modulatory role of catecholamines in the transsynaptic expression of c-fos in the rat medial...

Ž .Brain Research 749 1997 214–225

Research report

Modulatory role of catecholamines in the transsynaptic expression of c-fos inthe rat medial prefrontal cortex induced by disinhibition of the mediodorsal

thalamus: a study employing microdialysis and immunohistochemistry

Michael Bubser 1, Matthijs G.P. Feenstra ), Erna B.H.W. Erdtsieck-Ernste 2,Margriet H.A. Botterblom, Hannie F.M. Van Uum, Chris W. Pool

Graduate School Neurosciences Amsterdam, Netherlands Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam, The Netherlands

Accepted 24 September 1996

Abstract

Ž .We studied the interaction of catecholaminergic and thalamic afferents of the medial prefrontal cortex PFC by analyzing the effectsof catecholamine depletion on thalamus-induced c-fos expression in the PFC of freely moving rats. Thalamic projections to the PFC were

Ž .pharmacologically activated by perfusing the GABA-A receptor antagonist bicuculline 0.03 mM or 0.1 mM through a dialysis probeimplanted into the mediodorsal thalamic nucleus. Bicuculline perfusion induced Fos-like immunoreactivity in the thalamic projectionareas, including the PFC, and in the thalamic nuclei surrounding the dialysis probe. 6-Hydroxydopamine lesions of the ventral tegmentalarea causing a 70–80% depletion of catecholamines in the PFC did not influence the increase in the number of Fos-like immunoreactivenuclei in the prefrontal cortex in response to thalamic stimulation. However, densitometric image analysis revealed that the intensity ofFos-like immunoreactivity in the PFC of lesioned rats perfused with 0.1 mM bicuculline was higher than in correspondingly treated

Ž .controls. The behavioral activity to bicuculline perfusion, an increase of non-ambulatory activity 0.03 mM followed by locomotion andŽ .rearing 0.1 mM , was not changed in 6-hydroxydopamine-lesioned rats. It is suggested that the thalamically induced c-fos response is

directly mediated by excitatory, presumably glutamatergic, transmission and not indirectly by an activation of catecholaminergic afferentsof the PFC. The increase in the intensity of Fos-like immunostaining in strongly stimulated, catecholamine-depleted rats suggests thatcatecholamines modulate the degree to which thalamic activity can activate the PFC of awake animals.

q 1997 Elsevier Science B.V. All rights reserved.

Keywords: Bicuculline; Dopamine; Immediate early genes; Mediodorsal thalamic nucleus; Motor activity; Noradrenaline; Paraventricular thalamic nucleus;Ventral tegmental area

1. Introduction

Ž .The mammalian prefrontal cortex PFC is involved in anumber of processes related to the temporal organization

w xof behavior 28,40 . During the execution of PFC-relatedbehavioral tasks the electrophysiological and metabolic

w xactivity of the PFC increases 22,24,28,60,74 . As the PFC

) Ž .Corresponding author. Fax: q31 20 6961006; E-mail:[email protected]

1 Present address: Vanderbilt University School of Medicine, Depart-ment of Psychiatry, Psychiatric hospital at Vanderbilt, Suite 306, 160123rd Avenue South, Nashville, TN 37212, USA.

2 Present address: Department of Anatomy and Embryology, Facultyof Medicine, University of Amsterdam, Meibergdreef 15, 1105 AZAmsterdam, The Netherlands.

receives input from the mediodorsal and adjacent thalamicnuclei as well as from dopaminergic and noradrenergic cell

w xgroups 5,16,41 these afferents may play an important rolein regulating PFC activity. Insight into the mechanismscontrolling PFC activity is of particular interest sincestructural and functional deficits of the PFC and its cate-cholaminergic and thalamic afferents have been suggestedto contribute to the pathophysiology of schizophreniaw x1,7,13,22,57,67,74 .

Historically, the PFC has been defined as the projectionŽ .area of the mediodorsal nucleus MD of the thalamus

w x41,70 , but afferents from the midline and intralaminarthalamic nuclei also provide input to restricted subareas of

w xthe PFC 3,32,33,36,46 . In this paper we will use the termmediodorsal thalamus to designate the above-mentionedthalamic nuclei. The projections of the MD form asymmet-

0006-8993r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved.Ž .PII S0006-8993 96 01170-5

( )M. Bubser et al.rBrain Research 749 1997 214–225 215

w xric, presumably excitatory, synapses in the cortex 43,44 .Electrical stimulation of the MD elicits excitatory re-sponses in the PFC that are blocked by non-N-methyl-D-

w xaspartate glutamate receptor antagonists 21,27,68 . Thesefindings suggest that the thalamic afferents of the PFC useglutamate as their transmitter.

