals may reflect a homeo-static feedback mechanism of the glutamatergic system inresponse to cocaine.

Alternately, the function of VTA GluR1-containingAMPA receptor in mediating addictive behaviors may betransient. Behavioral sensitization is an animal model ofaddiction thought to parallel neuroadaptive changes asso-ciated with cocaine addiction. The VTA appears to beimportant in initiating, but not maintaining the neuroplas-ticity associated with behavioral sensitization (Wolf, 2003).Repeated cocaine administration leads to a progressiveenhancement of its stimulant effects that can be measuredby increases in locomotor activity across drug treatmenttrials (Stewart and Badiani, 1993) which persist even aftercocaine administration has ceased (Henry and White,1995). The enhanced locomotor activity, or behavioral sen-

sitization, is associated with an increase in GluR1 in theVTA (Carlezon and Nestler, 2002; Grignaschi et al., 2004;

Argilli et al., 2008). Local administration of a glutamatereceptor antagonist in the VTA prevents the develop-ment of behavioral sensitization (Karler et al., 1989) andconditioned place preference to cocaine (Harris and

Aston-Jones, 2003). However, once sensitization devel-ops, VTA injections of glutamate antagonists do notaffect locomotor sensitization (Kalivas and Alesdatter,1993). This suggests that AMPA receptor-dependentactivation of VTA dopaminergic neurons is transient andinvolved largely in initiating, but not maintaining, neuro-plasticity associated with addictive behavior. This may


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explain why there was little change, as compared tosaline controls, in subcellular GluR1 localization in do-paminergic dendrites within both VTA subregions follow-ing chronic cocaine treatment.

The observed absence of change in GluR1 distributionseen with chronic cocaine administration may also reflect a

change towards NMDA receptor-mediated transmission ora shift in AMPA receptor subunit composition in theseneurons following chronic cocaine administration. The re-cruitment of GluR1-containing AMPA receptors to sitesalong the plasma membrane is important for activation ofthe cell and potentially NMDA receptors, which are crucialfor the induction of LTP, a proposed mechanism of addic-tion. Cocaine has been shown to increase the AMPA:NMDA ratio in VTA dopaminergic neurons facilitating LTPinduction (Ungless et al., 2001; Saal et al., 2003; Borglandet al., 2004). AMPA receptors can rapidly move in and outof the synapse to mediate this type of neuronal activation(Heine et al., 2008). However, once activated, NMDA re-

ceptors may be more important in mediating neuroplastic-ity associated with addictive behaviors reducing the needfor AMPA receptor activation (Bredt and Nicoll, 2003).

Alternately, increases in synaptic activation following co-caine administration are attributed to increased GluR1-containing AMPA receptors at post-synaptic densities inthe VTA (Argilli et al., 2008). It has been shown that theseGluR1-containing AMPA receptors are displaced byGluR2-containing AMPA receptors 24 h after a single co-caine injection (Bellone and Luscher, 2006; Mameli et al.,2007) providing a potential rationale for the lack ofchanges in subcellular GluR1 localization seen followingchronic cocaine administration.

Subcellular GluR1 localization and cocaine-inducedtrafficking in non-TH dendrites

Acute cocaine administration resulted in a small increasein cytoplasmic GluR1 immunogold density in non-TH con-taining dendrites of both VTA regions. Most non-TH la-beled neurons in the VTA are GABAergic (Johnson andNorth, 1992), and exert tonic inhibition on VTA dopaminer-gic neurons (Yim and Mogenson, 1980; Xi and Stein,1998). Thus, internalization of GluR1-containing AMPAreceptors, as suggested by the increased cytoplasmic la-beling in non-TH dendrites, may result in decreased GABAinhibition of VTA dopaminergic neurons after a single in-

jection of cocaine.

