nigrostriatal collaterals to thalamus degenerate in parkinsonian animal models

Nigrostriatal Collaterals to ThalamusDegenerate in Parkinsonian Animal ModelsAmanda Freeman, BS,1 Brian Ciliax, PhD,1 Roy Bakay, MD,2 Joseph Daley, BS,1 R. Daniel Miller, MS,1

Glenda Keating, PhD,1 Allan Levey, MD, PhD,1 and David Rye, MD, PhD1

Movement, cognition, emotion, and positive reinforcement are influenced by mesostriatal, mesocortical, and mesolimbicdopamine systems. We describe a fourth major pathway originating from mesencephalic dopamine neurons: a mesotha-lamic system. The dopamine transporter, specific to dopamine containing axons, was histochemically visualized in tha-lamic motor and limbic-related nuclei and regions that modulate behavioral state as opposed to sensory nuclei in rats,nonhuman primates, and humans. Anatomical tracing established this innervation’s origin via axon collaterals from themesostriatal pathway. These findings implicate the thalamus as a novel site for disease specific alterations in dopamineneurotransmission, such as exist with nigral degeneration attending Parkinson’s disease. This was confirmed in hemipar-kinsonian animals where reduction of thalamic dopamine innervation occurred coincident with signs of active axonaldegeneration. Individual mesencephalic dopamine neurons therefore have the potential to modulate normal and patho-logic behavior not only through traditional nigrostriatal pathways but also by way of axon collaterals that innervate thethalamus.

Ann Neurol 2001;50:321–329

Dopamine is a neurotransmitter that modulates diversebehaviors including movement, motivation, cognition,reward, and feeding.1,2 The mesostriatal, mesocortical,and mesolimbic systems are the most conspicuous ofpathways employing dopamine (DA), and are commonto vertebrate species.3–5 Alterations of DA transmissionin these circuits are central to current heuristic modelsof disease.6–8 Pathologic behavior has been attributedto loss of normal DA influences upon one of parallel,multisynaptic pathways linking the striatum with func-tionally unique thalamocortical circuits via basal gan-glia output nuclei nuclei (ie, the internal pallidum[Gpi] and substantia nigra pars reticulata [SNr]).9 Do-pamine loss from the sensorimotor striatum, for exam-ple, is thought to account for the bradykinesia/akinesia,rigidity, and tremor of Parkinson’s disease (PD).8 Su-persensitivities to DA in associative or limbic subcir-cuits, on the other hand, are hypothesized to contrib-ute to obsessive-compulsive and tic disorders, andschizophrenia.7 Thus, DA’s effects upon pathologic be-haviors are thought to arise from indirect (via Gpi andSNr), rather than direct, actions upon thalamocorticalcircuits. This thinking does not easily explain theprominent disturbances in thalamocortical arousal thatattend PD.10–12 This study therefore examinedwhether thalamic DA innervation represented an addi-

tional substrate for DA’s modulation of thalamocorticalcircuitry.

Materials and MethodsTissue Preparation and ImmunohistochemistryFor complete details on tissue preparation and immunohis-tochemical procedures, see Miller and colleagues,13 and Cil-iax and colleagues.14,15 Briefly, rhesus monkeys (macaca mu-latta) (n 5 5) and Sprague Dawley and Brown Norway rats(Charles River, Raleigh, NC; n 5 54) were deeply anesthe-tized and perfused intracardially with 0.9% saline, 4% para-formaldehyde in 0.1M phosphate buffer (pH 7.4; PB) (fixa-tive), and an equal volume of ice-cold 10% sucrose. Humanbrain tissue was obtained at autopsy from four neurologicallynormal individuals (ages 21–68 years, 4–8 hours postmor-tem), and 1–1.5cm thick coronal tissue blocks cut from theright hemispheres were placed in fixative for 24 to 48 hoursat 4°C. Following equilibration with 30% sucrose, all tissuewas frozen sectioned in the coronal plane at 40 to 50mm ona sliding microtome and collected into adjacent series of sec-tions in PB. Individual series were processed to reveal dopa-mine transporter (DAT) immunoreactivity using rabbit poly-clonal14 and mouse monoclonal13,15 antisera directed at theDAT amino-terminus. Immunohistochemical visualization ofDAT employed the appropriate biotinylated secondary Vec-tastain Elite kits (Vector Laboratories, Burlingame, CA) andenzymatic detection of peroxidase with diaminobenzidine

From 1Emory University, Department of Neurology, Atlanta, GA;and 2Rush-Presbyterian Medical Center, Chicago, IL.

