Amphetamine acts within the lateral hypothalamic area to elicit affectively neutral arousal and ...

13 Pages·2013·0.39 MB·English

InternationalJournalofNeuropsychopharmacology(2014),17,63–75. ©CINP2013 ARTICLE
doi:10.1017/S1461145713000734
Amphetamine acts within the lateral
hypothalamic area to elicit affectively neutral
arousal and reinstate drug-seeking
BrookeE.Schmeichel andCraigW.Berridge
DepartmentofPsychology,UniversityofWisconsin,Madison,WI53706,USA
Abstract
Psychostimulants, including amphetamine (AMPH), exert robust arousal-enhancing, reinforcing and D
o
locomotor-activating effects. These behavioural actions involve drug-induced elevations in extracellular w
n
norepinephrine(NE)anddopamine(DA)withinavarietyofcorticalandsubcorticalregions.Thelateral lo
a
hypothalamicarea(LHA),includingthelateralhypothalamusproper,perifornicalareaandadjacentdor- d
e
d
sboymbeodthialNhEypanotdhaDlaAmpurso,jeisctiimonpslicaantdedsyinsteamppiceatiltlyivea-damnidniasrteoruesdalA-rMelaPtHedhparsobceesesnesd.eTmhoenLsHtrAateidsitnonaecrtviavtaetde from
LHAneurons.Combined,theseandotherobservationssuggesttheLHAmaybeasiteofactioninthebe- h
haviouraleffectsofpsychostimulants.Totestthishypothesis,weexaminedthedegreetowhichAMPH ttps
(10nmol, 25nmol) acts within the LHA to exert arousing, locomotor-activating and reinforcing actions ://a
c
in quietly resting/sleeping rats. Although intra-LHA AMPH robustly increased time spent awake, this a
d
e
occurredintheabsenceofpronouncedlocomotoractivationorreinforcingactions,asmeasuredinacon- m
ditionedplacepreference(CPP)paradigm.Arousingandstressfulconditionsordrugre-exposurecanelicit ic.o
relapseinhumansandreinstatedrug-seekinginanimals.GiventheLHAisalsoimplicatedinthereinstate- up
ment of drug-seeking behaviour, additional studies examined whether AMPH acts within the LHA to .co
m
rtheiantsAtaMtePaHnaecxttiionnguwisihtheidnCthPePLpHroAdiuscseudffiwciitehntsytostereminicstaAtMedPrHuga-dsemekininisgtrbateihoanv.iOouurr,raessumltesasduermedoninstrthaties /ijnp
pafafreacdtiivgemly.Cneoulltercatlivaerloyu,stahleasendobrseeinrvstaattioendsrudgem-seoenksitnragtebethhaatvpiosuyrc.hostimulantsactwithintheLHAtoelicit /article
-a
Received22April2013;Reviewed21May2013;Revised24May2013;Accepted29May2013; b
s
Firstpublishedonline29July2013 tra
c
Keywords:Arousal/waking,lateralhypothalamus,psychostimulants,reinforcement,reinstatement. t/17
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3
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Introduction effects of psychostimulants involve actions of both 21
3
DAinthenucleusaccumbensandNEintheprefrontal b
Psychostimulants are a widely abused class of drugs, y
cortex (Blanc et al., 1994; Darracq et al., 1998; g
representing a major public health concern (Gonzales u
Dickinson et al., 1988; Drouin et al., 2002a,b; Pascoli es
et al., 2010; Marshall and Werb, 2010). These drugs etal.,2005;SnoddyandTessel,1985)whileNEaction t on
exert robust arousal-enhancing and reinforcing effects within the medial septal and medial preoptic areas 05
thatcontributetotheiruseandabuse.Thebehavioural A
contributes to the arousal-promoting actions of these p
actions of psychostimulants, including amphetamine drugs(for review, Berridge,2006). ril 2
(AMPH), involve drug-induced elevations in extra- A significant challenge in the treatment of psycho- 01
cellular norepinephrine (NE) and dopamine (DA) 9
stimulant abuse is the prevention of relapse. Relapse,
within multiple cortical and subcortical regions. For
or the reinstatement of drug-seeking behaviour, can
example, the locomotor-activating and reinforcing
be triggered by re-exposure to the drug, stress and/or
contextually-conditioned cues in humansand animals
(Brown et al., 1995; Jaffe et al., 1989; Ludwig et al.,
Addressforcorrespondence:DrB.E.Schmeichel,TheScripps 1974; Shaham et al., 2000; Sinha, 2001). NE and DA
ResearchInstitute,10550NorthTorreyPinesRoad,SP30-2411,LaJolla,
also play a central role in the reinstatement of
CA92037,USA.
psychostimulant-seeking via actions within several
Tel.:[phone] Fax:[phone]
Email:[email protected] basal forebrain regions, including areas related to
64 B.E. SchmeichelandC.W.Berridge
arousal and autonomic processes, such as the medial The cannulae and EEG/EMG electrodes (when
septal area and bed nucleus of the stria terminalis applicable) were cemented into position using acrylic
(for review, Stewart, 2000; Weinshenker and cement(Esschem,USA).Animalsweregivenbuprenor-
Schroeder,2007). phine (0.01mg/kg; Reckitt Benckiser Pharmaceuticals
The lateral hypothalamic area (LHA; i.e. the lateral Inc., USA) post-surgically to alleviate pain and were
hypothalamus proper, adjacent perifornical area and allowed to recover for 5–7d before testing. All efforts
dorsomedial hypothalamus), has long-been hypoth- were made to minimize the number of animals used.
esized to participate in a variety of behavioural pro- All facilities and procedures were in accordance with
cesses, including arousal and appetitively-motivated guidelines regarding animal use and care put forth
behaviour (for review, Boutrel et al., 2010; Marchant by the National Institutes of Health and approved by
et al., 2012). Recent evidence further implicates the the Institutional Animal Care and Use Committee of
LHA in the reinstatement of drug-seeking behaviour theUniversityofWisconsin.