Ž .The ventral tegmental area VTA is the main source ofŽ .the dopamine DA neurons innervating the deep layers

Ž . w xlayers Vb and VI of the PFC 5,16,41 . By contrast,NAergic and serotonergic fibers terminate in the superfi-

Ž w x.cial layers of the PFC for review see ref. 79 . Stimula-tion of the VTA or iontophoretic administration of DA orDA agonists into the PFC inhibits spontaneous activity ofpyramidal neurons via a D2-like DA receptor-mediated

w xmechanism 54,61,68 . In accordance with the distributionof DA and its receptors in the deep cortical layersw x25,71,73 , inhibitory actions of DA are more easily en-

w xcountered in the deep layers of the PFC 9 . InhibitoryŽ .actions of noradrenaline NA have also been shown in the

w xPFC 9,68 .Studies on the interaction between thalamic and cate-

cholaminergic PFC afferents have shown that the excita-tion of the PFC that is induced by stimulation of the MD isblocked by prior stimulation of DAergic, but not ascending

w xNAergic, afferents to the PFC 21,68 . Furthermore, stimu-lation of the mediodorsal thalamus has been shown to

w x w xincrease DA utilization 39,53 , DA in vivo release 19w xand extracellular glutamate 20 in the PFC.

In order to get a better understanding of the regulationof PFC function by its thalamic and DAergic afferents, weinvestigated how the activity of PFC neurons, as reflectedby c-fos expression, is altered by thalamic and DAergicmanipulations in awake, freely moving rats. c-fos is animmediate early gene that is expressed by many neuralsystems in response to electrical or pharmacological stimu-

w xlation 48,58,62 . In a previous study we have shown thatactivation of the mediodorsal thalamus rapidly and tran-siently induces c-fos mRNA and Fos-like immunoreactiv-

Ž . w xity Fos-LI in its projection areas including the PFC 17 .In the present study we attempted to determine if and inwhich manner a depletion of prefrontal catecholaminesaffects basal and thalamus-evoked c-fos expression in thePFC.

Specifically, we tested whether catecholamines facili-tate thalamus-induced c-fos expression in the PFC becauseDA and NA receptor stimulation can induce c-fosw x14,47,65,77 or whether inhibitory actions of catechol-

w xamines 21,34,54,68 suppress thalamically induced c-fosexpression in the PFC by reducing the impact of thalamicinput. If the first hypothesis – catecholamine-facilitatedc-fos expression – is valid, depletion of prefrontal cate-cholamines should decrease thalamically induced c-fos ex-pression whereas, according to the latter hypothesis, reliev-ing the cortex of its catecholaminergic inhibition shouldincrease c-fos expression in the PFC.

w xThese data have been presented in preliminary form 8 .

2. Materials and methods

2.1. Subjects and housing

Ž .Male Wistar rats Harlan, Zeist, The Netherlands werehoused in groups of six animals per cage. They were

Žmaintained on a 12r12 h lightrdark schedule lights on at.07:00 a.m. with continuous access to food and water. At

least 4 days before the implantation of the dialysis probeŽ .rats were placed into Perspex cages 25=25=32 cm

where they were housed individually with food and waterfreely available. Following placement into the Perspex

w xcages rats were handled daily for 5 min 17 . All experi-ments were approved by the animal experimentation com-mittee of the Royal Netherlands Academy of SciencesŽ .NIH 94.09r00 .

2.2. 6-OHDA lesion

Ž .Rats 200–255 g, ns64 were anesthetized with Hyp-Ž .norm 0.08 mlr100 g, i.m. and placed into a Kopf

Ž .stereotaxic instrument. 6-OHDA hydrobromide 6 mgrmlŽwas dissolved in ice-cold vehicle physiological saline. w xcontaining 0.1 mgrml ascorbic acid 7 . Rats received

unilateral infusions of vehicle or 6-OHDA into the rostralŽ Ž .part of the right VTA 1.5 ml at AP y5.0 mm bregma , L

q0.8 mm, V y8.2 mm and 1.0 ml at AP y5.6 mmŽ .bregma , L q0.5 mm, V y8.3 mm; incisor bar 0.6 mm

. w xbelow the interaural line 51 .

2.3. Neurochemical analyses

For a detailed analysis of monoamine depletion in theforebrain, controls and lesioned rats were decapitated 2weeks after the VTA infusion, their brains were rapidlyremoved from the skull and cooled in ice-cold saline.Medial PFC, nucleus accumbens and striatum were dis-sected, tissue samples from the left and right hemisphereswere separated, frozen on dry ice and stored at y808C.Frozen samples were weighed, homogenized with a

Ž .glassrTeflon homogenizer in 1 ml perchloric acid 0.1 MŽ . Žcontaining EDTA 0.3 mM , centrifuged for 20 min 4000

.=g at 48C and analyzed by high-performance liquidchromatography with electrochemical detection. For the

Ž .analysis of DA, dihydroxyphenylacetic acid DOPAC ,Ž .serotonin and 5-hydroxyindoleacetic acid 5-HIAA , the

supernatant was directly injected into the chromatographysystem and separated at a flow rate of 0.8 mlrmin. The

Ž . Ž .mobile phase final pH 3.1 contained grl sodium nitrateŽ . Ž . Ž .3.5 , sodium acetate 3.0 , citric acid 12 , heptanesul-

Ž .fonic acid 0.26 and 15% methanol. For the analysis ofNA in the PFC, the remaining supernatant was extractedon Sephadex G-10 columns and eluted using a modifica-

w xtion of the method of Westerink 75 . The fraction contain-ing NA was injected into the chromatography system andseparated at a flow rate of 0.8 mlrmin with a mobile phase

( )M. Bubser et al.rBrain Research 749 1997 214–225216

Ž . Ž . Ž .final pH 5.0 containing grl sodium acetate 6.0 , citricŽ . Ž .acid 2.0 , heptanesulfonic acid 0.175 and 6% methanol.