Received Dec 15, 2000, and in revised form Feb 14 and Apr 20,2001. Accepted for publication Apr 20, 2001.

Published online Jun 28, 2001; DOI: 10.1002/ana.1119

Address correspondence to Dr Rye, Department of Neurology,1639 Pierce Drive, Suite 6000 WMRB, P.O. Drawer V, EmoryUniversity, Atlanta, GA 30322. E-mail: [email protected]

© 2001 Wiley-Liss, Inc. 321

(DAB). Control tissue was processed identically in the ab-sence of primary antisera, with equal concentrations of non-specific immunoglobulin ('0.4–0.8mg/cc), or with antiserapreadsorbed with an excess of DAT protein. The intensity ofthe DAB reaction product was enhanced following tissuemounting and air drying, by submersion in 1% silver nitrateat 56°C for 45 minutes, ddH2O, 2% gold chloride at roomtemperature for 5 minutes, and 5% sodium thiosulfate for10 minutes. Select sections were processed similarly withcommercially available antibodies to tyrosine hydroxylase(TH) (Pel-Freeze Biologicals, Rogers, AK) or dopamine-b-hydroxylase (DBH) (Protos Biotech, New York, NY). Oneseries was mounted and counterstained with thionin to revealcytoarchitectonic detail.

WGA-HRP Retrograde TracingExperimental tissue from a previously published series of ratswith thalamic injections of the retrograde tracer WGA-HRPin Hallanger and colleagues16 was carefully reexamined toidentify the neural source of thalamic DA innervation. Sevenadditional cases were similarly prepared and analyzed. Thus,a total of 32 cases with small (2–25 nanoliter) injections of 1to 2.5% WGA-HRP, that together included all major tha-lamic nuclei, were analyzed (see Table 1 and Fig 1 in Hal-langer et al16). Every fourth section was processed with tet-ramethylbenzidine for WGA-HRP visualization 48 hoursafter injection.

Fluorogold Retrograde Tracing Combined withTyrosine Hydroxylase ImmunohistochemistryPressure injections of 25 nanoliters of 3% Fluoro-gold (FG;Fluorochrome Inc., Denver, CO) were made in differentthalamic nuclei in 12 rats with reference to the atlas of Paxi-nos and Watson.17 Following perfusion 4 to 8 days after in-jection, individual one-in-six series of tissue sections wereprocessed immunohistochemically for Fluoro-gold immuno-reactive (FG-IR) and tyrosine hydroxylase immunoreactive(TH-IR) alone, and simultaneously with two different chro-mogens. Fluoro-gold immunohistochemistry (FG IHC) wasperformed first with a primary rabbit polyclonal-antisera(Chemicon International, Temecula, CA), a goat anti-rabbitVectaStain Elite kit (BD Transduction Laboratories, SanJose, CA), and visualization employing nickel intensifiedDAB (Ni-DAB), followed by tyrosine hydroxylase (TH)IHC employing a commercial rabbit polyclonal antisera, agoat anti-rabbit VectaStain Elite kit, and DAB as the chro-mogen. Contrast between the black, granular Ni-DAB de-posits at sites of transported FG, and the diffuse, brown, cy-toplasmic DAB indicative of TH-IR, made it easy todistinguish cells containing both markers, from cells exhibit-ing either marker alone.