(Boutrel et al., 2005; Harris et al., 2005). This dorsal
D
hypothalamic region receives both NE and DA input Experimental procedures ow
n
(Baldo et al., 2003; Yoshida et al., 2006) and lo
Sleep/wakeeffects ofintra-LHA AMPH a
systemically-administered AMPH activates neurons d
e
d
w20i0th2)in. MthoereLoHvAer,(ENstEabwroaoskereectenatll.y, 2d00e1m;oFnasdtrealteedt atlo., Tpreesvtiinogusplyro(cBeedrurirdegsewanerdeFsoimotiel,ar19t9o6)t.hBorsieefldye,socnribtehde from
act within the LHA to promote arousal (Schmeichel day prior to testing, rats were transferred to sound- http
asungdgeBsetrtrhidegLeH, A20m13a)y. bCeoamsbitieneodf,actthioenseinotbhseeravroatuiosanls- aspttreinnugatwedastetshtirnegadcehdamobnetros athned caanstnauinlalesvsiastepellacsotiicl s://ac
a
promoting and other behavioural effects of psycho- threaded sleeves (Plastics One, USA). The other end de
m
stimulants,including AMPH. ofthespringwasattachedtoacounterbalanceoutside ic
To test this hypothesis, we examined the degree theouterchamber.Animalswerehousedindividually .ou
p
towhichmicroinfusionofAMPHintotheLHAelicits overnightwith freeaccessto food andwater. .c
o
arousal-promoting, locomotor-activating and reinfor- Onthemorningoftesting,between0900–1100hours, m
cing (as measured in a conditioned place preference the 33ga infusion needle was connected to PE20 tub- /ijnp
pmairnaeddigmw;heCthPePr) AacMtioPnHs. aActdsdiwtioitnhainl stthuedieLsHdAetetro- iOnnge)praontedctleodadbeydawsittahinvleeshsicsletee(larctoifiilcisaplrienxgtra(Pcelallsutliacsr /article
reinstate previously-extinguished drug-seeking, as fluid (AECF); in mM: NaCl 147, CaCl2 1.3, MgCl2 0.9, -abs
measured by CPP. Our results indicate that AMPH KCl 2.5) or drug dissolved in AECF with a 50nl air tra
c
acts within the LHA to promote affectively neutral bubble separating drug or vehicle from water. The t/1
7
arousal andto reinstatedrug-seeking behaviour. infusionneedlewasinsertedintotheguide,extending /1
4.0mm beyond the end, and secured. EEG/EMG elec- /63
trodeswereconnectedtoafieldeffecttransistorhead- /82
7
Methods stage. Baseline EEG/EMG recordings were initiated at 21
3
a point where the animal had recovered from the b
Animalsand surgery y
arousing effects of needle insertion and had returned g
u
Sixty-eight male Sprague–Dawley rats (260–280g, to quiet-rest/sleep. Infusions of 250nl (125nl/min) es
Charles River, USA) were pair-housed for at least 7d were made using a microprocessor-controlled pump t on
priortosurgerywithadlibitumaccesstofoodandwater (Harvard Apparatus, USA), following at least 1h of 05
A
on an 11:13h light:dark cycle (lights on 0700 hours). quiet-rest/sleep. Behavioural data were collected con- p
Animals were anesthetized with isofluorane and 25 tinuously onto polygraph and video recording tape ril 2
0
gauge guide cannulae (Component Supply Company, foratleast1.5h followinginfusion. 1
9
USA; Plastics One, USA) were stereotaxically im- Cortical electroencephalograms (EEGs) and electro-
planted, aimed bilaterally at the LHA (flathead; in myograms (EMGs) were recorded as described pre-
mm, −2.8 A/P, range of ±1.8–2.15 M/L, −3.8 V/D; viously (Berridge and Foote, 1996). EEG/EMG
angled 6° from vertical). A subset of animals was recordings were scored for the following behavioural
alsoimplantedwithabipolarelectroencephalographic state categories: (i) slow-wave sleep (high-voltage
(EEG) electrode into the frontal cortex (+2.7 A/P, ±1.5 EEG, low voltage EMG); (ii) rapid-eye movement
M/L)andabipolarelectromyographic(EMG)electrode (REM) sleep (low-voltage EEG combined with EMG
intothedorsalneckmuscle.Allcoordinatesarerelative activity that was approximately 50% lower amplitude
to bregma. thanthatobservedinslow-wavesleep,withoccasional
Amphetamine action inthe lateralhypothalamicarea 65
Pre-infusion
EMG
mtt
3
EEG
v
o
pt
Post-infusion
fx
EMG
AP = –2.8 from Bregma EEG
60 s
Fig.1. Leftpanel:photomicrographdepictingplacementoftheinfusionneedlewithinthelateralhypothalamicarea(LHA).
Arrowindicatestheventralmostextentoftheneedletrack.Rightpanel:rawcorticalelectroencephalograph(EEG)and
electromyograph(EMG)tracesbeforeandafterintra-LHAamphetamine(AMPH).Shownare5minrecordingsofEEG/EMG
D
priorto(pre-infusion)andbeginning2minfollowingtheendofaunilateralintra-LHAinfusionofAMPH(25nmol; o
w
post-infusion).Priortotheinfusion,theanimalspentthemajorityoftimeasleep,reflectedinlarge-amplitude,slow-wave n
lo
EEGactivitycombinedwithlow-amplitudeEMGactivity.FollowingAMPHinfusion,theanimalspentamajorityoftime ad
awake,reflectedinadecreaseinEEGamplitudeandanincreaseinEMGamplitude,includinglargedeflectionsindicativeof ed
activemovement.3V=thirdventricle;fx=fornix;mtt=mammillothalamictract;opt=optictract. fro
m
h
ttp
s
short-duration, large-amplitude deflections due to by others to characterize the motor-activating effects ://a
c
a
muscle twitches); (iii) waking (low-voltage EEG, sus- of systemically-administered psychostimulants (Segal, d
e
m
tained high-voltage EMG of an average amplitude at 1975).