The chromatography system consisted of a Waters 600Epump, a WISP 712 autosampler, a Supelcosil 5C18-DB

Ž . Ž .column 250=4.6 mm with guard column 20=4.6 mmwhich were kept at 458C and an ANTEC electrochemicaldetector that was operated at an oxidation potential of 750mV.

2.4. Microdialysis and behaÕioral analysis

Following a 2 week recovery from VTA infusion, ratsŽ .240–315 g were implanted under chloral hydrate anes-

Ž .thesia 400 mgrkg, i.p. with a concentric dialysis probeŽ Ž .into the MD AP y2.5 mm bregma , L q1.5 mm, V

y6.5 mm with a mediolateral angle of 128; incisor bar 2.5. w xmm below the interaural line 17,51 . Following 4 h of

ŽRinger perfusion, the test compound Ringer or bicucullinemethyl chloride, Research Biochemicals International, Nat-

.ick, USA, in concentrations of either 0.03 mM or 0.1 mMwas perfused for 20 min, whereafter Ringer perfusion wascontinued for 65 min.

The behavioral activity of Ringer- or bicuculline-per-fused rats was recorded with a video system from 70 minbefore to 70 min after the test compound had entered thebrain. For each 20 min sampling period the duration ofnon-ambulatory behavior such as eating, drinking and

Ž .grooming activity I and the duration of ambulatory activ-ity, such as rearing or walking around the perimeter of the

Ž . w xcage activity II were determined 17 .

2.5. Immunohistochemistry

Seventy-five min after bicuculline had reached the brain,Žrats were deeply anesthetized with Nembutal 60 mgrkg,

.i.p. and intracardially perfused with 100 ml physiologicalsaline followed by 4% paraformaldehyde in 0.1 M phos-

Ž .phate buffer pH 7.4 . Further procedures for tissue pro-cessing and immunohistochemical staining for Fos-LI were

w xas described previously 17 . Briefly, sections were incu-bated with a sheep polyclonal antiserum raised against

w x Žhuman Fos 2–17 peptide Cambridge Research Biochem-.icals, OA-11-823, lot 09046, diluted 1:10 000 . As it was

not possible to stain the sections of all rats in one batch,the following precautions were taken to enable compar-isons between different stimulation conditions and lesionconditions. Batches containing sections of five to six ani-mals from control and lesion groups were processed withthe same staining solution and each batch consisted of

Ž . Žeither: I only one stimulation condition for comparing. Ž .controls and lesion groups , or II of different stimulation

condition for the analysis of concentration-dependent ef-fects of bicuculline.

One set of sections was stained for tyrosine hydroxylaseŽ .TH immunoreactivity to verify the 6-OHDA lesion inrats used for Fos-LI staining. Sections were incubated with

Žrabbit polyclonal serum against tyrosine hydroxylase In-stitute Jacques Boy, Reims, France, 1:3000, 48C,

.overnight . Incubations with the second and the third anti-body were carried out at room temperature and TH im-munoreactivity was visualized by 0.2% nickel-enhancedreaction of 0.05% DAB and 0.01% H O .2 2

2.6. Image analysis

For the analysis of Fos-LI levels in the medial PFC,three coronal sections per animal at the levels indicated inFig. 1 were selected. They were analyzed using a com-

Žputer-assisted image analysis IBAS KAT image analysis.system, Kontron Elektronik, Munich, Germany that was

Fig. 1. Schematic representation of coronal sections through the rat prefrontal cortex showing the prefrontal subareas outlined for quantitative imageŽ . Ž . Ž . Ž . Ž . Žanalysis. I Dorsal prelimbic area superficial layers , II dorsal prelimbic area deep layers , III ventral prelimbic arearinfralimbic area superficial

. Ž . Ž . w xlayers , and IV ventral prelimbic arearinfralimbic area deep layers were outlined freehand. Figures are redrawn from the atlas of Swanson 66 .


connected to a Bosch TYK9B TV camera equipped with aw xchalnycon tube 59 . The camera was mounted on a Zeiss

microscope that was equipped with planapo objectives. Allmeasurements were performed with a small band filter at

Ž .460 nm the absorbance maximum of DAB .Following shading correction and setting for 100%

Žtransmission in the background, 3=4 images 768=512.pixel per image covering the right medial PFC were

loaded at 10= magnification. For each section the PFCwas outlined in a reconstructed 2.5= magnification imageand divided into subareas representing the superficial and

Ž .the deep layers of the dorsal prelimbic area dPL and theŽ . Žventral prelimbic arearinfralimbic area vPLrIL see Fig.

.1 . As indicated in Fig. 1, the deep layers roughly encom-passed cortical layers Vb and VI.

An algorithm was used to distinguish Fos-LI-positivenuclei from background staining. The threshold of detec-tion was determined by comparing for a representativesample of sections the loaded images with the mask calcu-lated by the algorithm until the algorithm recognized bothstrongly and weakly stained Fos-LI-positive nuclei. Foreach outlined area the weighted mean of the number ofFos-positive nucleirmm2 was calculated. Furthermore,densitometric analysis provided an index of the intensity ofFos-LI staining which was calculated as: area of themask=optical densityrsize of the outline area.