Cell CountingCell counts were performed in three representative cases inwhich the injection sites were centered upon thalamic nucleiwith different intensities of DAT terminal staining, and thatwere nonoverlapping. Perikarya staining only for TH-IR,FG-IR, and cells exhibiting both markers in a one-in-six se-ries including the ipsilateral and contralateral SNc werecounted independently by two observers blinded to the FG

injection site, and aided by an eyepiece reticle. Positive stain-ing required visualization of one or both chromogens in theperikarya and at least one proximal dendrite. Retrogradelylabeled neurons were counted in two additional thalamicprojecting brainstem nuclei, the pedunculopontine tegmentalnucleus (PPN) and locus coeruleus (LC), to assess the rela-tive strength of the SNc projections. Total number of retro-gradely labeled cells was then calculated from raw cell countsemploying the formula of Konigsmark.18 To control fordouble counting, Abercrombie’s correction factors were thenapplied as follows: 0.72 (SNc); 0.67 (PPN); and 0.71(LC).19 Extent of retrograde labeling is presented as a pro-portion of the total nuclear population with denominatorsderived from published values for SNc,20 PPN,21 and LC.22

Fluorescent Triple LabelingPressure injections of 25 to 40 nanoliters of 4% True Bluechloride (TB; 5-benzofurancarboximidamide, 2,29-(1,2-ethene-dyl) bis,dihydrochloride; Molecular Probes, Eugene, OR)were stereotaxically centered upon nuclei in the rostral one-half of the thalamus in Sprague Dawley rats (n 5 4). Thesame animals received ipsilateral striatal injections (8 3 25nLeach) of red fluorospheres (RF; Molecular Probes, EugeneOR). Rats were perfused 2 days later and their brains sec-tioned. Tissue was immunohistochemically labeled for thepresence of TH using a secondary antibody labeled withCy-5 (Jackson ImmunoResearch Laboratories, Inc., WestGrove, PA). Control tissue was processed identically in theabsence of primary antisera. Sections were mounted and cov-erslipped with Vectashield (Vector Laboratories, Burlingame,CA) and examined simultaneously with confocal microscopyand two-photon microscopy using a laser scanning micro-scope (LSM 510, Carl Zeiss, Inc., Thornwood, NY) coupledto a modelocked titanium-sapphire laser (Coherent Inc.,Santa Clara, CA).

MPTP and 6-OHDA LesioningUnilateral, intracarotid, or intrastriatal administration of do-paminergic neurotoxins were performed to lesion the nigro-striatal pathway unilaterally in nonhuman primates and rats;the contralateral hemisphere serving as an internal control.1-methyl, 1-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)was administered at 0.4mg/kg to rhesus monkeys via an in-tracarotid injection (n 5 3) as described in Ellis and col-leagues23 or intrastriatally (n 5 2) via an osmotic minipumpas detailed in Aosaki and colleagues.24 Rats (n 5 6) receivedunilateral, striatal injections of 6-hydroxydopamine (6-OHDA) (5uL of 4.3mg 6-OHDA/mL 0.9% NaCl and0.2% ascorbate) at stereotaxic coordinates (AP 5 -0.8,ML 5 -3.5, DV 5 -6.0) via pressure injection over 15 min-utes. Control animals (n 5 2) received a 5mL injection of0.9% saline containing 0.2% ascorbate. The integrity of tha-lamic DA innervation in hemiparkinsonian animals was as-sessed by TH IHC and DAT IHC, the latter having provenreliable in our previous assessments of the nigrostriatal path-way integrity in PD and the MPTP nonhuman primatemodel of PD.13

Staining for Axonal DegenerationDegenerating axonal elements were identified with two dif-ferent silver impregnation protocols: the FD NeuroSilver Kit

322 Annals of Neurology Vol 50 No 3 September 2001

I (FD NeuroTechnologies, Ellicott City, MD) and theamino/cupric/silver methodology (Neuroscience Associates,Knoxville, TN). These findings were confirmed by IHC vi-sualization of activated microglia employing a primary mousemonoclonal antisera (clone EBM11) for the microglial spe-cific, transmembrane glycoprotein CD68 (Dako Inc., Carpin-teria, CA) and a rabbit anti-rat VectaStain Elite kit, and visu-alization employing DAB.