ic
least twice that observed in slow-wave sleep, often .o
u
with large-amplitude movement deflections; Fig. 1). p.c
CPPtesting withsystemic andintra-LHAAMPH o
Tobescoredasadistinctstate,theEEG/EMGactivity m
patterns neededto persistfor515s. The reinforcing actions of systemic and intra-LHA /ijnp
30Tmimineesppoecnht, iinncleuadcihngstatwteoweapsocshcsoreimdmfoerdiaetaeclhy AmMalsPHusiwngeraeCasPsPesaspedpairnattuwsocosnespiasrtiantge ogfrotuwposdoifstainnci-t /article
prior to infusions (PRE1=0–30min; PRE2=30–60min) chambers (each 34×24.5×30cm; black walls/textured -ab
s
and three epochs immediately following infusions floor; black and white striped walls/smooth floor) tra
(POST1=0–30min; POST2=30–60min; POST3=60–90 and a neutral, free-choice entry zone (24.5×17× ct/1
min). EEG/EMG recordings were scored by an ob- 10cm). Animals were tested over three phases: pre- 7/1
server blindto experimental conditions. conditioning (1d; 10min session, access to both /63
/8
chambers), conditioning (8d; 20min session, access 2
7
restricted to one chamber) and post-conditioning (1d; 21
Locomotoranalyses 3
10minsession,accesstobothchambers).Forsystemic b
y
Locomotor activity (i.e. quadrant entries and rears) administration, conditioning consisted of eight con- g
u
was scored in animals that received the highest dose secutivetreatmentdaysduringwhichanimalsreceived es
of AMPH infused unilaterally and bilaterally. s.c. injections of a wake-promoting dose of AMPH t on
Behaviour was recorded continuously using black (0.25mg/kg) and vehicle (saline) on alternating days. 05
A
and white, low-level illumination video camera, start- Forintra-LHAAMPH,conditioningconsistedofeight p
ing after insertion of the infusion needle and continu- consecutive treatment days during which animals ril 2
0
ing for at least 2h following infusion. Locomotor received bilateral intra-LHA infusions (250nl/120s) 1
9
activity was scored from videotape by a trained ob- of a wake-promoting dose of AMPH (25nmol) and
serverblindtotreatmentconditionsusingacomputer- vehicle (AECF) on alternating days. All treatments
based event recorder (The Observer, Noldus were administered in a transfer cage (similar to the
Information Technology, The Netherlands). The fre- home cage) 15min prior to placement in the CPP
quency of quadrant entries and rears (both wall and apparatus.Testingsessionswererecordedonadigital
free)wasscoredforthree30minepochs,includingone video recorder (JVC Enverio, USA). Time spent in
epoch prior to infusion (PRE1=0–30min) and two each chamber for both pre- and post-conditioning
epochsfollowinginfusion(POST1=0–30min;POST2= test days was scored by a trained observer using
30–60min). These measures are similar to those used a computer-based event recorder (The Observer,
66 B.E. SchmeichelandC.W.Berridge
NoldusInformationTechnology).Animalsweretested Drugs
in a biased design paradigm, with drug paired to the
D-Amphetaminesulfate(AMPH;Sigma,USA)wasdis-
non-preferred chamber as determined by the pre-
solved in either AECF for intra-tissue infusions or
conditioning test.
saline for systemic administration. Doses used in the
intra-tissue infusion studies were based on previous
studies demonstrating this dose range elicited robust
CPPreinstatementwithsystemic AMPH
waking when infused into the medial septal and
A separate group of animals was used to examine medial preoptic areas (Berridge et al., 1999) and
the effects of systemic AMPH on reinstatement of locomotor-activating and reinforcing effects when
CPP. In these studies, a CPP was first established infused into the nucleus accumbens (Carr and White,
using systemically-administered AMPH (0.25mg/kg 1983; Gerdjikov and Beninger, 2005, 2006; Newman
s.c.), as described above. Following establishment of etal.,2013;Schildeinetal.,1998).ForCPPstudies,sys-
a CPP, animals underwent extinction training during temicdoseswereusedthathavebeendemonstratedto D
o
w
which they received s.c. saline for six consecutive elicit pronounced arousal-promoting actions in the n
lo
days in both the AMPH- and saline-paired chambers absence of a strong locomotor activation (Berridge a
d
(alternating days; 20min session, access restricted to et al., 1999; Berridge and Stalnaker, 2002). In the case ed
onechamber).Forthenexttwodays,animalsreceived of systemic AMPH reinstatement of CPP, a lower fro
m
asaline injection followedbyafree-explore extinction dose of AMPH (0.10mg/kg for reinstatement vs. 0.25 h
test (10min session, access to both chambers). Time mg/kg for conditioning) was used similar to previous ttp
s
spent in the drug-paired chamber was measured. studies of psychostimulant-induced reinstatement in ://a
c
If extinction criterion was not met (<50% of post- animals(Cruzetal.,2008;MuellerandStewart,2000). a
d
e
conditioning test value for two consecutive days), m
extinction training continued with a saline injection ic.o
prior to free-explore extinction tests every 1–3d. Histologyanddataselection up
After meeting extinction criteria (range 12–25d, aver- .co
Following experimentation, animals were deeply m
age 16.6±1.6d), animals received systemic AMPH anesthetized and perfused transcardially with at least /ijn
p
(0.10mg/kg s.c.) and were tested for reinstatement of 60ml of 4% formaldehyde. The brain was removed /a
CPP 15min later (10min session, access to both and placed in fixative for at least 24h, frozen and rtic
le
chambers). 40μM sections were collected throughout the rostral- -ab
caudal extent of the LHA. The sections were stained stra
CPPreinstatementwithintra-LHA AMPH wexittehnNt eoufttrhael-Rneededdlyee.trTahckelwocaastioidneonftitfiheedv.eDntartaal-mweorset ct/17
/1
An additional group of animals was used to examine included in the analyses for those cases in which /63
the effects of intra-LHA AMPH on reinstatement of both the EEG/EMG recordings were electrically ad- /82
7
CPP. In these studies, CPP (AMPH; 0.25mg/kg s.c.) equate(whenapplicable)andthehistologicalanalyses 21
and extinction were established as described above. verified accurate placements of infusion needles 3 b
Aftermeetingextinctioncriteria(range8–28d,average (Fig. 1). y g
u
15.1±2.5d), animals received a bilateral intra-LHA es
infusion (250nl/120s) of AMPH (25nmol) or vehicle t on
(AECF) and were tested for reinstatement 15-min Statisticalanalyses 05
A
following infusion (10-min session, access to both AMPH-inducedchangesinEEG/EMG-basedmeasures p
chambers). The order of intra-LHA infusions (AMPH of sleep/wake state and locomotor activity were ana- ril 2
0
or AECF) was counterbalanced across animals, with lysedstatisticallywithatwo-waymixed-designanaly- 1
9
extinction training occurring prior to each treatment. sis of variance (ANOVA) with drug treatment as the
Limited pilot studies indicated that initial intra-LHA between-subjects variable and time as the within-
needleinsertionshadthepropensitytoalterreinstate- subjects variable. Where between-subjects omnibus
ment outcomes. Thus, before receiving intra-LHA in- tests proved significant, pairwise post-hoc group com-
fusions, a mock infusion/needle-drop was performed parisons for each time epoch were made using a one-
on the day prior to reinstatement testing. This per- way ANOVA. CPP-based measures of reinforcement
mitted acclimation to mild infusion restraint and were analysed statistically with a matched-pair t test.