2.7. Statistics

All data are presented as means"S.E.M. For statisticalcomparisons only raw data were used which were submit-

Ž .ted to a multifactorial analysis of variance MANOVAfollowed by Tukey’s t-test or to Student’s t-test whereappropriate. A P value -0.05 was considered to besignificant.

3. Results

3.1. 6-OHDA-induced transmitter depletion

Unilateral infusion of 6-OHDA into the VTA reducedDA and NA levels in the ipsilateral PFC to a large extent

and almost completely depleted DA in the striatal com-Ž .plex. As shown in Table 1, the depletion of DA y70%

Ž .and NA y85% in the ipsilateral PFC was more pro-Ž .nounced than serotonin depletion y41% . NA was also

Ž .reduced in the contralateral PFC of lesioned rats y18%and in the ipsilateral vs. the contralateral PFC of controlsŽ .y25% . Neither prefrontal DOPAC nor 5-HIAA wereaffected by VTA infusions. DA depletion on the lesionedside was accompanied by an increase in DOPACrDA

Ž .ratio q208% . In the nucleus accumbens and the dorsalstriatum, VTA lesion reduced DA and DOPAC by morethan 89% on the ipsilateral side, as opposed to a reductionof -45% on the contralateral side. Serotonin depletionwas confined to the ipsilateral striatal complex and did not

Ž .exceed 50% data not presented .TH-immunoreactive fibers were reduced throughout the

PFC. In the superficial layers there was a pronounced lossof TH-positive fibers running parallel to the pial surface.There was also a loss of TH-positive fibers radiallytraversing all cortical layers and a reduction of TH-positive

Ž .fibers in the deep layers of the PFC data not presented .

3.2. Probe placements

The distribution of the probe placements in themediodorsal thalamus is shown in Fig. 2. The probes werelocated along the antero-posterior axis of the MD and the

w xparaventricular thalamus as outlined by Swanson 66 . Inthe mediolateral extent, the majority of the probes werelocated in the medial part of the MD while some probesencroached on the border of the paraventricular and theintermediodorsal nuclei. The tips of the cannulae justentered the centromedial nucleus.

3.3. BehaÕior

Thalamic perfusion with bicuculline caused a concentra-tion-dependent increase of behavioral activity that was not

Žaffected by 6-OHDA lesion of the VTA data not pre-.sented . During thalamic perfusion with Ringer, controls

and lesioned rats were most of the time inactive, i.e., theywere asleep or awake, exhibiting neither non-ambulatory

Table 1Ž .Effects of 6-hydroxydopamine infusion into the right ventral tegmental area on tissue levels means"S.E.M. in pgrmg of biogenic amines, and some of

their metabolites, and on metabolitertransmitter ratios in the left and right medial prefrontal cortex) ))Control-contra Control-ipsi 6-OHDA-contra 6-OHDA-ipsi

a a a,b,cNA 573"28 432"29 471"30 85"18a,b,cDA 76" 9 83"16 78"10 23" 5

DOPAC 32" 6 37" 9 47"10 23" 55-HT 404"36 338"28 438"44 240"345-HIAA 152"13 151"15 189"24 135"22

abcDOPACrDA 0.430" 0.043 0.441" 0.076 0.559" 0.106 1.273" 0.2505-HIAAr5-HT 0.380" 0.019 0.468" 0.045 0.125" 0.023 0.698" 0.164

) Ž . Ž .Controls ns12 were injected with 1.5 mlq1.0 ml vehicle saline containing 0.1 mgrml ascorbic acid .)) Ž . Ž .6-Hydroxydopamine-lesioned rats ns13 were injected with 6-OHDA HBr 9 mgr1.5 mlq6 mgr1.0 ml .a b c Ž .P-0.01 vs. control-contra; P-0.01 vs. control-ipsi; P-0.01 vs. 6-OHDA-contra MANOVA followed by Tukey’s t-test .


Fig. 2. Schematic representation of the probe placements in the mediodorsal thalamus. A: overview of the mediodorsal thalamus showing the dimensionsŽ .of the implanted dialysis probe. B–E: individual probe placements B, y1.8, C, y2.0, D, y2.45, and E, y2.85 mm from bregma with lines indicating

the positions of the exchange surfaces of the dialysis probes. All probes were implanted into the right hemisphere but for reasons of clarity the probeŽ . Ž .placements in the controls ns18 and the lesioned rats ns19 are depicted in the right and left hemispheres, respectively. Figures are redrawn from the

w xatlas of Swanson 66 .

Ž Ž ..activity eating, drinking or grooming activity I norlocomotor activity and rearing. Perfusion of the dialysisprobe for 20 min with bicuculline 0.03 or 0.1 mM in-creased activity I from 10–20% to 70–80% of the time inboth controls and lesioned rats. Thalamic perfusion ofbicuculline 0.1 mM induced a pronounced, short-lasting

Ž .increase in locomotion and rearing activity II from 0% to10–15% of the time in both controls and lesioned rats.

3.4. Fos-like immunohistochemistry

Bicuculline perfusion increased Fos-LI in the mediodor-sal thalamus of controls and lesioned rats in particular in


the paraventricular, intermediodorsal and centromedial nu-Ž .clei Fig. 3A,B . Bicuculline perfusion also increased Fos-

LI in the vPLrIL and dPL of the PFC of controls andŽ . Žlesioned rats Fig. 3C,D . Comparison of bicuculline 0.1

.mM -perfused controls and lesioned rats suggests thatdespite of an apparently more pronounced expression ofFos-LI in the mediodorsal thalamus of a control vs. a

Ž .lesioned rat Fig. 3A,B , Fos-LI expression in the PFC ofŽthe lesioned rat was higher than in the control Fig.

