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ocaine-induced homeostatic regulation and dysregulation of nucleusccumbens neurons
anhua H. Huanga, Oliver M. Schlüterb, Yan Donga,∗
Program in Neuroscience, Washington State University, Pullman, WA 99164, USADepartment of Neurobiology, European Neuroscience Institute, Grisebachstr. 5, 37077 Göttingen, Germany
r t i c l e i n f o
rticle history:eceived 16 June 2010eceived in revised form 22 July 2010ccepted 30 July 2010vailable online 11 August 2010
Homeostatic response is an endowed self-correcting/maintaining property for living units, ranging fromsubcellular domains, single cells, and organs to the whole organism. Homeostatic responses maintain sta-ble function through the ever-changing internal and external environments. In central neurons, severalforms of homeostatic regulation have been identified, all of which tend to stabilize the functional outputof neurons toward their prior “set-point.” Medium spiny neurons (MSNs) within the forebrain region thenucleus accumbens (NAc) play a central role in gating/regulating emotional and motivational behaviorsincluding craving and seeking drugs of abuse. Exposure to highly salient stimuli such as cocaine adminis-tration not only acutely activates a certain population of NAc MSNs, but also induces long-lasting changesin these neurons. It is these long-lasting cellular alterations that are speculated to mediate the increasinglystrong cocaine-craving and cocaine-seeking behaviors. Why do the potentially powerful homeostaticmechanisms fail to correct or compensate for these drug-induced maladaptations in neurons? Based on
recent experimental results, this review proposes a hypothesis of homeostatic dysregulation inducedby exposure to cocaine. Specifically, we hypothesize that exposure to cocaine generates false molecu-lar signals which misleads the homeostatic regulation process, resulting in maladaptive changes in NAcMSNs. Thus, many molecular and cellular alterations observed in the addicted brain may indeed resultfrom homeostatic dysregulation. This review is among the first to introduce the concept of homeostaticneuroplasticity to understanding the molecular and cellular maladaptations following exposure to drugs of abuse.
Homeostasis is central for living organisms [1]. With appro-riate homeostatic responses, individual cells, tissues, organs andrganisms are able to maintain relatively stable functional outputhroughout the ongoing internal and external challenges [1]. Forentral neurons, homeostatic plasticity is a powerful endogenousechanism that functions to maintain stable neuronal function.
hus far, several forms of homeostatic neuroplasticity have beenharacterized in different brain regions, all of which act to sta-ilize the overall functional output of the neurons toward theirrior “set-point” [2]. With homeostatic plasticity, neurons mobi-
ize available resources to restore or functionally compensate forhe altered cellular components throughout developmental regu-ations, metabolic turnovers, and even pathological insults.
A stable function of nucleus accumbens (NAc) medium spinyeurons (MSNs) is essential for proper behavioral outputs of emo-ional and motivational drives. When measured at the cellularevel, natural rewards or modest incentive stimuli rarely produceong-lasting biochemical and biophysical changes in the NAc [3].owever, following exposure to drugs of abuse, a large numberf adaptive changes occur, affecting presynaptic terminals, postsy-aptic receptors, voltage-gated ion channels on the membrane, andeuromodulators, resulting in profound alterations in NAc MSNs3]. Apparently, normal function of NAc MSNs fails to be restoredn drug-exposed animals because of, hypothetically, either a shift ofhe homeostatic “set-point” or malfunctional homeostatic mecha-isms.
Dysregulation of the reward system characterized by a chroniceviation of reward set-point is termed allostasis [4]. It is a processf maintaining apparent stability around a new set-point throughhanges, but at a price [5]. From a reductionist point of view, allosta-is results from inadequate local and global feedback regulations.lthough more complex than homeostasis, it typically involves thehole brain and body instead of simply local feedbacks, it is medi-
ted by the same molecular and cellular mechanisms that underlieomeostasis, or rather, malfunctional homeostasis.
This review summarizes several forms of homeostatic neu-oplasticity and their potential roles in cocaine-induced cellularlterations of NAc MSNs. Based on these observations, we hypoth-size that the key signaling substrates controlling homeostaticlasticity are altered by exposure to cocaine, and the consequentalse signals mislead homeostatic plasticity. Thus, a large numberf molecular and cellular alterations observed in NAc MSNs fromocaine-exposed animals are results of homeostatic dysregulation.
. Concept of homeostatic neuroplasticity
.1. Regulated vs. homeostatic plasticity
Depending on the induction mechanism, expression speci-
city/pattern, and functional roles, neuroplasticity can be catego-ized as regulated or homeostatic neuroplasticity (Fig. 1). Regulatedeuroplasticity, also called experience- or activity-dependent plas-icity, refers to a large category of plasticity that occurs uponpecific/contingent stimulations. Two well-characterized forms of
regulated plasticity are long-term potentiation (LTP) and long-term depression (LTD) of excitatory synaptic transmissions. Thetwo defining properties of regulated neuroplasticity are contin-gency and specificity [6]. For example, a successful induction ofLTP at excitatory synapses within the CA1 region of the hippocam-pus requires a contingent pre- and post-synaptic activation (thus,pre- and post-synaptic contingency), and expression of LTP occursexclusively within the activated projection afferent (thus, afferent-specificity). Accordingly, regulated plasticity has been proposedas a cellular mechanism mediating the formation of contingentmemory following learning processes [6,7]. In general, regulatedneuroplasticity follows positive feedback loops which shift the neu-ron from its original functional state to a new functional state inorder to gain new, or modified, behavioral outputs.