minimized tissue damage associated with insertion of For CPP reinstatement, a one-way within-subject
aneedleon thedayof testing. ANOVA was used (4 levels: pre-conditioning,
Amphetamine action inthe lateralhypothalamicarea 67
post-conditioning, extinction, reinstatement) followed 1800 Waking
AMPH Bilat.
byplanned pairwisepost-hoc groupcomparisons.
1500 AMPH 25 nmol
AMPH 10 nmol **
1200 Vehicle ** * **
s)
Results e ( 900
m
Ti 600
Effectsof intra-LHAAMPH onwaking
300
Toassessthearousal-promotingeffectsofAMPH,rats
0
received 250nl intra-LHA infusions of either vehicle
PRE2 PRE1 POST1 POST2 POST3
(AECF; n=8), 10nmol AMPH (n=7) or 25nmol
AMPH (n=8) and EEG/EMG-indices of sleep/wake 1800 Slow-wave sleep
state were analysed. In prior work with subcortical 1500
infusions of AMPH or NE receptor agonists, we 1200 D
observed that unilateral infusions were sufficient to e (s) 900 own
elicit waking (Berridge et al., 1999; Schmeichel and m lo
Ti 600 a
Berridge, 2013). Thus, to minimize the number of d
** * * e
animals needed for these studies, all infusions were 300 ** d fro
unilateral,witheachanimalreceivingadifferenttreat- 0 m
ment in each hemisphere on separate days, except for PRE2 PRE1 POST1 POST2 POST3 http
oAnMePgHrou(np=t5h).atArsecreeipvoerdtebdilaptreervailouinstlrya-(LBHerArid2g5enmanodl 600 REM sleep s://ac
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Foote, 1996; Berridge and O’Neill, 2001), animals de
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supnedNneotrtashigemnseiafijtocearsinttityngdoicfffoentridemnitecioesnassil.neetpimperisoprentot aiwnfaukseionosr Time (s) 300 ic.oup.co
asleepwereobservedpriortoanytreatmentorfollow- * m
ingvehicleinfusionsintotheLHA(Fig.2).Incontrast, ** * /ijnp
intra-LHA infusion of AMPH elicited dose-dependent 0 PRE2 PRE1 POST*1* POST2 POST3 /artic
increases in waking and decreases in slow-wave and le
REM sleep (Fig. 2; Awake: treatment, F(3,24)=19.49, Fig.2. Intra-lateralhypothalamicarea(LHA)infusionsof -abs
p<0.001; time, F(4,96)=22.47, p<0.001; treatment×time, amphetamine(AMPH)dose-dependentlyincreaseswaking tra
c
F(12,96)=3.92, p<0.001; Slow-Wave Sleep: treatment, anddecreasesslow-waveandREMsleep.Shownarethe t/1
7
F(3,24)=11.54, p<0.001; time, F(4,96)=21.13, p<0.001; effectsofunilateralinfusionofvehicle,10nmolAMPH, /1
tmreeanttm, eFn(3t,×2t4i)m=5e.,76F,(1p2,<960).=013;.5t7i,mep,<F0(.40,0961);=4R.2E1M, :p<t0re.0a1t-; 2in5funmseodliAntMoPthHe,LaHndAboilnatteirmaleisnpfuensitoinnof25nmolAMPH /63/827
treatment×time, F(12,96)=1.10, p=0.37). When infused e(EleMctGro)e-dnecfiepnhedalowgarkapinhg,(EsEloGw)-/weleacvterosmleyepog,raanpdhyREMsleep 213
unilaterally at 25nmol, the average latency for b
AMPH-induced waking was 265±54s from the start states.Symbolsrepresentmean(±S.E.M.)time(s)spent y g
awakeper30minepoch.PRE1andPRE2represent u
oignfetrtrhai-enLc1Hr2eA0asseinisnffuiunssioiwonnaskwoinfitg2h5aannrdmanodgleeAcroMefaP9seH6s–4pi9nr4ossdl.ouBwciel-dawtealarvare-l peapnreoim-cinhafslus..s*ipo<n0e.0p5o;c*h*sp.<P0O.0S1Tc1o–mPOpaSrTe3drteoprveesheinctlep-torseta-tiendfusion est on 05
A
andREMsleepthanthatseenwithunilateralinfusions p
of this dose(Fig.2). less effective/consistent at eliciting waking beyond ril 20
As shown in Fig. 3, unilateral AMPH elicited 1
levelsobservedineitherpre-infusionepochsorfollow- 9
waking when infused into the majorityof the medial-
ing vehicle treatment (i.e. <1200s, assigned values of
lateral extent of the dorsal hypothalamus also known
1and2;seeFig.3).Bilateral25nmolAMPHinfusions
to contain neurons implicated in the modulation of
wereplacedcentrallywithintheLHA(i.e.perifornical
sleep/wakestate(i.e.HCRTandmelanin-concentrating
region; not shown).
hormonesynthesizingneurons;(Swansonetal.,2005),
including the lateral hypothalamus proper, adjacent
Effectsof intra-LHA AMPHonlocomotoractivity
perifornical area, and dorsomedial hypothalamus. In
contrast, infusions outside of this region, including Theabove-describedstudiesobservedmaximalwaking
ventrally, dorsally and laterally, were substantially with bilateral intra-LHA infusion of 25nmol AMPH.