.3C,D,G,H . Fos-LI was also observed in the contralateralPFC where it was less pronounced and more variable than

Ž .ipsilateral to the stimulation data not presented .

3.4.1. Cell countsThalamic perfusion with bicuculline caused a concentra-

tion-dependent increase in the number of Fos-LInucleirmm2 in the PFC, that was not changed by 6-OHDAlesion of the VTA. Fig. 4 shows that within each treatmentgroup the number of Fos-LI nucleirmm2 did not differacross the rostro-caudal extent of the PFC when prelimbic

Žand infralimbic PFC areas I through IV as outlined in Fig..1 were combined. This was confirmed by MANOVA

showing that the number of Fos-LI nucleirmm2 did notŽ .differ between leÕels F s1.32, P-0.27 . There was,2, 4

Žhowever, a significant main effect of concentration F2,.s85.0, P-0.0001 . At each rostro-caudal level the4

concentration-dependent increase in the number of Fos-LInucleirmm2 was significant only for 0.1 mM bicuculline.There was also a non-significant trend for a lesion effectŽ .F s2.60, Ps0.11 , but no significant interactions1, 4

Ž .between any of these factors F-0.43, P)0.66 .The increase in the number of Fos-LI nucleirmm2 that

was induced by bicuculline perfusion varied between deepand superficial layers as well as between dPL and vPLrIL,but this response was not modified by 6-OHDA lesion of

Ž .the VTA Fig. 5 . These subareas of the PFC showed alow number of Fos-LI nuclei in response to Ringer perfu-

Ž 2 .sion 16.3–20.7 nucleirmm and responded to bicu-Ž .culline perfusion with a 2- to 4-fold 0.03 mM and 7- to

Ž .9-fold 0.1 mM increase of the number of Fos-LI

Fig. 3. Photomicrographs of coronal sections showing the distribution of Fos-LI in the ‘‘mediodorsal’’ thalamus and the medial prefrontal cortex inresponse to thalamic perfusion of Ringer or 0.1 mM bicuculline. A, B, E: Fos-LI distribution in the mediodorsal thalamus of a 0.1 mM bicuculline-per-

Ž . Ž . Ž .fused control A , a 0.1 mM bicuculline-perfused 6-OHDA-lesioned rat B and a Ringer-perfused control E . For each case the depicted section wasequidistant from the implanted probe which is not shown. C, D: overview of Fos-LI distribution in the prefrontal cortex of the 0.1 mM bicuculline-perfused

Ž . Ž .control C and lesioned rats D whose thalamus was shown in A and B, respectively. F, G, H: detailed view of Fos-LI in the superficial layers of theŽ . Ž . Ž . Ž .ventral prefrontal cortex vPLrIL of a Ringer-perfused control F , a 0.1 mM bicuculline-perfused control G and lesioned H rats. Note the limited

Ž .thalamic activation in B that induced a pronounced expression of Fos-LI in the PFC of the lesioned rat D as compared to the extensive thalamicŽ . Ž .activation A which induced less Fos-LI in the PFC of the control C . Abbreviations: CM, centromedial thalamic nucleus; IMD, intermediodorsal

thalamic nucleus; MD, mediodorsal thalamic nucleus; MDm, medial segment of the mediodorsal thalamic nucleus; PV, paraventricular thalamic nucleus.Ž .Scale barss500 mm A–H .


2 Ž .nucleirmm Fig. 5 . In controls the effect of 0.1 mMbicuculline on the number of Fos-LI nucleirmm2 was

Ž .weakest in the dPL basal and increased in the followingŽ . Žrank order: dPL deep, 410"54 -dPL superficial, 491

. Ž . Ž"62 -vPLrIL deep, 593"67 -vPLrIL superficial,.621"75 . Only in the lesioned rats stimulated with 0.1

mM bicuculline there was a non-significant 10–25% in-crease in the number of Fos-LI nucleirmm2 as compared

Ž .to the corresponding controls Fig. 5 . MANOVA con-Žfirmed significant main effects of ‘‘concentration’’ F2,

.s99.0, P-0.0001 . In all subareas the dose-dependent12

increase was significant at 0.1 mM bicuculline, but only inŽ .the vPLrIL superficial it was significant at 0.03 mM

bicuculline. There was also a significant effect of ‘‘struc-Ž .ture’’ F s4.01, P-0.01 , but only a non-significant3, 12

Ž .trend for the factor ‘‘lesion’’ F s3.04, Ps0.0841, 12

and there were no significant interactions between any ofŽ .these factors F-1.36, P)0.24 .