Homeostatic plasticity often occurs “spontaneously” upon per-sistent internal or external drive. It follows negative feedback loopsto restrain neural activity within a normal operating range, a rangeso narrow that it typically becomes a “set-point.” Homeostatic plas-ticity is one of the few important mechanisms that function tomaintain the stability of neurons and neural circuits. One formof homeostatic neuroplasticity that has been well characterizedis homeostatic synaptic compensation at the neuromuscular junc-tion. It functions to maintain stable synaptic transmission againstpre- or post-synaptic alterations. For example, when the sensi-tivity of postsynaptic neurotransmitter receptors was chronicallydecreased, presynaptic neurotransmitter release is gradually andpersistently increased, such that the synaptic depolarization ofmuscle is restored to precisely the levels observed in the absenceof the perturbation [8,9]. As such, the altered synaptic transmissioncan be fully restored through homeostatic mechanisms. Althoughnot clearly defined, it appears that contingency and specificity arenot the two essential properties for homeostatic plasticity. Indeed,several lines of evidence support a notion that homeostatic plas-ticity is triggered by non-coincidental events, and is more likely anon-specific, global effect [2].
Functionally, the key difference between regulated and home-ostatic plasticity is that regulated plasticity tends to drive thefunctional output of a neuron away from the prior “set-point,”whereas homeostatic plasticity tends to maintain the overall func-tional output of a neuron around the “set-point.” Thus, regulatedand homeostatic plasticity functionally oppose and complementeach other. Without regulated plasticity, new forms of neural activ-ity, new memories, and behavioral patterns may not be formed,whereas without homeostatic plasticity, neural activity may even-tually run out of control, and behavioral output may become highlyunpredictable.
2.2. Different forms of homeostatic plasticity
The functional output of a neuron is defined by its actionpotential firing. Two major factors that determine in vivo action
potential firings are integrated synaptic input and intrinsic mem-brane excitability. Synaptic input drives the membrane potentialtoward, or away from, the threshold for action potential firing. Theintrinsic membrane excitability not only sets the action potentialthreshold, but also determines how often to fire action potentials
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Fig. 1. Using synaptic plasticity as an example, the diagram shows the differences between regulated and homeostatic plasticity. (A) A postsynaptic neuron receives multiplepresynaptic inputs (1, 2, and 3). These presynaptic terminals may project from different neurons and thus may also represent synapses from different pathways. (B) Regulatedsynaptic plasticity exhibits two defining properties, contingency and specificity. A successful induction of regulated synaptic plasticity requires a contingent activation ofp naptice postp tivateC esyna
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re- and postsynaptic terminals (diagramed as action potential firing at both presyxpressed in the affected pathway (e.g., only synapse 2 is potentiated by enhancedostsynaptic neuron, are not altered because they are not within the contingently achronic decrease of postsynaptic receptor sensitivity induces a global increase in pr
nce the membrane potential moves beyond the threshold. It haseen recognized that homeostatic plasticity plays a role in regulat-
ng both synaptic transmission and membrane excitability, as wells the interaction of the two. These different forms of homeostaticlasticity may play a profound role in stabilizing the functionalutput of neurons.
.2.1. Two-way homeostatic regulation of synaptic transmissionHomeostatic plasticity at synapses has been demonstrated
n several preparations as a two-way communication betweenhe pre- and post-synaptic sites. The most clear-cut results arerom studies of the neuromuscular junction, in which alterations
and postsynaptic terminals at synapse 2; affected synapse is shaded), and is onlysynaptic responsiveness). Other synapses (1 and 3), although located on the samed pathway. (C) Homeostatic synaptic plasticity is induced and expressed “globally”.ptic release such that the action potential firing of postsynaptic neurons is restored.
of presynaptic release cause opposing changes on postsynapticacetylcholine receptors, such that the postsynaptic responsivenessis altered to functionally compensate for the presynaptic changes[10–12]. On the other hand, a reduction in the number and/or func-tion of postsynaptic receptors induces a compensatory increase inpresynaptic release [11,13,14]. Such two-way homeostatic trans-synaptic crosstalks appear to also exist in central neurons. In
cultured cortical, hippocampal, or spinal neurons, a decrease in net-work (presumably synaptic) activity induces a global increase inexcitatory synaptic strength, whereas an increase in the networkactivity, achieved by partial inhibition of inhibitory synaptic input,results in a homeostatic decrease in excitatory synaptic strength
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15–21]. Thus, synaptic homeostasis is achieved, at least in part, byntra-synaptic homeostatic signaling.
.2.2. Homeostatic membrane plasticityHomeostatic mechanisms also exist around the somatic mem-
rane to gauge and stabilize the intrinsic membrane excitabilityf neurons in response to changes in membrane activity. In cor-ical neuronal cultures, chronic prevention of action potentialring induces an increase in the density of voltage-gated sodiumhannels and a decrease in potassium channels, effects that collec-ively function to compensate for the decreased membrane activity22,23]. In dorsal root ganglion neurons, chronic membrane excita-ion (achieved by chronic electrical stimulation) induces a gradualecrease in voltage-gated calcium conductances, a change thatends to dampen membrane excitability [24]. Thus, membrane-esiding voltage-gated ion channels may be selectively targeted ashe local functional domains for homeostatic regulation.