ResultsPresumptive thalamic DA innervation was visualizedby tyrosine hydroxylase-immunohistochemistry (THIHC) because in many vertebrate species, most TH-immunoreactive axons prove to be dopaminergic ratherthan noradrenergic.5,25 To differentiate which TH-IRfields were dopaminergic rather than adrenergic or nor-adrenergic, monoclonal13,15 and polyclonal14 antibod-ies to the dopamine transporter (DAT), an exquisitelyspecific presynaptic marker for DA containing axons,26

were substituted for anti-TH antibodies in immunohis-tochemical protocols. TH-IR axons were widely dis-tributed in the thalamus and included limbic, motor,and principal sensory relay nuclei, the thalamic reticu-lar nucleus (RTn), as well as the midline and intralami-nar cell groups, in rat, rhesus monkey, and humanbrains. DAT-IR axon profiles and presumptive termi-nal fields differed in being sparse to absent in the an-terior and mediodorsal nuclei (limbic), the sensory re-lay nuclei, and the caudal one-half of the RTn.Moderate-to-dense DAT staining corresponded to nu-clear subdivisions defined in part by their innervationby basal ganglia efferents, including the ventral anterior(VA) and ventral lateral (VL) nuclei and the centrome-dian/parafascicular (CM/Pf) complex in each species.In the human and monkey brain, DAT-IR axonal pro-files were densest within nuclear subdivisions project-ing upon the dorsolateral prefrontal (VApc) and or-bitofrontal (VAmc) cortices, and the homologousanterior one-third of the RTn27 (Fig 1A, B, and E, F).The central medial nucleus (CeM) midline nuclei(paraventricular, rhomboid, and reuniens nuclei), theventral and lateral aspects of the mediodorsal thalamus(MD), and the central lateral and paracentral in-tralaminar nuclei exhibited moderate-to-dense DAT-IRstaining. Moderate DAT-IR axon profiles were ob-served in the dorsal aspect of the anteroventral nucleus(AV), middle one-third of the RTn, the cerebellar re-cipient zone (VPLo), the lateral habenula (less dense inhuman and monkey vs rat), and anterior pole of thepulvinar.

Three anatomical tracer experiments were conductedin rats to determine the source of thalamic DA inner-vation. In the first set of animals, the retrograde tracerwheatgerm-agglutinin conjugated horseradish peroxi-dase (WGA-HRP) was injected into different thalamicnuclei and histochemically visualized. The most con-

spicuous retrogradely labeled dopaminergic cell popu-lations were the A8-A9-A10 mesencephalic groups,with minimal labeling evident in the A11 group. In asecond set of experiments, simultaneous immunolabel-ing for retrogradely transported Fluoro-gold (FG) andTH verified the neurochemical signature of a majorityof thalamic projecting SNc neurons as dopaminergic(Fig 2C and D). Quantification of retrograde labelingin three representative cases whose injection sites of

Fig 1. Dark, silver intensified, benzidine reaction productindicating dopamine transporter immunoreactive (DAT-IR)axon elements in coronal hemisections through the rostral thal-amus of human (A) and rat (E) brain. Higher magnificationof DAT-IR in the thalamic reticular nucleus (RTn)(B), ven-tral lateral nucleus pars oralis (VLPo)(C) and ventral one-half of the anteroventral thalamic nucleus (AV)(D) in thehuman, and the ventral lateral nucleus in the rat (F). Aster-isk in (E) denotes area shown at higher magnification in (F).AD 5 anterodorsal thalamic nucleus; Ca 5 caudate; CeM 5central medial thalamic nucleus; ic 5 internal capsule; mt 5mamillothalamic tract; VA 5 ventral anterior thalamic nu-cleus, magno and parvocellular divisions. Bars 5 1.0mm (A),0.01mm (B–D, F), and 0.3mm (E).