68 B.E. SchmeichelandC.W.Berridge
mtt 2 ZI
2 ZI
2
2 2 c p d
AHN fx 5 4DMH 4 fx 3 4 o pt
V3
LHA V3 23 LHA
M H 4 o pt
V
3 1
VMH
500 µm
ARH 2 ARH
D
o
AP = –2.3 from Bregma AP = –2.8 from Bregma w
n
lo
a
d
Fig.3. Schematicdepictionofthelocationofunilateralinfusionof25nmolamphetamine(AMPH)andtheamountoftime e
d
spentawakeassociatedwitheachinfusion.Symbolsindicatethepositionoftheventraltipoftheinfusionneedledetermined fro
fromhistologicalsections.Numeralsspecifytherangefortimespentawakeinthefirst60minfollowingtheinfusion m
(1=0–600s;2=601–1200s;3=1201–1800s;4=1801–2400s;5=2401–3000s).Noanimalswereawake>3000sfollowinginfusion. http
Vspeohnictalen-etroeuastelydwraatksedfiosprlraeyleadtivaelmyebarnieftimpeeriaowdsaktoeignrotohmis,peeartioadndofd7r3in9k±8a1nds,trheeflnecrteintugrnthteofsalcetetph.aAtMtyPpHica-ilnlyduanceimdawlsakwinilgl s://ac
wasobservedwithinfusionsplacedwithinthemajorityofthemedial–lateralextentofthedorsalhypothalamus.Infusions ad
e
outsideofthisdorsalregionweresubstantiallylesseffectiveatelicitingwakingbeyondlevelsobservedineitheruntreated m
ic
pre-infusionepochsorfollowingvehicletreatment.AHN,anteriorhypothalamicnucleus;ARH,arcuatenucleus .o
u
hypothalamus;cpd,cerebralpeduncle;DMH,dorsomedialnucleushypothalamus;fx,columnsofthefornix;LHA,lateral p
.c
hypothalamicarea;mtt,mammillothalamictract;opt,optictract;V3,thirdventricle;VMH,ventromedialnucleus o
m
hypothalamus;ZI,zonaincerta.Numbersbelowpanelsrefertocoronallevelrelativetobregma(modifiedfromSwanson, /ijn
1992). p/a
rtic
le
To assess whether AMPH-induced waking is associ- infusions in the lateral vs. perifornical vs. medial por- -ab
s
atedwithlocomotoractivation,wescoredthenumber tions of the dorsal hypothalamus. Importantly, the tra
c
of quadrant entries and rears in these same animals low level and pattern of locomotor activity observed t/1
7
prior to and following unilateral vehicle (AECF; n=7) in this study is similar to that seen with spontaneous /1
or 25nmol AMPH (n=8), or bilateral 25nmol AMPH waking(BerridgeandFoote,1996)andissubstantially /63
/8
(n=5) infusions. One case was dropped from the below levels seen with behaviourally-activating 2
7
vehicle group due a video-recording malfunction. As doses of systemically-administered psychostimulants 21
3
described above, animals were tested during the rest- (Kuczenski et al., 1991, 1997; Segal, 1975; Segal and b
y
ing phase and thus spent the majority of time asleep Kuczenski, 1997) or with AMPH infusion into the g
u
prior to the infusion, resulting in low locomotor nucleus accumbens at doses similar to those used in es
activity. the current studies (Newman et al., 2013; Schildein t on
Despite the robust arousal-promoting effects of et al., 1998). No evidence of stereotypy was observed 05
A
intra-LHA AMPH (Fig. 2), infusion of AMPH into with eitherunilateralor bilateralAMPH infusions. p
the LHA did not elicit a pronounced locomotor acti- ril 2
0
vation. The only increase in locomotor activity was a 1
Effectsof intra-LHAAMPH onreinforcement (CPP) 9
modest, yet statistically significant, increase in the
number of quadrant entries during the first 30min TodeterminethedegreetowhichAMPHactionwithin
following bilateral 25nmol AMPH infusion (Table 1; the LHA is reinforcing, we examined whether intra-
Quadrant entries: treatment, F =8.42, p<0.01; LHAAMPHinfusionelicitsaCPP.Wefirstconfirmed
(2,17)
time, F =8.81, p<0.01; treatment×time, F =3.22, thatsystemically-administeredAMPH(0.25mg/kgs.c.)
(2,34) (4,34)
p<0.05; Rears: treatment, F =3.67, p<0.05; time, elicits a CPP under these testing conditions. Prior
(2,17)
F =5.73, p<0.01; treatment×time, F =1.65, studies in our laboratory demonstrated that this sys-
(2,34) (4,34)
p=0.19). There were no differences in the magnitude temic dose of AMPH produces sustained increases in
of locomotor activity observed following AMPH waking in the absence of a prominent locomotor
Amphetamine action inthe lateralhypothalamicarea 69
Table1. Locomotoractivityasmeasuredbynumberofquadrantcrossesandrearspriortoandfollowingvehicle,unilateral
25nmolamphetamine(AMPH)andbilateral25nmolAMPHintra-lateralhypothalamicarea(LHA)infusions
Quadrantcrosses Rears
Time(min) PRE1 POST1 POST2 PRE1 POST1 POST2
Vehicle 0.3±0.3 0.3±0.2 1.4±0.8 0±0 0±0 0±0
AMPHunilateral 0.4±0 6.4±2.7 1.6±0.7 0±0 1.0±0.1 0±0
AMPHbilateral 0±0 13.8±5.3* 7.0±3.3 0±0 1.8±1.0 0.4±0.4
Meannumber±S.E.M.ofquadrantcrossesandrearsinone30minepochpriorto(PRE1)andtwo30minepochsfollowinginfusion
(POST1andPOST2).Animalsreceivedeitheraunilateralintra-LHAinfusionofvehicleor25nmolAMPHorabilateralinfusion
of25nmolAMPH.UnilateralvehicleorunilateralAMPHhadnosignificanteffectsonlocomotoractivityasmeasuredbyquad- D
rantentriesorrears.Themodest,yetsignificant,increaseinquadrantcrossesobservedinPOST1inthebilateralAMPH-treated ow
n
animalsissimilartothatseenwithspontaneouswaking(BerridgeandFoote,1996;Españaetal.,2001).*p<0.05vs.vehicletreated lo
a
animals. d
e
d
fro
m
600 PRE COND 600 PRE COND http
POST COND POST COND s
side (s) 400 ** 400 ://acade
d m
aire ic.o
p u
me drug- 200 200 p.com/ijn
Ti p
/a
0 0 rtic
le
s.c. AMPH (0.25 mg/kg) LHA AMPH (25 nmol) -a
b
s
Fig.4. Reinforcingeffectsofs.c.amphetamine(AMPH)butnotintra-lateralhypothalamicarea(LHA)AMPH,asmeasuredby tra
c
conditionedplacepreference(CPP).Leftpanel:awake-promotingdoseofs.c.administeredAMPH(AMPH0.25mg/kgs.c.) t/1