.2.3. Homeostatic synapse-membrane crosstalkIt is now evident that homeostatic mechanisms not only exist
ocally within relatively independent subcellular domains (i.e.,ynapses or somatic membrane), but often function between sub-ellular domains, such as between synapses and the non-synapticembrane. This synapse-membrane crosstalk may occur to con-
ribute critically to whole-cell homeostasis. Some of the firstvidence for synapse-membrane crosstalk was demonstrated intomatogastric ganglion (STG) neurons from the spiny lobster Pan-lirus and the crab Cancer. STG neurons in vivo fire in bursts, which
s driven by rhythmic excitatory synaptic input. When the synapticnput is pharmacologically inhibited or anatomically removed, STGeurons become silent but over time (∼70 h) regain the rhythmicring patterns [25]. Further evidence shows that this homeostaticecovery is mediated by expression and rearrangement of a setf voltage-gated ion channels [25]. Thus, synaptic alteration (lossf synaptic innervations) is sensed and transferred to a regula-ory mechanism related to ion channels on the somatic membrane,hich results in a restoration of the functional output of STG neu-
ons.In the mammalian CNS, it has been observed similarly in the
isual cortex that lowering visual drive from the retina inducescompensatory increase in the intrinsic membrane excitability,
everal synapses away, in layer 2/3 pyramidal neurons [26]. In addi-ion, the reverse is also true; changes at the somatic membrane cane sensed and transferred to synapses to induce global scaling ofynaptic strength (so-called synaptic scaling). In cultured corticaleurons, selective inhibition of postsynaptic action potential fir-
ng by locally blocking voltage-gated sodium channels at the somas sufficient to increase synaptic glutamatergic receptors throughhe dendrite [27]. Furthermore, changes in the membrane activityaction potential firing) are capable of inducing homeostatic regu-ations of presynaptic release mechanisms, as has been shown athe neuromuscular junction [8] and in cultured cortical neurons28].
Of particular interest is a phenomenon observed in the rat NAcSNs, which is phenotypically identical to the above-mentioned
estoration of oscillating behaviors in cultured STG. A commonn vivo physiological property of many types of central neuronss the rhythmic oscillation of the membrane potential between ayperpolarized downstate potential (e.g., −75 mV) and a relativelyepolarized upstate potential (e.g., −55 mV) [29,30]. The upstate
s the functionally active state for most of the oscillating neurons
o fire action potentials [29–31]. This upstate–downstate oscilla-ion is well exemplified in the dorsal striatal and NAc MSNs inivo [29,30,32,33]. It is driven by rhythmically synchronous acti-ation of excitatory synaptic inputs [34–36]. When NAc slices arecutely prepared, however, the cell bodies of most glutamater-
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gic projections are removed, and upstate events become rare inNAc MSNs. However, after ∼24 h of “recovery” in vitro, ∼50% ofNAc MSNs regain the upstate–downstate cyclings and fire actionpotentials during the upstate [37]. In contrast to STG neurons [25],recovery of upstate–downstate cycling is not mediated by postsy-naptic ion channels. Instead, it is likely mediated by the recoveryof synchronous activity of existing glutamatergic terminals, asthe spontaneous presynaptic release, but not the efficacy of indi-vidual excitatory synapses, was significantly up-regulated duringthe course of slice culture [37]. Although it remains to be deter-mined how those glutamatergic terminals severed from the somaachieve synchronous activity, it is clear that under different condi-tions, very different cellular resources are utilized by different celltypes to achieve a common biological purpose, to homeostaticallyregain the lost function. Nonetheless, the self-governing homeo-static mechanisms must exist (1) to “remember” the set-point, (2)to continuously compare the current functional output with theset-point so as to detect the altered functional output, (3) to triggerappropriate molecular cascades once a change is detected, and (4)to suspend the homeostatic responses once the functional output ismodified close to the set-point. Thus, homeostatic responses are notrandom events; they are highly organized and sophisticated self-ruling cascades including several levels of regulatory substrates.As such, tweaking the critical components within the homeostaticcascade can be a way for either pathogenic insults to twist thefunctional output of neurons or therapeutic approaches to enhancehomeostatic recovery of the neuronal function.
3. Homeostatic plasticity in NAc
A large portion of drug-induced adaptive cellular changes arelikely homeostatic [38,39]. However, understanding homeostaticplasticity and its role in addiction is still in its infant phase. Thusfar, only a small number of studies directly address this issue inaddiction-related brain regions including the NAc.
3.1. Synaptic scaling, in vitro studies
Synaptic scaling is a relatively well-defined form of homeo-static synaptic plasticity, expressed as global scaling of synapticweights up or down to compensate for altered neural activity. Itcan be induced globally or locally, and is mediated by adjustmentsof presynaptic release and/or postsynaptic responsiveness [2].