Freeman et al: Nigrostriatal Collaterals to Thalamus 323

equal volumes were nonoverlapping provided confir-mation (Fig 3). This analysis substantiated the differ-ential quality of thalamic dopamine innervation evi-dent by DAT-IR described above. Retrograde labelingwas greatest, for example, with an injection centeredon a thalamic region exhibiting relatively denseDAT-IR axonal staining; ie, the basal ganglia and cer-

ebellar recipient nuclei. Retrograde labeling was mini-mal or absent with injections centered on the anteriornuclear group and principal sensory nuclei, consistentwith their sparsity of DAT-IR axonal staining (see Fig3). Several organizational features of this novel me-sothalamic pathway were evident. First, mesothalamicdopamine projections were exclusively ipsilateralwhereas thalamic innervations originating from alter-nate brainstem nuclei such as the cholinergic peduncu-lopontine nucleus (PPN) and noradrenergic locus co-eruleus (LC) were bilateral (see Fig 3). Second,collateralization of mesothalamic dopaminergic axonsbetween thalamic nuclei of a single hemisphere—ifpresent—was not extensive, in contrast to thalamic in-nervation taking origin from the PPN and LC. For ex-ample, only 27% of all dopaminergic SNc neuronswere labeled by the three nonoverlapping tracer injec-tions, as compared to nearly 100% or more of the totalpopulation of PPN neurons, and 65% of all LC neu-rons in the same cases (see Fig 3). Third, parallel non-dopaminergic mesothalamic projections from the SNcwere observed (see Fig 3). A third set of experimentssought to determine whether collateral branches of stri-atal dopaminergic axons targeted the thalamus employ-ing paired injections of different fluorescent tracers. In-jections into the thalamus (TB) and striatum (RF),combined with TH-immunofluorescence employing aCy-5 labeled secondary antibody revealed 15 to 20SNc cells/section containing each of the fluorescentmarkers (triple labeled) (Fig 4). Thus, our results pro-vide direct evidence that a substantial number of indi-vidual dopaminergic nigrostriatal neurons possess axoncollaterals that innervate the thalamus. A more detailedtopographic mapping of these novel pathways, an as-sessment of their neurochemical signatures, and a de-termination of the proportion of nigrostriatal neuronsthat collateralize to the thalamus, will be the topic of afuture report.

To determine if thalamic DA innervation might berelevant to disorders known to affect mesencephalicDA neurons, such as PD, hemiparkinsonism was in-duced in animals and thalamic DA innervation was as-sessed. Unilateral, intracarotid administration of thedopaminergic toxin MPTP to rhesus monkeys pro-

Š Fig 2. Examples of retrograde cell labeling in the SNc. (A andB) WGA-HRP labeling of numerous SNc cells following injec-tion in the paraventricular thalamic nucleus (neutral redcounterstain). (C and D) Fluoro-gold retrograde filling (blackgranules) of tyrosine hydroxylase immunoreactive (TH-IR) SNccell bodies (brown cytoplasmic staining) following injectionincluding the anteroventral, ventral anterior and ventral lat-eral thalamic nuclei, and adjacent thalamic reticular nucleus.SNc 5 substantia nigra pars compacta; SNr 5 substantianigra pars reticulata. Bars 5 0.1mm in A and C; 0.03mmin B and D.