elicitedasignificantincreaseintimespentinthedrug-pairedside.Rightpanel:incontrast,awake-promotingdoseofAMPH 7/1
bilaterallyinfusedintotheLHA(25nmol)didnotincreasetimespentinthedrug-pairedside.Barsdisplaymean(±S.E.M.)time /63
(s)spentinthedrug-pairedsideovera10mintestingperiod.**p<0.01vs.PRECOND. /82
7
2
1
3
b
y
activation (Berridge and Stalnaker, 2002). AMPH AMPH did not elicit a CPP (Fig. 4, Right panel; g
u
aindmthienitsimtraetisopnen(nt=in18th)epdrorduug-cpeadiraedsicghnaifimcbaenrt,idnecmreoanse- tin8=tr0a.-7L3H; Ap=0A.4M9)P.HThiisnfluascikonofarlesionfocrocnetmraesntst fwoliltohwitnhge est on
strating reinforcing actions of this systemic dose of robust reinforcing effects of similar doses of AMPH 05
AMInPHas(eFpiagr.a4te, Lgerfotuppanoefl;atn1i7m=−al9s.7(n0;=p9<),0w.0e01th).enexam- i1n9f8u3s;edCuinntnointhgheanmucalenuds aKceclulemy,be1n9s92(;CaGrerradnjidkoWvhainted, April 2
0
ined the degree to which intra-LHA AMPH elicited a Beninger, 2005, 2006; Kelley and Throne, 1992; 1
9
CPP. For these studies, we targeted the central region Schildeinet al.,1998).
of the LHA (i.e. perifornical area), a region within
which AMPH acts to promote arousal (see above)
Effectsof intra-LHA AMPHonreinstatement
and that has been implicated in reward-related pro-
ofdrug-seeking (CPP)
cesses.Animalsreceivedalternatingbilateralinfusions
ofAMPH(25nmol)andvehicle(AECF)intotheLHA The above-described studies demonstrate that AMPH
during the conditioning phase (see Methods above). acts within the LHA to exert arousal-promoting but
Incontrasttothatseenwithsystemically-administered not reinforcing actions. High arousal conditions that
AMPH, intra-LHA infusion of an arousing dose of are not reinforcing (e.g. stress) reinstate drug-seeking
70 B.E. SchmeichelandC.W.Berridge
600 was an overall significant treatment effect (Fig. 6;
PRE COND
POST COND F =7.88, p<0.001) and all animals showed a sig-
(5,30)
s) EAXMTPINH CRTEIIONNST nificant increase in time spent in the drug-paired
ed side ( 400 ** % cdhitaiomnbinerg fo(tl6lo=w12in.6g6;thpe<0in.0i0ti1a)l asynsdtemaicsigAnMifiPcHantcorne--
air ## duction in time spent in the drug-paired chamber
p
g- followingextinction(extinctionprecedingvehicleinfu-
u
e dr 200 sion: t6=6.50; p<0.001; extinction preceding AMPH
m infusion: t =6.82; p<0.001). Following extinction,
Ti intra-LHA v6ehicle infusion did not significantly alter
0 time spent in the drug-paired chamber (t6=0.04;
p=0.97). In contrast, intra-LHA infusion of AMPH
Fig.5. Systemicallyadministeredamphetamine(AMPH) reinstated drug-seeking, producing a significant
D
reinstatesanextinguishedAMPH-inducedconditionedplace increase in time spent in the drug-paired chamber ow
preference(CPP).SystemicallyadministeredAMPH(0.25 n
mg/kgs.c.)elicitedasignificantCPP(PRECONDvs.POST compared to extinction (t6=2.53; p<0.05). The magni- loa
tude of intra-LHA AMPH reinstatement was similar d
COND).Followingextinctiontraining,significantlylesstime ed
wasspentinthedrug-pairedside(EXTINCTION). to thatseenwith systemicAMPH (Figs 5and6). fro
m
SubsequenttreatmentwithAMPH(0.10mg/kgs.c.) h
reinstatedtheCPP(AMPHREINST).Barsdisplaymean Discussion ttp
s
(t±esS.tEin.Mg.)pteimrioed(.s)**spp<e0n.t01invtsh.ePRdrEuCg-OpNairDe;d##spid<e0.o0v1evrsa.P1O0mSTin The dorsal hypothalamus, including the lateral hypo- ://ac
a
COND;%p<0.05vs.EXTINCTION. thalamusproper,perifornicalregionandadjacentdor- de
m
somedial hypothalamus (i.e. LHA, collectively), has ic
behaviour in humans and animals. Therefore, ad- long-been suggested to play a role in the regulation .ou
p
ditionalstudiesexaminedwhether AMPHacts within of behavioural state and state-dependent processes .c
o
theLHAtoreinstateapreviouslyextinguishedAMPH- (Marchant et al., 2012; Olds, 1962; Stellar, 1954). m
induced CPP. For these studies, animals underwent More recently, the discovery of the LHA hypocretin /ijnp
cboynedxittiionnctiinognwtraitihninAgM. WPHe fi(0r.s2t5cmongfi/krmgesd.c.t)hafotlalowloewd, (pHarCtiRcTul)arslyystienmthheacsonrtee-xfotcoufsaerdouinsatel,rersetwoanrdtahnedLrHeiAn-, /article
but arousing dose of systemic AMPH (0.10mg/kg statement/relapse (Boutrel et al., 2010). Thus, it is of -ab
s
s.c.) would reinstate a previously-extinguished CPP interest that systemically-administered psychostimu- tra
c
under the current testing conditions (n=7). In this lants activate LHA neurons (Estabrooke et al., 2001; t/1
experiment, there was an overall significant effect of Fadel et al.,2002). Collectively,these andotherobser- 7/1
testing stage (e.g. pre- and post-conditioning, extinc- vations suggest a potentially prominent role of the /63
tion,reinstatement;F(3,18)=13.87,p<0.001).Allanimals LHA in the behavioural actions of psychostimulants, /827
showed an initial CPP (Fig. 5; t6=10.20; p<0.001) and including AMPH. The current studies provide the 21
a significant reduction in time spent in the drug- first evidence that AMPH acts directly within the 3 b
y
paired chamber following extinction (AMPH extinc- LHAtoelicitaffectivelyneutralarousalintheabsence g
u
(tCiornu:zt7e=t1a4l..,4270;0p8<;0M.0u0e1l)l.eArasnhdasStbeewenarsth,2o0w0n0)e,lssyeswtehmeriec oalfthaoupgrhonAouMnPceHdinlofucosmedotinortoatchtievaLtHioAn.faFiulerdthetormeolirceit, est on
AMPH reinstated the extinguished CPP (Fig. 5; reinforcing effects as measured by CPP, intra-LHA 05