The involvement of synaptic scaling in drug-induced cellu-lar adaptations was first proposed in NAc MSNs as a potentialmechanism mediating cocaine withdrawal-induced up-regulationof synaptic AMPA receptors (AMPARs) [40]. The rationale was thatfollowing repeated cocaine exposure, the NAc and its source of glu-tamatergic innervations from the medial prefrontal cortex (PFC)both become metabolically hypoactive [41–43]. The decreasedactivity in NAc MSNs is hypothesized to trigger synaptic scalingat excitatory synapses to increase the postsynaptic responsiveness[40,44]. The ability of NAc MSNs to undergo synaptic scaling wassubsequently demonstrated in vitro. In these studies, NAc MSNs,which are GABAergic neurons, are co-cultured with PFC gluta-matergic neurons to restore excitatory synapses [45,46]. It wasshown then that prolonged (1–3 days) pharmacological blockadeof glutamate AMPARs causes an increase in the number of surfaceand synaptic AMPARs. Reciprocally, blocking GABAergic trans-mission leads to a decrease in the surface and synaptic AMPARs
[46]. Furthermore, the increased synaptic AMPARs appear to beGluR1/2-containing AMPARs, as the surface and total levels ofGluR1 and GluR2, but not GluR3, are bi-directionally regulated asMSNs undergo synaptic scaling [46,47]. Interestingly, repeated pre-treatment with dopamine, which acutely increases the surface level
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f GluR1 and GluR2 subunits of AMPARs, occludes the homeostaticp-regulation of postsynaptic AMPARs in NAc MSNs [46]. Becausecommon acute pharmacological effect of drugs of abuse is to
ncrease the level of dopamine in the NAc, this occlusion effect sug-ests that dopamine may be one substrate for cocaine exposure torigger homeostatic scaling of AMPARs in NAc neurons. It is worthoting that unlike the effect of dopamine [46], repeated exposureo cocaine does not significantly alter the surface levels of GluR1nd GluR2 are not altered when measured during short-term with-rawal [40,44]. These seemingly discrepant results may reflect the
mpact of the short-term withdrawal even it is short (i.e., 1 day), theifference between in vitro and in vivo conditions, or the differenceetween acute and homeostatic effects of dopamine and cocaine.
More recently, we demonstrated a novel form of homeostaticrosstalk in NAc MSNs between excitatory synapses and membranexcitability, so-called homeostatic synapse-driven membrane plas-icity (hSMP; Fig. 2) [48]. hSMP is triggered by a chronic increase orecrease in the strength of excitatory synaptic transmission and
s expressed as a persistent decrease or increase in membranexcitability [48]. Given that the functional output of a central neu-on is largely determined by the integration of synaptic input andembrane excitability, hSMP functions to stabilize the output ofAc MSNs by offsetting the impact of altered synaptic activity onction potential firing. Furthermore, hSMP also exists in hippocam-al neurons (Schlüter et al., unpublished data), suggesting thatSMP is not limited to NAc MSNs and may be a general regulatoryechanism for central neurons.In theory, hSMP requires a sensor to gauge the intensity of
ynaptic activity, an executive component to translate the sensednformation into the regulation of membrane component, and aet of ion channels to express hSMP. Our results suggest thatynaptic NMDA receptors (NMDARs) may act as sensors becauseanipulations of synaptic NMDARs alone are sufficient to induce
i-directional modulations of membrane excitability of NAc MSNs48]. Synaptic NMDARs have long been known to function as co-ncidence detectors for regulated synaptic plasticity [6], wherehe NMDAR subunit compositions as well as the level of activ-
ig. 2. A schematic view of homeostatic synapse-driven membrane plasticity in NAceurons. On the postsynaptic membrane, AMPARs mediate most of postsynapticurrent, and NMDARs are also activated during synaptic transmission. The activityf synaptic NMDARs is positively correlated with the activity level of excitatoryynapses. As such, the constitutively active NMDAR-coupled intracellular signalinge.g., the CaMKII-mediated signaling) can be up- or down-regulated accordingly onreal-time base, which in turn modulates ion channels (e.g., SK channels) locatedn the somatic membrane.
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ity drive synaptic transmission toward different destinies, LTP orLTD, depending on specific experimental conditions [49–53]. Ourresults show that in addition to be co-incidence detectors, synap-tic NMDARs may also function to detect the overall activity levelof excitatory synapses [48]. Identification of synaptic NMDARsas the trigger for hSMP provides significant insight in identi-fying the potentially coordinative and cooperative subcellularnetwork that carries out homeostatic regulation. Using a pharma-cological approach, our preliminary studies show that NR2A- andNR2B-containing NMDARs may differentiate their roles in medi-ating bi-directional regulation of membrane excitability via hSMP.More specifically, persistent activation of NR2B-, but not NR2A-containing, NMDARs induces a homeostatic decrease in membraneexcitability of NAc MSNs (unpublished data). Given that NR2A- andNR2B-containing NMDARs are coupled to different sets of intracel-lular signaling cascades or to the same signaling cascades but withdifferent intensity [49–52,54–58], targeting NMDAR subunits andtheir differentially coupled intracellular signaling cascades may bea feasible starting point to tackle the cellular logic underlying hSMP.
The expression of a hSMP-based decrease in the membraneexcitability of NAc shell MSNs is mediated by regulation of calcium-activated SK type potassium channels; an increase in the activity ofsynaptic NMDARs induces a gradual up-regulation of SK channel-mediated after hyperpolarization (AHP), resulting in dampening ofthe membrane excitability [48]. This result is intriguing because SKchannel activity is otherwise not typically detectable by electro-physiology in NAc shell neurons [48]. As such, hSMP may initiatecertain protein expression and/or delivery mechanisms de novoto achieve the overall balancing act. On the other hand, hSMP-mediated increase in the membrane excitability of NAc MSNs doesnot involve SK channels [48]. Thus, other unidentified substratesare employed for the expression of hSMP-mediated increase inmembrane excitability. Taken together, the available results sug-gest that although the intrinsic membrane excitability is the finaltarget of hSMP, different repertoires of membrane-located ionchannels are involved in different directions of hSMP-based mod-ulations.