Chronic cocaine administration resulted in VTA region-specific differences in synaptic GluR1 labeling in dendriteswithout TH-immunoreactivity. In the PB VTA, these den-drites showed a non-significant decrease in synapticGluR1 labeling, which may allow for disinhibition of VTAdopaminergic neurons that project to mPFC. This is ofparticular interest because loss of GABAergic inhibitionhas been shown to facilitate induction of LTP to chroniccocaine (Liu et al., 2005). Conversely, there was a rela-tively large increase in the number of synapses labeledwith GluR1 in non-TH-containing dendrites within the PNVTA, although this change was not significant. Increasedinhibition of VTA dopaminergic neurons results in de-

creased dopamine release in the nucleus accumbens (Ka-livas and Duffy, 1990; Xi and Stein, 1998). Together, thefindings provide anatomical evidence for decreased GABAinhibition of PB, but increased GABA inhibition of PN do-pamine neurons within the VTA following repeated cocaineadministration, which may be a potential mechanism for

increased drug seeking and declining reward that occurswith repeated cocaine use.

Neither acute nor chronic cocaine administration pro-duced significant changes in AMPA GluR1 distributions innon-TH-containing dendrites of the VTA. Conceivably, theheterogeneity of VTA GABAergic neurons (Bayer andPickel, 1991; Van Bockstaele and Pickel, 1995; Om-elchenko and Sesack, 2009) and potential inclusion ofglutamatergic neurons (Yamaguchi et al., 2007) may resultin differential GluR1 changes in distinct neuronal subpopu-lations explaining the muted cocaine-induced changesseen in non-TH-containing dendrites of the VTA.

Effects of injection on subcellular GluR1 localization

Overall, there were no significant changes in GluR1 immu-nogold labeling between saline injected and non-injectedmice, with one exception. Mesocortical projecting TH-la-beled dendrites of the PB VTA showed a significant in-crease in both cytoplasmic and plasmalemmal GluR1 im-munogold density following both single and multiple injec-tions of saline, as compared to non-injected mice (Fig. 2D,E). These cortical-projecting dopaminergic neurons havebeen shown to be more vulnerable to stress (Horger andRoth, 1996) and to play an important role in stress-inducedreinstatement of cocaine use (McFarland and Kalivas,2001; Park et al., 2002). Acute stress is also known toincrease AMPA mediated synaptic excitability (Saal et al.,2003), and stress from saline injections upregulates VTAGluR1 (Fitzgerald et al., 1996). Our findings support theseprevious studies and are the first to demonstrate PB VTA-specific changes in AMPA receptor GluR1 subcellular dis-tributions in dopaminergic dendrites potentially induced bystress from i.p. injection of saline. The changes in AMPAGluR1 labeling did not significantly differ with one or mul-tiple saline injections.

Implications

The findings in the present study provide ultrastructuralevidence for transient acute-cocaine-induced increase incytoplasmic AMPA GluR1 in mesocortical (PB VTA) and

increase in dendritic surface expression in mesolimbic (PNVTA) dopamine neurons. These findings are consistentwith the respective differential anatomical connectivity ofthese regions (Van Bockstaele and Pickel, 1995; Sesackand Carr, 2002; Sesack et al., 2003) and the known differ-ential glutamatergic control of mesocortical and mesolim-bic projection neurons (Takahata and Moghaddam, 2000).Moreover, we show that in contrast with dopaminergicneurons, putative GABAergic neurons located preferen-tially in the PN VTA show enhanced synaptic GluR1 label-ing following repeated, but not single, injections of cocaine.These neurons are likely among those that received mono-synaptic input from the PFC and project to the NAc (Carr


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and Sesack, 2000; Omelchenko and Sesack, 2009). Theincreased activation of these neurons, suggested by theincreased surface expression of GluR1, reveals a novelmechanism for enhanced VTA inhibition of the limbic re-ward circuit such that increasingly higher doses areneeded following repeated administration of cocaine.

AcknowledgmentsThis work was supported by grants NIH-

NIDA P60 05130 to MJK and VMP, NIH-NIDA R01 004600 to

VMP, and NIH-NIDA T32 007274 to Charles Inturrisi.

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(Accepted 24 May 2010)(Available online 27 May 2010)


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