A
t6=W2.e67t;hpe<n0.e0x5a).mined the degree to which bilateral ACoMmPbHinendo,neththeesleesosbrseeirnvsattaitoends adnemexotninstgruatisehethdatCPthPe. pril 2
0
intra-LHA infusion of vehicle (AECF) and AMPH LHAisasiteinvolvedinthearousingandreinstating 1
9
(25nmol)reinstatedanextinguishedCPPinaseparate effects,butnotthereinforcingorlocomotor-activating
group of animals (n=7) that underwent conditioning effectsofAMPHand,presumably,otherpsychostimu-
with systemic AMPH (0.25mg/kg) and extinction as lants.Theseobservationsprovidenewinsightintothe
described above. For intra-LHA reinstatement testing, neurobiology of psychostimulants and the LHA, and
each animal received vehicle and AMPH infusions provide further support for a role of the LHA in the
intothecentralregionoftheLHA,anareaimplicated reinstatement of psychostimulant use. Furthermore,
inthearousal-promotingactionsofAMPH(seeabove) given narcolepsy is associated with dysregulation of
as well as reinstatement of drug-seeking behaviour the LHA (Nishino et al., 2000; Thannickal et al.,
(Boutrel et al., 2005; Harris et al., 2005). There 2000),thecurrentobservationsalsosuggestthisregion
Amphetamine action inthe lateralhypothalamicarea 71
600 600 600
PRE COND EXTINCTION EXTINCTION
POST COND 0.25 mg/kg REINST VEH REINST AMPH 25 nmol
s)
side ( 400 ** 400 400 %
d
e
air
p ## ##
ug- 200 200 200
dr
e
m
Ti
0 0 0
Systemic Intra-LHA Intra-LHA
Fig.6. Amphetamine(AMPH)directlyinfusedintothelateralhypothalamicarea(LHA)reinstatesanextinguished
D
conditionedplacepreference(CPP).Leftpanel:systemicallyadministeredAMPH(systemic;0.25mg/kgs.c.)eliciteda o
significantCPP(PRECONDvs.POSTCOND0.25mg/kg).Afterconditioning,thesesameanimalsunderwentextinction wn
lo
training(EXTINCTION).Followingextinction,one-halfoftheseanimalsthenreceivedbilateralintra-LHAvehicleinfusions a
d
(REINSTVEH)whiletheotherhalfreceivedbilateralintra-LHAAMPH(REINSTAMPH25nmol).Followingeachtreatment, e
d
animalswerere-testedforextinction,andthenreceivedtheothertreatment(e.g.AMPHiftheyhadfirstreceivedvehicle). fro
Middlepanel:bilateralinfusionofvehicleintotheLHAhadnoeffectontimespentinthedrug-pairedsideinextinguished m
animals.Rightpanel:incontrast,bilateralintra-LHAAMPH(25nmol)reinstatedtheextinguishedCPP,significantlyincreasing http
tpiemrieodsp.e*n*pt<in0.t0h1evds.ruPgR-EpaCirOedNsDid;e##.pB<a0rs.0d1ivsps.laPyOmSTeaCnO(N±SD.E.;M%.)pt<im0.0e5(sv)s.spreesnpteicntitvheeEdXrTuIgN-pCaTirIeOdNs.ideovera10mintesting s://ac
a
d
e
m
ic
may participate in the therapeutic, arousal-promoting havedescribedthatspontaneouswakinginratsunder .o
u
p
actions of psychostimulants in the treatment of this testingconditionsidenticaltothoseusedinthecurrent .c
o
disorder. studies is associated with low levels of locomotor m
activity interspersed with bouts of eating, grooming /ijn
p
AMPH actswithintheLHAto elicit a(Bnedrriqdugieet-raensdtingFoboetefo,re19a9n6;anEismpaalñareteutrnasl.t,o2s0l0e1ep). /article
arousal-enhancing butnot locomotor-activating Following intra-LHA AMPH, we observed a compar- -ab
or reinforcing effects s
able level and pattern of locomotor activity that both tra
c
The arousal-enhancing effects of psychostimulants qualitatively and quantitatively differs from that seen t/1
contribute to their widespread use. Previous studies with behaviourally-activating doses of systemically- 7/1
indicate that psychostimulant-induced arousal in- administered psychostimulants (Kuczenski et al., /63
volves enhanced noradrenergic signaling within the 1991, 1997; Segal, 1975; Segal and Kuczenski, 1997). /82
7
medial septal and medial preoptic areas (for review, Assuch,webelievethelowleveloflocomotoractivity 21
Berridge, 2008). The current studies demonstrate that observed in the current studies largely reflects the 3 b
y
the LHA is also a site of action in AMPH-induced wake-promotingeffectsofintra-LHAAMPHinfusion. g
u
arousal. Moreover, recent studies demonstrate addi- Despite the LHA being implicated in appetitive pro- es
tive wake-promoting actions of noradrenergic α1- and cesses (Harris et al., 2005; Marchant et al., 2012), t on
β-receptors within the LHA, similar to that seen in when infused directly into the LHA, 25mnol AMPH 05
the medial septal and medial preoptic areas lacked reinforcing properties, as measured by CPP. Ap
(Schmeichel and Berridge, 2013). Collectively, these Although testing only a single dose of AMPH is a ril 2
0
observationssuggestalikelyroleforNEinthearousal- potential limitation of the current study, these results 1
9
promotingeffectsofAMPHwithintheLHA.However, are in contrast to the robust reinforcing effects of a
DA also likely contributes to the arousal-promoting similar dose of AMPH when infused into the nucleus
actions of psychostimulants (Isaac and Berridge, accumbens (Carr and White, 1983; Cunningham and
2003; Monti et al., 1990; Trampus et al., 1991; Wisor Kelley, 1992; Gerdjikov and Beninger, 2005, 2006;
et al., 2001). The degree to which DA acts within Kelley and Throne, 1992; Schildein et al., 1998).
the LHA to modulate arousal state remains to be Combined, these observations provide further evi-
determined. dence that the arousing, reinforcing and locomotor-
Evenatthehighestdose,intra-LHAAMPHhadno activating effects of AMPH can be anatomically
majorimpactonlocomotoractivity.Inpriorwork,we dissociated.