4. Homeostatic regulation and dysregulation in NAcfollowing exposure to cocaine
Following exposure to cocaine, both excitatory synaptic inputand membrane excitability of NAc MSNs undergo substantialchanges. These changes are dependent on the timing (e.g., expo-sure vs. withdrawal, short-term vs. long-term withdrawal, andwithdrawal vs. re-exposure), and the specific procedures (e.g.,self-administration vs. i.p. injection by experimenter). Two keyquestions this review considers are whether these cocaine-inducedcellular changes are homeostatically linked, and whether home-ostatic plasticity acts to maintain the functional stability of NAcMSNs in cocaine-treated animals.
are obtained using the sensitization procedure (e.g., i.p. injection ofcocaine for 5–10 days, 10–20 mg/kg/day). During short-term with-drawal (on withdrawal days 1–3), results about the strength ofexcitatory synapses in NAc MSNs come from biochemical measure-
ments of surface receptor protein levels and electrophysiologicalrecordings of excitatory transmissions onto NAc neurons. Using across-linking technique to label surface AMPAR subunits, Wolf andcolleagues demonstrated that surface AMPARs, mostly enrichedat synapses of NAc MSNs, remain unchanged at this time point
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40,44]. Nevertheless, Thomas and colleagues measured AMPAR-nd NMDAR-mediated excitatory postsynaptic currents (EPSCs) inAc shell MSNs and found that the ratio of AMPAR EPSCs to NMDARPSCs (AMPAR/NMDAR ratio) is decreased at this withdrawal timeoint [59]. Although the decrease was initially interpreted as aown-regulation of synaptic AMPARs and no change in NMDARs,ore recent evidence suggests that new NMDARs, especiallyR2B-containing NMDARs, are inserted into postsynaptic loca-
ions, resulting in generation of nascent, NMDAR-only synapses inAc MSNs [60]. As such, the overall synaptic level of NMDARs isp-regulated at this withdrawal time point and thus may presentcoherent explanation for a lack of change in surface AMPARs
40,44] and a decrease in the AMPAR/NMDAR ratio [59] during thishort-term withdrawal time point.
Approximately in parallel to synaptic alterations mentionedbove, the intrinsic membrane excitability is decreased in NAchell MSNs at the same withdrawal time point, as measured byction potential firing upon somatic current injections [48,61–63].he decreased membrane excitability is likely mediated by aombination of changes in voltage-gated sodium channels [64], cal-ium channels [65,66], and potassium channels [62,65], as well asalcium-activated SK type potassium channels [48]. These cocaine-nduced membrane adaptations, among others, may act in sync toeduce the responsiveness of MSNs to synaptic excitation. Indeed,n vivo recordings from NAc MSNs revealed that their action poten-ial firings upon iontophoretically applied glutamate is decreaseduring short-term withdrawal from repeated exposure to cocaine67].
What may be the underlying homeostatic regulations at thisithdrawal time point? Apparently, AMPAR-mediated synaptic
caling has not yet kicked in to play [40,44]. This may be sur-rising given that NAc MSNs undergo synaptic scaling relativelyuickly (within 3 days) when overall activity level drops or whenepeated exposure to dopamine occurs [68]. The lack of synapticcaling after 5 days of cocaine exposure may suggest the processeing suppressed or temporarily suspended. On the other hand,
t is relatively clear that homeostatic crosstalk between excitatoryynapses and membrane excitability is involved in cocaine-inducedembrane adaptations at this withdrawal time point. As described
n Section 3.2, in NAc MSNs, hSMP functions to increase or decreasehe intrinsic membrane excitability in response to a decrease orncrease in excitatory synaptic activity, respectively. Furthermore,ynaptic NMDARs act as the sensor for the activity level of excita-ory synapses [48]. As such, even surface or synaptic AMPARs doot appear to be altered; an increased overall activity of synap-ic NMDARs may trigger the hSMP system with a false signaldescribed in Section 3.2) and initiate a homeostatic decrease inhe membrane excitability of MSNs. Indeed, our preliminary resultsuggest that up-regulation of synaptic NR2B-containing NMDARs,hich mediates generation of silent synapses at this stage [60],
nduces a homeostatic decrease in membrane excitability of NAcSNs (unpublished data). In addition, hSMP-triggered decrease inembrane excitability of NAc MSNs is mediated in part by the
unctional expression of SK channels, and inhibition of SK chan-els partially restores the intrinsic membrane excitability of NAcSNs following exposure to cocaine [48]. Thus, during short-termithdrawal, hSMP as an effective synapse → membrane homeo-
tatic mechanism may be usurped by drugs of abuse to produceomeostatic dysregulation in NAc.
.1.2. Repeated self-administration of cocaine
After short-term (1 day) withdrawal from repeated self-
dministration of cocaine, a slight but significant decrease in theurface level of AMPARs in NAc MSNs is detected [69], whereas theverall synaptic NMDARs are likely to be up-regulated due to gen-ration of silent synapses (Lee and Dong, unpublished data). At a
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similar withdrawal time point, the intrinsic membrane excitabilityis decreased [63]. Similar to what happens following i.p. injec-tions of cocaine, hSMP may contribute to the decreased membraneexcitability of NAc MSNs as a result of increased NMDAR-signalingat this withdrawal time point.