duced parkinsonism in the contralateral hemibody. Se-vere striatal DAT depletion was confirmed in the cor-responding hemisphere in agreement with previousfindings.13 Closer inspection revealed a coincident lossof DAT and TH-IR axons in multiple thalamic nuclei,most notably the basal ganglia and cerebellar recipientnuclei and the RTn (Fig. 5A). The contralateral, con-trol hemispheres exhibited a normal pattern of DATand TH-IR (see Fig 5A). As noradrenergic axons orig-inating in the locus coeruleus and expressing the nor-epinephrine transporter are known to be susceptible,albeit inconsistently, to MPTP,28 relative preservationof thalamic dopamine-b-hydroxylase-IR confirmed thespecificity of the findings. Impregnation of damagedaxons with traditional silver staining in a pattern mir-roring that of DAT-IR loss in the MPTP treated hemi-spheres (see Fig 5C) but not the contralateral controlhemispheres (see Fig 5B) demonstrated active axonaldegeneration rather than an adverse effect of MPTP orits mode of delivery upon IHC sensitivity or DAT pro-tein expression. Histochemical detection of activatedmicroglia in the affected hemispheres confirmed theseobservations (see Fig 5D) and extends recent neuroim-aging of microglial specific ligands in the thalami ofPD patients.29 As many nigrostriatal neurons collater-alized to the thalamus, we hypothesized that their de-generation known to occur with intrastriatal injectionsof dopaminergic neurotoxins30,31 would deplete tha-lamic DA innervation coincidentally. Unilateral, intra-striatal MPTP infusions in two rhesus monkeys indeedproduced histochemically evident decrements in tha-

lamic DAT and TH-IR (see Fig 5A). These findingswere replicated in six rats following unilateral, intrastri-atal injections of the dopaminergic neurotoxin

Fig 4. Retrograde filling of SNc cells projecting to the thala-mus (True Blue chloride) and striatum (red fluorospheres),and tyrosine hydroxylase (TH) immunohistochemistry (Cy-5).The merged image reveals two neurons, labeled 1 and 2, ex-hibiting each marker (ie, triple-labeled). Arrowheads indicateadjacent TH-positive thalamic projecting SNc cell not back-filled from the striatum. Bar 5 0.01mm

Fig 3. Quantification of ispi- and contralateralretrograde labeling of dopaminergic (red) andnondopaminergic (red cross-hatching) SNc cells,and other brainstem nuclei (pedunculopontinetegmental nucleus [PPN] and locus coeruleus[LC]) in three representative cases with nonover-lapping Fluoro-gold injection sites. Data pre-sented as estimates of the percent of total nuclearpopulation retrogradely labeled by each injection(see Materials and Methods). Note that retro-grade labeling of SNc included dopaminergic andnondopaminergic neurons and was exclusivelyipsilateral.


6-OHDA. Profiles of most remaining TH-IR axonswere swollen and dystrophic suggesting active degener-ation (see Fig 5E and F).

DiscussionThe concept that the thalamus receives DA innervationhas previously lacked extensive experimental support.Localization of thalamic DA innervation has beenproblematic because of previous inabilities to distin-guish it from noradrenergic terminals by catecholaminehistofluorescence or immunohistochemical visualizationof tyrosine hydroxylase (TH), an enzyme common to

dopamine, norepinephrine, and epinephrine synthesiz-ing neurons.32 Our findings confirm and extend de-scriptions of DA thalamic innervation suggested by thepresence of DA,33,34 DAT,35,36 and DA receptors,37–39

and the origin of this innervation from the A8 andA10 dopaminergic cell groups.40,41 This is the firststudy to identify such an extensive projection of theSNc (A9) upon the thalamus. Previous lack of appre-ciation for the full extent of thalamic DA innervationmay reflect a differentially lower expression of DAT insubsets of midbrain dopaminergic neurons,15,42 poten-tially those projecting upon the thalamus. Limitationsin the specificity and sensitivity of techniques were alsolikely contributors. Positron emission tomographic im-aging with selective ligands has estimated thalamicDAT levels to approximate those in frontal cortex andapproach 20% of those in caudate/putamen.35,36 Rela-tively low levels of synaptic markers such as DAT andDA receptors may simply reflect the relative paucity ofdendritic spines and low cell-packing density of manythalamic nuclei. The paucity of thalamic dopamine in-nervation suggested by neuroimaging is also misleadingbecause the heterogeneous distribution of DAT withfocal concentrations in small thalamic nuclei such asthe VApc/mc, CeM, and RTn (see Fig 1) are beyondthe limits of its resolution. Light microscopic visualiza-tion of DAT, on the other hand, unambiguously dif-ferentiated DA from other monoamine axon profilesand clearly delineated presumptive thalamic targets.