3.4.2. Densitometric analysisThalamic perfusion with bicuculline caused a concentra-

tion-dependent increase in the intensity of Fos-LI stainingwhich was enhanced in 6-OHDA-lesioned rats perfusedwith bicuculline 0.1 mM. The intensity of Fos-LI staining

Ž . Žincreased 2- to 6-fold 0.03 mM and 7- to 14-fold 0.1. Ž .mM in response to bicuculline perfusion Fig. 6 . In

lesioned rats stimulated with 0.1 mM bicuculline the inten-

ŽFig. 4. Effect of 20 min perfusion of Ringer or bicuculline BIC, 0.03 or.0.1 mM through a dialysis probe implanted into the right mediodorsal

2 Žnucleus of the thalamus on the number of Fos-LI nucleirmm mean".S.E.M. at different rostro-caudal levels of the right medial prefrontalŽ .cortex combined prelimbic and infralimbic areas of controls or rats with

6-OHDA lesion of the ventral tegmental area. For the number of animalssee legend of Fig. 3. ) P-0.01 vs. control-Ringer; a P-0.01 vs.

Ž .lesion-Ringer MANOVA followed by Tukey’s t-test .


2 Žnucleus of the thalamus on the number of Fos-LI nucleirmm mean".S.E.M. in the right medial prefrontal cortex of controls or rats with

Ž .6-OHDA lesion of the ventral tegmental area. Upper panel: left dorsalŽ . Ž .PL – superficial and right basal layers; lower panel: left ventralŽ .PLrIL – superficial and right basal layers. The means"S.E.M. of the

Ž .control-Ringer group were 16.3"2.8 dorsal PL superficial , 16.8"1.9Ž . Ž .dorsal PL basal , 16.8"2.1 ventral PLrIL superficial and 20.7"4.4Ž .ventral PLrIL basal . For the number of animals see legend of Fig. 3.) P-0.05 vs. control-Ringer; a P-0.05 vs. control-BIC 0.03 mM;$ P - 0.05 vs. lesion-Ringer; q P - 0.05 lesion-BIC 0.03 mMŽ .MANOVA followed by Tukey’s t-test .

sity of Fos-LI staining was 30–40% higher than in thecorresponding controls. MANOVA revealed significant

Žmain effects of ‘‘concentration’’ F s73.6, P-2, 12. Ž .0.0001 , ‘‘structure’’ F s7.8, P-0.0001 and ‘‘le-3, 12Ž .sion’’ F s4.50, P-0.04 . Subsequent MANOVAs1, 12

with ‘‘lesion’’ and ‘‘structure’’ as factors indicated thatsignificant effects of ‘‘lesion’’ were only present in rats

Ž .perfused with 0.1 mM bicuculline F s5.6, P-0.021, 3Ž .but not for 0.03 mM bicuculline F s0.14, Ps0.711, 3

Ž .or Ringer perfusion F s3.3, Ps0.08 .1, 3

Although great care was taken to perform the stainingfor Fos-LI identical from one batch to another, it might beargued that the results of densitometric analysis werebiased by the different outcome of immunohistochemicalstaining in different batches. To check this, we comparedfirst the optical density of the background in the medial



Žnucleus of the thalamus on the intensity of Fos-LI staining mean".S.E.M. in the right medial prefrontal cortex of controls or rats with

Ž .6-OHDA lesion of the ventral tegmental area. Upper panel: left dorsalŽ . Ž .PL – superficial and right basal layers; lower panel: left ventralŽ .PLrIL – superficial and right basal layers. For the number of animals

see legend of Fig. 3. ) P-0.05 vs. control-RINGER; a P-0.05 vs.control-BIC 0.03 mM; $ P-0.05 vs. lesion-RINGER; q P-0.05 le-

) Ž .sion-BIC 0.03 mM; — — designates a significant difference P-0.05Žbetween control-BIC 0.1 mM and lesion-BIC 0.1 mM MANOVA fol-

.lowed by Tukey’s t-test .

PFC and found that it did not differ between controls andŽlesioned rats stimulated with 0.1 mM bicuculline P)0.54

.for each subarea . Second, we re-analyzed the data derivedfrom one staining batch that contained two controls andthree lesioned rats which were treated with 0.1 mM bicu-

Ž .culline. Compared to stimulated controls 100% the inten-sity of Fos-LI staining was increased in the lesioned rats to

Ž Ž .. Ž Ž ..212"9.5% dPL deep , 163"93% dPL superficial ,Ž Ž .. Ž191"29% vPLrIL deep , and q220"11% vPLrIL

Ž ..superficial . Although this low number of rats precludeda valid statistical comparison it was evident that rats of thelesion group showed a clear increase in the intensity ofFos-LI staining.

Due to a high unspecific staining that was present at thelevel of probe insertion in the thalamus, but occasionallywas present throughout the mediodorsal thalamus, noquantitative analysis of thalamic c-fos expression could beperformed.

4. Discussion

DA and NA inhibit thalamus-evoked andror basal neu-w xronal activity of PFC neurons 21,34,54,61,68 but both

neurotransmitters can also increase PFC activity as indi-w xcated by increased levels of c-fos 14,65 . In the present

study we examined in which manner catecholamine deple-tion affects basal and thalamus-induced c-fos expression infreely moving rats. We investigated whether DA or NAfacilitate stimulation-induced Fos levels in the medial PFCor whether the inhibitory actions of catecholamines reducethe impact of thalamocortical activation and thus suppressthalamocortical induction of Fos-LI. The present data showthat disinhibition of the mediodorsal thalamus by theGABA-A receptor antagonist bicuculline induces an in-creased amount of Fos-LI in the PFC of rats with unilateral6-OHDA lesion as compared to bicuculline-perfused con-trols.