72 B.E. SchmeichelandC.W.Berridge
Previous work suggests a topographical organiz- et al., 2000). This raises the question of whether
ationofLHAHCRTneurons,withneuronsinthedor- HCRT is involved in the arousal-promoting or re-
somedialandperifornicalhypothalamicareaslinkedto instating actions of intra-LHA AMPH. In recent work
arousal and neurons in the lateral hypothalamus we demonstrated that activation of noradrenergic
associated with reward processes (for review, Harris α -receptors within the LHA promotes waking,
1
andAston-Jones, 2006). In the current studies,wedid althoughthiswasnotassociatedwithHCRTneuronal
not obtain evidence for a topographic organization activation (Schmeichel and Berridge, 2013). These
for either the wake-promoting effects or the lack of observations could suggest the arousal-promoting
reinforcing actions of intra-LHA AMPH. This is con- actions of intra-LHA AMPH are independent of
sistent with previous work that failed to observe a HCRTactivation.However,givenAMPHalsoelevates
topographic organization in the activation of HCRT DAsignaling,itremainspossiblethatthebehavioural
neurons under high-arousal conditions (España et al., effects of intra-LHA AMPH involve actions of HCRT
2003). These observations could either suggest that a neurons. Alternatively, the LHA contains melanin-
D
topographicorganizationoftheLHAisnotauniversal concentrating hormone (MCH) synthesizing neurons ow
n
featureofthisregionorthatthearousingandreinstat- implicated in the induction/maintenance of sleep lo
a
ingeffectsofintra-LHAAMPHdonotinvolveHCRT. (Adamantidis and de Lecea, 2008; Modirrousta et al., d
e
d
2005). Thus, psychostimulant action within the LHA fro
AMPHacts withintheLHAto reinstatean may involveinhibition of MCH neuronal signaling. m
extinguishedCPP Finally, recent evidence indicates a role for HCRT http
Re-exposure to a drug can elicit relapse, or the rein- nseeeukriontgra.nFsomrisesxioanmpilne, cthenetrarelinHsCtaRteTmeandtmionfistrdartuiogn- s://ac
statement of drug-seeking behaviour. Reinstatement a
reinstates previously extinguished drug-seeking de
can be also be triggered by non-reinforcing, arousing/ m
stressfulconditions(Shahametal.,2000).Stress-related (tBreoautmtreelntetwiatlh., a2n005H; CHRaTr-rRis1 eatntaalg.,on2is0t05)p,rewvehniltes ic.ou
relapseisconsistentwithevidenceindicatingapromi- p
stressor-induced reinstatement of cocaine-induced .c
nent role of negative affect in addiction (Baker et al., o
drug-seeking (Boutrel et al., 2005). Interestingly, m
2004;KoobandVolkow,2010).Alternatively,theabil- unpublished studies in our laboratory indicate that /ijnp
ietlyevoaftesdtreasrsotuosarleinpesrtatsee,dirnugr-esleaepksien/grecinosutladteimmepnlti.caItne arousal-enhancing doses of HCRT fail to produce /artic
either a CPP or conditioned place aversion. Thus, the le
support of this latter hypothesis, the current studies ability of intra-LHA AMPH to promote reinstatement -ab
demonstratethatalthoughintra-LHAAMPHinfusion s
while lacking reinforcing/rewarding actions is similar tra
failed to elicit either a CPP or conditioned place aver- c
sion, these infusions were potently arousing and to that seen with HCRT administration and could t/17
suggest HCRT participates in the behavioural effects /1
reinstated drug-seeking. These observations are con- of intra-LHAAMPH. /63
sistent with the participation of arousal/autonomic- /8
2
related neurocircuitry in stress-related reinstatement 7
2
of drug-seeking behaviour (Highfield et al., 2000; Methodologicalconsiderationsfor 13
b
Leri et al., 2002). As such, these observations suggest CPP reinstatement y
g
arousal-related mechanisms, independent of affective u
state, may be sufficient to reinstate drug-seeking. Tanhdecurerirnesnttatsitnugdieasctuiosends CoPfPitnotraas-sLeHssAtheArMeiPnHfo.rciAnng est on
Moreover, these observations suggest that prevention alternative approach to assessing the reinforcing and 05
of relapse could involve pharmacological targeting of A
reinstating actions of a drug is self-administration. p
arousal-relatedneural systems. As reviewed elsewhere (Aguilar et al., 2009), there ril 2
0
are key differences in the type of learning, route 1
PotentialLHAneuronal populations involved 9
of administration and degree of motivation/effort
inthebehaviouralactionsof AMPH
associatedwiththetwoparadigms.Forexample, self-
HCRTneuronsareimplicatedinappetitivebehaviour, administration involves instrumental learning and
including drug-seeking (Aston-Jones et al., 2010; active drug taking and likely evaluates the primary,
Harris et al., 2005; Zhou et al., 2008, 2012) and or innately, rewarding/reinforcing properties of a
psychostimulant-induced synaptic plasticity (Rao drug (Bardo and Bevins, 2000; Di Chiara, 1999). CPP,
et al., 2013; Yeoh et al., 2012). Furthermore, HCRT on the other hand, involves passive Pavlovian drug-
has been demonstrated to robustly promote arousal conditioning and likely measures the incentive
(España et al., 2001, 2002; Hagan et al., 1999; Piper motivationalpropertiesofthedrug-associatedcontext.