The specificity of the pattern of thalamic DA inner-vation described here is supported by several indepen-dent facts, including (1) recognition of similar patternsacross three species; (2) recognition that a relative ab-sence from principal sensory nuclei conforms to a gen-eral construct evident across disparate vertebrate spe-cies5; and (3) independent confirmation by an alternatemethodology in anatomical tracing. Failure of tradi-tional retrograde tracers to identify mesencephalic DAinnervation of the thalamus may also have resultedfrom insensitivity of the methodologies. Perikaryal andproximal dendritic filling of retrogradely labeled neu-rons was more consistently observed with FG, which isacknowledged to be a more sensitive tracer than WGA-HRP. Anterograde labeling of dopaminergic mesotha-lamic pathways, on the other hand, is evident in theresults of numerous investigators. Incorporation of dif-fused tracer by more well-accepted thalamic projectingneurons in the adjacent reticular formation and SNrinterfered with correct assignment of their origin to theSNc. Mesencephalic projections to the RTn, for exam-ple, have been attributed to the SNr43,44 rather thanthe SNc as suggested by our findings. Reconstructionsof individually labeled nigrostriatal axons have recentlyconfirmed collaterals that ascend into the thalamus butprovide little detail on their destination or preva-lence.45 By multiple independent methodologies in

Fig 5. Dopamine transporter immunoreactive (DAT-IR) incontrol (left) and experimental (right) hemisphere of a rhesusmonkey subjected to 1-methyl, 1-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) by intrastriatal delivery. Arrow-heads designate equivalent regions of the two thalamic reticu-lar nuclei to facilitate comparisons of DAT-IR densitiesbetween the control and experimental hemispheres. Darkfieldphotomicrograph of silver impregnated, degenerating axon ele-ments in the RTn and adjacent VLo ipsilateral (C) to anintracarotid MPTP injection and in the contralateral, controlhemisphere (B). CD68 immunoreactive, activated microglia(arrowheads in D) in the thalamus of a rhesus monkey receiv-ing unilateral, intracarotid MPTP. Fine TH-IR thalamicterminals in the control hemisphere opposite to intrastriatalinjection of 6-OHDA in the rat (E). Swollen, irregularTH-IR axon profiles (arrowheads in F ) in the thalamus ofthe hemisphere receiving intrastriatal 6-OHDA. Ca 5 Cau-date; fi 5 fimbria; ic 5 internal capsule; RTn 5 thalamicreticular nucleus; 3v 5 3rd ventricle. Bars 5 1.0mm in (A);0.2mm in B and C; and 0.02mm in D to F.


several species we have unequivocally established that alarge proportion of individual dopaminergic SNc neu-rons simultaneously innervate striatum and thalamus.

These observations have important implications fortheories about the functional role of DA and mesence-phalic DA neurons in normal and pathologic behav-iors. According to prevailing views, mesostriatal DAneurons modulate behavior via direct effects on distinctstriatal regions that are linked with correspondingthalamocortical circuits via the basal ganglia output nu-clei8,9 (Fig 6A). Functionally distinct cortical regions inturn reinnervate partially overlapping striatal subdivi-sions, thereby describing a series of parallel yet highlysegregated circuits centered upon oculomotor, limbic,associative, and sensorimotor functions. Within thesensorimotor domain, the SNc is hypothesized to rein-force motor learning.31 We identified here two alterna-tive routes by which mesencephalic DA neurons canmodulate neural activity and motor behavior (see Fig6B). The first is one in which DA directly modulatesthalamic nuclei with an apparent predilection for sub-regions with known motor related functions. The sec-ond is one in which DA modulates the RTn, which inturn provides feedback inhibition to functionally ho-mologous thalamic nuclei. This arrangement affordsindividual mesencephalic DA neurons the potential tosimultaneously modulate striatal, thalamic, and RTnsynapses. Because several synapses need to be traversedbefore striatally mediated effects of dopamine engagethe thalamus, however, collaterals of nigrostriatal neu-rons may “prime” thalamic nuclei, thereby contribut-ing to a reinforcing role for SNc neurons. Collateralprojections appear to target functionally homologousstriatal and thalamic subdivisions,44 providing the po-tential for subpopulations of SNc neurons to modulatebehaviors independently within restricted domains. Al-

ternatively, collaterals might diverge and innervatefunctionally distinct striatal, thalamic, and RTn sites,thus providing a framework more suited to coordinateactivity between parallel circuits, as appears the case forother ascending brainstem pathways.44