4.2. Long-term withdrawal
4.2.1. Repeated i.p. injections of cocaineAfter 7–21 days of withdrawal from repeated i.p. injections of
cocaine, excitatory synapses onto NAc MSNs appear to be strength-ened. On the presynaptic side, glutamate release is presumablyincreased by reduced presynaptic group 2/3 metabotropic gluta-mate receptor (mGluR2/3) signaling [70]. mGluR2/3 located onthe presynaptic terminals normally sense the extrasynaptic levelof glutamate and inhibit presynaptic release upon activation [71].Following long-term withdrawal, the basal level of non-synaptic,extracellular glutamate is reduced [72–76], presumably due todecreased activity of cystine–glutamate exchanger [72] and glu-tamate transporter-1 (GLT-1) on the glial cell membrane [77],which together decrease presynaptic mGluR2/3 activation [70].Meanwhile, mGluR2/3 receptors and the downstream signalingare also down-regulated [78]. Both alterations in mGluR2/3 activa-tion and signaling result in disinhibition of presynaptic glutamaterelease [79]. On the postsynaptic side, AMPARs are up-regulated atboth protein and functional levels [40,44,59]. Specifically, GluR1/2-containing AMPARs are up-regulated at excitatory synapses ontoNAc MSNs [40,44]. In addition, an increase in the number and sizeof the dendritic spines is also observed at this time point [80,81],suggesting a coordinated remodeling process that functions tostrengthen the excitatory transmission onto NAc MSNs.
In parallel to increased excitatory synaptic transmission, theintrinsic membrane excitability of NAc shell MSNs is decreasedduring long-term withdrawal from repeated i.p. injections ofcocaine [48,61–63]. Thus, it appears that the decreased membraneexcitability of NAc MSNs is a consequence of increased excitatorysynaptic strength via hSMP. Consistent with this notion, SK typecalcium-activated potassium channels, which are one of the keyexpression substrates for hSMP, continue to be up-regulated inNAc MSNs at this withdrawal time point [48,63]; selective inhibi-tion of SK channels partially restores the membrane excitability ofNAc MSNs in cocaine-pretreated rats [48]. Nonetheless, the reversecould also be true, that the decrease in membrane excitationleads to a homeostatic compensatory increase in the postsynapticresponsiveness, namely synaptic scaling. This may occur espe-cially since a decrease in the membrane excitability already existsduring short-term withdrawal, in the absence of changes in synap-tic strength. The causal relationships between these two sets ofchanges during exposure or withdrawal remain to be determined.
The homeostatic regulations underlying the above-mentionedchanges include glutamate exchanger- and transporter-mediatedglutamate homeostasis [82], mGluR-mediated modulation ofpresynaptic release, AMPAR-mediated postsynaptic scaling, andpotentially, hSMP. Alterations in the NAc at this stage can be trig-gered by reduced upstream (e.g., mPFC) activity. However, althoughenhanced transmission efficacy may partially compensate for thereduced presynaptic activity, it also increases the dynamic rangeupon which subsequent cocaine exposure could exert its effect;decreased membrane excitability of NAc MSNs may reduce thebasal firings and, in effect, enhances the signal-to-noise ratio when
cocaine challenge occurs (see Section 4.3 re-exposure). There-fore, the above-mentioned homeostatic regulations in the NAcmay have been taken advantage of by addictive drugs followinglong-term withdrawal to refine cocaine-related signal transmis-sion/processing in the NAc.
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Other potential homeostatic mechanisms regulating synap-ic transmission in the NAc include endocanabinoid-mediatedegulation of glutamate release. Activity in NAc MSNs inducesonic release of endocanabinoids, which activates presynapticB1 receptors and inhibits presynaptic release of glutamate83]. Decreased postsynaptic responsiveness and thus decreasedostsynaptic activity may induce lower levels of CB1-mediated
nhibition of presynaptic release. As such, the retrograde CB1-ignaling may function as a homeostatic mechanism balancingresynaptic release and postsynaptic responsiveness. This poten-ial homeostatic mechanism appears to be disrupted by cocainexposure, as one i.p. injection of cocaine completely abolishesB1 receptor-mediated synaptic modulation in NAc MSNs [84].hus, CB1-signaling as a potential homeostatic regulator for pre-s. postsynaptic coordination [85] can be targeted by drugs ofbuse to induce homeostatic dysregulation. Another set of potentialolecular candidates mediating homeostatic regulation between
re- and postsynaptic responses in NAc MSNs is neurexins andeuroligins. Neurexins and neuroligins are expressed pre- andost-synaptically, respectively. They bind to each other and their
nteractions affect both presynaptic release and postsynapticesponsiveness [86,87]. An increasing body of evidence suggestshat neurexin–neurligin complex is subject to dynamic regulationollowing exposure to drugs of abuse [88–91]. Though lacking spe-ific results, it is speculated that the neurexin–neuroligin complexs tweaked by exposure to drugs of abuse, disrupting the pre- andostsynaptic homeostasis.
.2.2. Repeated self-administration of cocaineAMPAR-mediated synaptic transmission in the NAc is enhanced
fter long-term withdrawal from extended access to cocaine self-dministration, and this enhancement is achieved by synapticncorporation of calcium-permeable, GluR2-lacking AMPA recep-ors (CP-AMPARs) [69]. It remains a controversy whether suchynaptic recruitment of these atypical AMPARs also occurs dur-ng long-term withdrawal from repeated i.p. injections of cocaine;ne lab shows rectification of AMPAR EPSCs during long-term with-rawal [92], suggesting the recruitment of CP-AMPARs at synapses,hereas others do not detect such a change [44,59]. Nonethe-
ess, CP-AMPARs are different from the “regularly” expressedluR2-containing AMPARs in that CP-AMPARs have higher sin-le channel conductance and a higher permeability to calciumons [55,93]. As such, synaptic expression of CP-AMPARs notnly increases the overall AMPAR-mediated transmission (synap-ic strength), but also activates calcium-based signals. Collectively,he overall strength of excitatory synapses at NAc MSNs isp-regulated during long-term withdrawal from cocaine self-dministration.