4.1. GABAergic regulation of thalamocortical neurons

The activity of the mediodorsal thalamus is controlledby intrinsic thalamic afferents from the reticular nucleus aswell as by afferents from the ventral pallidum and parsreticulata of the substantia nigra and several other fore-brain structures that provide strong GABAergic inhibitionw x11,12,31,38,39,53,55,64 . Blockade of GABAergic trans-mission by bicuculline perfusion reliably induced Fos-LI

w xin the mediodorsal thalamus 17 . We extended these re-sults by showing that these effects are concentration de-pendent. The transsynaptic response in the PFCw x19,20,39,76 is in accordance with the excitatory, gluta-matergic nature of the mediodorsal thalamus-PFC projec-

w xtion 25,27,68 since depolarization and subsequent cal-cium influx are among the factors known to induce neu-

w xronal Fos expression 62 . In this respect it must be notedthat strong transsynaptic expression of Fos-LI was also

Žobserved in the shell of the nucleus accumbens see alsow x.ref. 17 which also receives input from the paraventricu-

w xlar thalamus 33,46 , but these data will not be discussed inthe present study.

Irrespective of the mediolateral or rostro-caudal place-ment of the dialysis probe within the boundaries of themediodorsal thalamus, the paraventricular, intermediodor-sal and centromedial nuclei showed a more pronouncedFos-LI than the MD itself, even at levels where nosurgery-induced tissue damage was observed in the MD.The strong activation of the midline and intralaminarthalamic nuclei, especially the paraventricular nucleus,which provides a dense innervation of the basal and super-

w xficial layers of the vPLrIL 3,33,35,46 and the corre-spondingly strong expression of Fos-LI in the vPLrILcortical areas points to a prominent role of these midlinenuclei in thalamocortical c-fos expression. An involvementof activated MD projections to layers I and III of the PFC


w x31,41 is indicated by the high level of Fos-LI in superfi-cial layers of the dPL and vPLrIL, in spite of the weakc-fos response in the MD itself. Fos-LI in the basal layersmay also in part be induced via MD activation, becauseMD afferents synapse onto dendritic spines of pyramidal

w xneurons located in these basal layers 43,44 . Interestingly,others have also reported that both in naive and in phar-macologically challenged rats the levels of Fos-LI in themidline thalamic nuclei are higher than in the MDw x15,26,49 .

4.2. Catecholaminergic inÕolÕement in thalamocortical Fosexpression

In confirmation of previous studies, 6-OHDA infusionsinto the VTA, the main source of cortical DA innervationw x5 , caused a stronger reduction of DA levels in the

w xstriatum and nucleus accumbens than in the PFC 35,72 .This depletion pattern occurred although most DA neuronsof the VTA and medial substantia nigra had been de-

Ž .stroyed data not presented . Therefore, it is likely thatsome of the remaining DA innervation of the PFC derivedfrom DAergic projections of the zona incerta to the PFCw x w x45 . Like in previous studies 35,72 a pronounced deple-tion of prefrontal NA was observed following 6-OHDAinfusion into the VTA. The DA depletion was accompa-nied by an increase of DOPACrDA ratios in PFC, nucleusaccumbens and striatum. Although we did not measure NAmetabolites in the PFC, previous data indicate that asimilar increase of NA metabolism may have occurred inthe lesioned NA system. By contrast, the lesser degree ofserotonin depletion did not cause an increase of 5-HIAArserotonin ratios. Increased DOPACrDA ratiossuggest a higher metabolic activity in remaining DA termi-nals in order to compensate for loss of DAergic afferentsw x78 . Thus, one might argue that the degree of prefrontalDA depletion was insufficient to affect DAergic regulation

w xof PFC function. However, Bean and Roth 4 have shownthat in the PFC a mean tissue depletion of 68%, which weachieved in the present study, is sufficient to reduce basallevels of extracellular DA by 50%. As the response of thepartially lesioned mesocortical DA system to challenges,such as DA receptor blockade or footshock, is blunted or

w xeven abolished 4,18 , a similar impairment of prefrontalDA function is to be expected under the challenge of

w xmediodorsal thalamus stimulation 19 .In the present study basal levels of Fos-LI in the PFC

were not changed by 6-OHDA lesion. Thus, partial cate-cholamine depletion of the PFC does not lead to the samelong-lasting increase of Fos-LI that occurs in a subset of

w xstriatal neurons of the DA-depleted striatum 37 . Usingthe deoxyglucose method others have shown that the basalmetabolic activity of the PFC is also not affected by

w xprofound catecholamine depletion 42 .In our study catecholamine depletion did not reduce

thalamus-induced Fos expression in the PFC, suggesting

that thalamocortical excitation suffices to induce Fos-LIeven when catecholamine function of the PFC is compro-mised. Similar data have been obtained in the striatum ofanesthetized rats where electrical stimulation of the cortexincreased Fos levels both in intact and DA-depleted striataw x23 . Based on our data, the hypothesis that the increase of

w xprefrontal DA levels induced by thalamic stimulation 19might be sufficient to facilitate thalamus-induced Fos ex-pression in the PFC has to be rejected. Furthermore, thesedata imply that glutamate-mediated Fos induction is funda-mentally different from DA-mediated Fos inductionw x47,50,77 which requires the concurrent activation of glu-

w xtamate receptors 10,69 .Our finding that in response to strong thalamic stimula-