The physiological effects of DA on thalamic neuronsand function have received only marginal attention.Dopamine enhances membrane excitability and facili-tates low threshold spike (LTS) activity primarily viaD2 versus D1-like receptors in mediodorsal thalamicneurons,46 and can modulate visually evoked activity inthe lateral geniculate by suppressing or enhancing neu-ral discharge via D1-like or D2-like receptors, respec-tively.47 Physiological responses to DA have not beenassessed in the RTn, although neurons here expressD148 and D4 receptors.49 The physiological and be-havioral effects of DA in thalamus are likely to be com-plex given the multiplicity of DA receptor subtypes(D1–D5), their presynaptic or postsynaptic locationrelative to inhibitory interneurons and cortically pro-jecting neurons, and their differential effects on ionconductances and second messenger systems.50

Some features of the dopaminergic mesothalamicpathway bear remarkable semblance to the monoam-inergic and cholinergic components of the ascendingreticular activating system. All originate from themesopontine junction, exhibit widespread ascendingprojections that collateralize extensively,32 and modu-late the responsiveness of their target neurons to otherstimuli, often in the context of behavioral state (eg,wake vs sleep).51 Strict lateralization and less intratha-lamic collateralization of mesothalamic dopamine path-ways, however, argue for their participation in morespecialized, even lateralized, behaviors. Alternatively,these novel pathways might influence thalamocorticalarousal state given the diffuse thalamocortical dysrhyth-mia,11 impairments of wakefulness and REM sleep,12

and reductions in cellular firing in thalamic motor nu-clei (eg, VLo and VPLo)10 observed in PD. A shiftfrom tonic to burst firing is also observed, similar tothat driven by calcium-mediated LTS, which promotelow-frequency, SWS-like, synchronized oscillations.52,53

This perturbation likely reflects generalized hyperpolar-ization brought about by excessive inhibition or disfa-cilitation, both reasonable consequences of DA loss inthe RTn and principal thalamic nuclei, respectively.Loss of DA from the VPLo may also explain the suc-cess of systemic dopaminomimetics, but not embryonicSN-to-striatum grafts, in reversing parkinsonian trem-or.54 Together with our findings, these considerationsargue strongly that the integrity of thalamic DA inner-vation is compromised in PD, and this will requireconfirmation. Collateralization of the nigrostriatalpathway to thalamus also has relevance to a broaderspectrum of DA-mediated neuropsychiatric disorders.Innervation of the VApc, for example, will need to be

Fig 6. (A) Schematic of basal ganglia nuclei and their princi-pal connections that is central to prevailing heuristic models ofnormal and pathologic behavior (modified from DeLong55).(B) A revised model highlighting those novel pathways reportedherein (bold lines and arrows).


considered in attention deficit disorder where dimin-ished dopaminergic tone in dorsolateral prefrontal-striatal circuits that traverse the VApc has been hypoth-esized.6 In a similar vein, innervation of the AV, MD,and VAmc may be alternate sites at which hypothe-sized supersensitivities to DA in the associative or lim-bic striato-pallidal-thalamocortical subcircuits contrib-ute to obsessive-compulsive and tic disorders, andschizophrenia.7 Thus, popular models of disease thathave until now ascribed DA mediated pathologic be-haviors solely to DA influences upon the basal gangliaor cerebral cortex will require critical reappraisal andrevision.

This work was supported by the United States Public Health Service(NS-36697 and NS-40221).

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nigrostriatal collaterals to thalamus degenerate in parkinsonian animal models

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