Similar to repeated i.p. injections of cocaine, cocaine self-dministration also reduces cystine–glutamate exchange and thusecreases the extracellular glutamate level in NAc during long-erm withdrawal [72,94,95]. The potential homeostatic regulationsmong decreased extracellular glutamate, potentially up-regulatedresynaptic release of glutamate, and increased postsynapticesponsiveness are discussed in the previous section.
Unlike what follows repeated i.p. injections of cocaine, follow-ng cocaine self-administration, the membrane excitability of NAc
SNs returns to the baseline level during long-term withdrawal63]. The increased excitatory synaptic strength and the lack ofhange in membrane excitability of NAc MSNs taken together raisehe question of why hSMP and/or synaptic scaling lose their effi-
acy under this withdrawal condition. As summarized above, thenly known difference in the increased excitatory synaptic strengthetween i.p. injections and self-administration of cocaine is,rguably, the increase in CP-AMPARs in self-administering animals.or hSMP, postsynaptic NMDARs act as the detector of synaptic
n Research 216 (2011) 9–18 15
strength and initiate the first step of signaling (calcium influx)toward regulation of membrane excitability [48]. CP-AMPARs aresimilar to NMDARs in that they also conduct calcium ions and thusmay augment calcium-based signaling in hSMP-based regulation.However, it has been known that calcium-based signaling is alsounder stringent compartmentational regulation; a calcium wavegenerated from one signaling cascade may not functionally diffuseinto other calcium-mediated signaling cascades [96,97]. In MSNs(in either the NAc or dorsal striatum), calcium-signaling, depend-ing on its source, differentially regulates voltage-gated ion channelsand thus exerts different impact on the membrane excitabilityof these neurons [98–101]. Furthermore, in addition to conduct-ing calcium ions, CP-AMPARs are also permeable to zinc ions,which are co-released with glutamate from presynaptic termi-nals and, in turn, trigger zinc-dependent intracellular signalingcascades that are not activated by the NMDAR-calcium pathway[102–104]. Nonetheless, the physiological consequences of Ca2+
signalings following Ca2+ influx from NMDARs and CP-AMPARscan be different. If NMDARs and CP-AMPARs play opposite rolesin hSMP, the lack of membrane alteration in rats withdrawn fromcocaine self-administration may be explained as a cancellationof these two effects. At the theoretical level, if CP-AMPARs playa role opposite to that of NMDARs, it once again suggests thatthe detector/sensor of homeostatic mechanisms is targeted bydrugs of abuse and the resulting false signal induces homeostaticdysregulation.
4.3. Re-exposure
4.3.1. Following long-term withdrawal from repeated i.p.injections
Twenty-four hours after a single re-exposure to cocaine orcocaine-related cues during long-term withdrawal from repeatedi.p. cocaine injections, the surface levels of AMPARs are substan-tially decreased to a degree comparable to or lower than that innaïve or saline-treated animals [44]. In addition, re-exposure tococaine substantially increases the level of non-synaptic, extracel-lular glutamate in the NAc [72]. On the other hand, re-exposureto cocaine from long-term withdrawal brings the decreasedmembrane excitability of NAc MSNs back to “normal” (as in saline-treated control rats) [63]. To some extent, re-exposure can beregarded as an acute stimulation. It is likely that effects observedafter re-exposure are a combination of regulated and homeostaticprocesses.
4.3.2. Following long-term withdrawal from repeatedself-administration of cocaine
Thus far, no results are available about regulation of excita-tory synaptic strength of NAc MSNs upon re-exposure to cocaineduring long-term withdrawal from cocaine self-administration,but one study provides a hint suggesting that the function ofsynaptic AMAPRs may be up-regulated [105]. Similar to thatfollowing i.p. injections of cocaine, re-exposure to cocaine dur-ing long-term withdrawal from cocaine self-administration alsoabruptly increases extracellular glutamate [75], which may leadto a decrease in presynaptic release of glutamate onto NAc MSNs.On the other hand, upon re-exposure, the intrinsic membraneexcitability of NAc MSNs becomes higher than that in saline-treated control rats [63]. Again, although there may be homeostatic
links among the putative increased postsynaptic responsiveness,decreased presynaptic release, and increased membrane excitabil-ity of NAc MSNs upon re-exposure to cocaine, regulated plasticitythat acutely occurs upon re-exposure must be taken into consider-ation for these effects.
16 Y.H. Huang et al. / Behavioural Brain Research 216 (2011) 9–18
Table 1Summary of cocaine-induced alterations at excitatory synapses and intrinsic membrane excitability of NAc neurons.
. A hypothesis of homeostatic dysregulation followingxposure to cocaine
We have summarized cocaine-induced alterations in presynap-ic release of glutamate, postsynaptic responsiveness, and intrinsic
embrane excitability of NAc MSNs (Table 1). Using the twoasic forms of homeostatic plasticity, one between pre- and post-ynaptic activities, and the other between synaptic input andembrane excitability as a conceptual basis, we attempt to pro-
ide a homeostatic point of view in understanding cocaine-inducedubcellular alterations in NAc MSNs (Fig. 3).