tion neither the number of Fos-LI neurons nor their re-gional and laminar distribution differed significantly be-tween controls and catecholamine-depleted rats suggeststhat the same population of PFC neurons was transsynapti-cally activated in both experimental groups. Thus, theincreased intensity of Fos-LI staining in strongly stimu-lated lesioned rats was in part caused by an enhanced c-fosresponse per activated neuron. It is noteworthy that suchchanges would have been overlooked if we had onlycounted Fos-positive cells instead of measuring the inten-sity of Fos-LI staining. The increased Fos response instrongly stimulated rats could be due to reduced cate-cholaminergic inhibition which may have rendered corticalneurons more susceptible to thalamocortical excitation. Asthis response only occurred in strongly stimulated rats it issuggested that deficient DA function may have becomeevident only under a condition where prefrontal DA re-

w xlease 19 and thus DAergic inhibition was increased incontrols but not in lesioned rats. The increase in theamount of Fos-LI was significant in the superficial layersof vPLrIL but there was a tendency for increased Fos-LIin the basal layers of the PFC as well. Given the restrictionof DA terminals and DA receptors to the basal layers of

w xthe PFC 5,25,71 , one might have expected an increase ofFos-LI exclusively in the basal layers, but it has beenshown that 86% of PFC neurons are inhibited by stimula-

w xtion of the mesocortical DA system 54 . Thus, prefrontalDA depletion may have played a prominent role in theobserved effects which is in line with the inhibitory action

w xof prefrontal DA on thalamocortical excitation 21,68 .These putative actions of prefrontal DA could be due todirect inhibitory actions of DA on cortical pyramidal cellsw x21,61,68 or they could be mediated via inhibitoryGABAergic interneurons which are stimulated by DAw x6,30,52,56 . Although NA does not block thalamus-evoked

w xPFC activity in anesthetized rats 68 , it can not be ex-cluded that a loss of NAergic innervation may have af-fected the prefrontal c-fos response to afferent stimulationin freely moving rats, especially when the important bal-ance between NA and DA in regulating PFC function is

w xtaken into account 67 . Finally, it must be mentioned thatdestruction of DAergic afferents of the dorsal and ventral


striatum which changes the activity of striatal projectionw xneurons to the ventral pallidum and substantia nigra 29,63

may have affected basal ganglia output to the thalamusw x12 and such a change could affect the responsiveness ofmediodorsal thalamus neurons to GABAergic blockade inlesioned animals. The extent to which such a mechanismcould influence thalamocortical activity is difficult to de-termine since the relative contribution of the thalamicreticular nucleus and the basal ganglia to inhibition of themediodorsal thalamus is not precisely known.

4.3. Thalamocortical actiÕity, behaÕioral actiÕation andprefrontal c-fos expression

The reticular nucleus of the thalamus provides crucialw xinhibitory input to dorsal thalamic nuclei during sleep 64 .

In accordance with this notion, relieving the mediodorsalthalamus of its inhibitory GABAergic input led to anawakening reaction of unrestrained, sleeping rats. As the

w xprocess of awakening 2 and high levels of motor activityw x17 are sufficient to induce c-fos in the motor and pre-frontal cortex, it was an important finding that behavioralscores of controls and lesioned rats were similar and couldthus not account for differences in prefrontal expression ofFos-LI. The most likely explanations for the failure of the6-OHDA lesion to influence the thalamus-induced behav-ioral activation are the partial catecholamine depletion ofthe PFC and the almost complete, but only unilateral,depletion of DA in the dorsal and ventral striatum, sincemany behavioral processes are only impaired followingbilateral severe, more than 80–90% catecholamine deple-

w xtion 7,78 .

4.4. Conclusions

It is concluded that the combination of microdialysisand intracerebral injection of neurotoxins to pharmacologi-cally manipulate transmitter function and the use of c-fosexpression to demarcate metabolically activated brainstructures makes it feasible to study interactions betweenneural systems impinging onto common target areas whileat the same time the behavioral relevance of such interac-tions can be investigated. We used these methods to showthat stimulation of the mediodorsal thalamus induces Fos-LIin the PFC as well as behavioral activation and that onlyc-fos expression but not the behavioral response to thala-mic stimulation is enhanced by prior depletion of DA andNA. In confirmation of electrophysiological studies carriedout in anesthetized rats, our data, obtained in freely mov-ing rats, demonstrate that catecholamines are involved inthe control of postsynaptic neuronal activity of the PFC bymodifying, i.e., reducing the impact of thalamic input tothe PFC. Furthermore, it is concluded that DA release inthe PFC elicited by thalamic stimulation does not facilitatec-fos expression induced by thalamic activation. An in-

volvement of subcortical DA depletion in the lesion-in-duced changes of c-fos expression cannot be excluded.

Acknowledgements

We would like to thank Prof. Dr. Ariel Y. Deutch, Dr.Corbert G. Van Eden, Prof. Dr. Henk J. Groenewegen andProf. Dr. Harry B.M. Uylings for discussions and helpfulcomments on this manuscript. The expert technical assis-tance of Alice Rinkens is gratefully acknowledged. Wethank Henk Stoffels for drawing the figures and GerbenVan der Meulen for photography. This work was sup-ported by the Van den Houten Foundation.

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modulatory role of catecholamines in the transsynaptic expression of c-fos in the rat medial...

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