Homeostatic plasticity expresses in multiple forms, whichogether with other homeostatic responses normally are suffi-ient to maintain normal functional output of neurons against
variety of internal/external environmental changes, develop-ental/metabolic turnover, and even pathogenic insults. However,
n drug-exposed animals, the brain reward system continuouslyrifts away from the previously set homeostatic point (so-calledllostasis), resulting in addiction-related behavioral alterations.he functional output of neurons in addiction-associated brainegions (e.g., the NAc) is substantially altered, raising the key ques-
ion of why homeostatic mechanisms fail in drug-exposed animals.ased on the results from cocaine-treated animals (summarized
n the early sections of this manuscript), we hypothesize thathe key components determining homeostatic plasticity/responsesre targeted by drugs of abuse to produce false signals, result-
ig. 3. Summary of synaptic and membrane alterations in NAc neurons followingon-contingent exposure to cocaine. A typical non-contingent cocaine procedure
s to treat the animal with 5-day i.p. injections of cocaine (15–20 mg/kg/day), fol-owed by different withdrawal periods. An important synaptic alteration in NAceurons during the late phase of cocaine exposure and short-term withdrawal ishe appearance of silent synapses enriched in NR2B-containing NMDARs. Theseilent synapses decrease over time during long-term withdrawal. Synaptic/surfaceMPARs in NAc neurons are altered minimally if any (i.e., a slight decrease) duringhort-term withdrawal, but are greatly up-regulated during long-term withdrawal.he intrinsic membrane excitability of NAc neurons is decreased throughout short-nd long-term withdrawal. Based on the timing of these cocaine-induced synapticnd membrane alterations, we hypothesize that these cellular alterations are home-statically linked. For example, although synaptic AMPARs are not up-regulateduring short-term withdrawal, the increased NMDAR-mediated activity may cre-te a false signal of increased synaptic strength to trigger hSMP, resulting inbserved decrease in membrane excitability (?1). Furthermore, newly generatedilent synapses provide extra synaptic slots that may facilitate synaptic recruit-ent of new AMPARs during long-tem withdrawal (?2). The synaptic recruitment
f AMPARs during long-term withdrawal may result from regulated or homeostaticlasticity. For homeostatic plasticity, a potential mechanism is that the decreasedembrane excitability may trigger another round of membrane-to-synapse home-
static response, resulting in an increase in excitatory synaptic strength in NAceurons (?3).
ing in homeostatic dysregulation. This notion is exemplified in thepotential hSMP-mediated regulation of membrane excitability ofNAc MSNs in cocaine-exposed rats. During short-term withdrawalfrom repeated i.p. injections of cocaine, although the excitatorysynaptic strength (synaptic AMPARs) of NAc MSNs appears notto be changed, synaptic NMDARs as the sensors of hSMP areup-regulated. This projects a false image that excitatory synapticstrength is increased; the membrane excitability of NAc MSNs maythus be decreased via hSMP.
Whereas this hypothesis is formulated mainly based on resultsfrom cocaine-treated animals, we speculate that homeostatic regu-lation and dysregulation are common phenomena occurring uponother drugs of abuse as well, although the specific molecular andcellular machinery mediating these homeostatic responses maydiffer. Nonetheless, this hypothesis suggests that despite the enor-mous number of drug-induced cellular and subcellular alterations,the key substrates of homeostatic responses should be focusedon with emphasis because alterations of these key homeostaticsubstrates may be the primary targets for drugs of abuse to trig-ger cascades of secondary changes. This hypothesis also suggestsa homeostatic point of view in correcting/restoring drug-inducedneuronal alterations. Studies using gene chip or high through-put screening indicate that more than 20,000 molecules in thebrain are altered during exposure to drugs of abuse, and evenmore during withdrawal from chronic exposure [106–108]. Appar-ently, individual correction of all these identified and possibly evenmore unidentified drug-induced alterations is not a feasible clinicalapproach for treating addiction. Instead of focusing on individualtargets, an alternative approach is to manipulate a small number ofkey homeostatic molecules, to tweak the endogenous homeostaticmechanisms, and thus to restore the normal functions of the asso-ciated brain regions at the functional level. This homeostasis-basedapproach is not uncommon in medical practice, especially for treat-ing mental disorders. For example, for most of the antidepressantsto achieve therapeutic efficacy, it takes a long latent period duringwhich cascades of homeostatic regulations occur. The monoaminesystem that these antidepressants interact with is related but notlikely to be the direct substrates mediating depression, whereas thehomeostatic responses triggered by monoamine-antidepressantinteraction might.
6. Concluding remarks
In summary, based on cocaine-induced cellular alterations, wepropose a homeostatic plasticity-based viewpoint in understand-ing addiction-related homeostatic regulation and dysregulation.This viewpoint brings both opportunities and challenges tothe behavioral studies of drug abuse. On one hand, complexdrug-taking/seeking behaviors often involve complex molecularand cellular processes. The viewpoint of homeostatic regula-tion/dysregulation may provide a logical link that brings together
individual molecules as a homeostatic network in understandingbehavioral alterations. On the other hand, with this viewpoint inmind, behavioral alterations induced by manipulation of a singlemolecule may not be readily interpreted as an independent effectof this molecule; molecular and cellular homeostatic responses
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econdary to the manipulation of this molecule must also be con-idered.
It is also important to note that this viewpoint does not explainll drug-induced molecular and cellular adaptations. An importantut not fully discussed topic in this manuscript is drug-inducedegulated plasticity, such as LTP and LTD of excitatory synapticransmission. Indeed, we believe that it is the interaction betweenomeostatic and regulated plasticity that produces the multiplicityf cellular and molecular alterations observed in the addicted brain.o understand the neuronal basis underlying drug addiction, futuretudies must differentiate as well as relate regulated plasticity andomeostatic plasticity upon exposure to drugs of abuse.
cknowledgements
We thank Dr. Marina Wolf, Dr. Rob Malenka, and Ms. Jenny Bay-on for suggestions on the manuscript. Research of the authors haseen supported by Alcohol and Drug Abuse Research Program ofashington State, NIH DA023206, and the Alexander von Hum-
oldt Foundation.
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