A population of cholinergic neurons is present in the macaque monkey thalamus

European Journal of Neuroscience, Vol. 10, pp. 2346–2352, 1998 © European Neuroscience Association

A population of cholinergic neurons is present in themacaque monkey thalamus

Beatriz Rico and Carmen CavadaDepartamento de Morfologıa, Facultad de Medicina, Universidad Autonoma de Madrid, Arzobispo Morcillo s/n, 28029 Madrid,Spain

Keywords: acetylcholine, ChAT, intralaminar thalamic nuclei, paracentral nucleus, VAChT

Abstract

Three new cholinergic markers were employed to study the cholinergic innervation in the thalamus of adultmacaque monkeys. They were: two antibodies against choline acetyltransferase (ChAT), one polyclonal and onemonoclonal; and a polyclonal antibody against the vesicular transporter of acetylcholine (VAChT), a powerfulnew marker that colocalizes with ChAT. This approach led to an unexpected finding: the three antibodiespositively immunostained a population of neurons in the paracentral nucleus. The immunostained cells areconfined to the dorsal region of this nucleus along its rostrocaudal extent. Measurement of the somatic areas ofthe immunostained neurons indicated that they correspond to a population of large neurons thought to beprojection neurons. Because dorsal paracentral neurons are known to project to the dorsal striatum and specificcortical areas involved in visual and visuomotor mechanisms, these structures might be modulated by cholinergicthalamic neurons.

Introduction

Precise identification of central cholinergic neurons and their projec-tions has relied on modern immunohistochemical tools. Antibodiesdirected against choline acetyltransferase (ChAT), which catalysesthe synthesis of acetylcholine (ACh), were instrumental in the earliestdescriptions of brain cholinergic groups in primates (Hedreenet al.,1983; Mesulamet al., 1984). More recently, the vesicular AChtransporter (VAChT) has been cloned. This protein, which is respons-ible for storing ACh in vesicles, has proven to be a powerful novelcholinergic marker: VAChT shares a single genetic locus with theChAT gene and colocalizes with ChAT (Ericksonet al., 1994; Gilmoret al., 1996; Ichikawaet al., 1997).

We undertook an investigation aimed at mapping the cholinergicinnervation of the macaque thalamus using immunohistochemistryfor ChAT and VAChT. In the course of the study we had anunanticipated finding: a group of ChAT- and VAChT-immunopositiveneurons in the paracentral nucleus (Pcn). Because of the non-existenceof previous reports on cholinergic neurons in the mammalian thalamus,we made innumerable efforts to check whether the immunostainingwas genuine or an artefact. All the evidence we have gatheredindicates that the Pcn nucleus of adultMacaca nemestrinaholds apopulation of cholinergic neurons.

The present study has been reported previously in abstract form(Rico & Cavada, 1997).

Materials and methods

Experimental proceduresSix adult Macaca nemestrinaof both sexes were used. Adequatemeasures were taken to minimize pain or discomfort of the animals

Correspondence: C. Cavada, as above. E-mail: [email protected]

Received 29 October 1997, revised 11 February 1998, accepted 11 March 1998

in accordance with European and Spanish guidelines (86/609/EEC:European Communities Council Directive of 24 November 1986; andBOE of 18 March 1988). Two animals were experimentally naive.Four animals had received cortical injections of axonally transportedtracers for purposes unrelated to those of the present study. Specifically,injections of fluorescent tracers (fast blue, diamidino yellow andfluoro ruby), biotinylated dextran amine, a horseradish peroxidase(HRP) mixture (HRP1 HRP-wheat germ agglutinin conjugate) anda cholera toxin B subunit-colloidal gold conjugate, were made inprefrontal, posterior parietal and inferior temporal areas. Because weroutinely used a peroxidase-antiperoxidase (PAP) protocol to developimmunopositive elements, it is unlikely that any of these tracers, withthe exception of HRP, could have induced false positive labelling.The HRP mixture was injected in the right hemispheres of the fourmonkeys in the rostral orbitofrontal cortex, dorsolateral prefrontalcortex and superior temporal sulcus (two animals), respectively. Allanimals were deeply anaesthetized with sodium pentobarbital andperfused through the ascending aorta with saline, 4% paraform-aldehyde in phosphate buffer (PB, 0.1M, pH 7.4) and a series of PBsucrose solutions (5–20%). The brains were stereotaxically blockedin the coronal plane, removed and cryoprotected by successiveimmersion in 20 and 30% sucrose solutions until sunk. The brainblocks were frozen sectioned into 40-µm-thick serial sections.

Parallel series of sections were processed to reveal the cyto-,myelo- and chemoarchitecture of the thalamus using cresyl violetstaining, silver myelin staining (Gallyas, 1979), and acetylcholinester-ase (AChE) histochemistry, respectively. The AChE activity, whichis particularly useful for identifying the thalamic nuclei, was revealedusing the protocol described in Cavadaet al. (1995).

Cholinergic neurons in the monkey thalamus 2347

The sections to be used in the immunohistochemical or controlexperiments were stored at –20 °C in an ethylene–glycol solution forseveral weeks or months until these procedures were run. Theethylene–glycol solution was removed from the sections throughseveral rinses in PB at 4 °C over 3 days.

Immunohistochemical procedures

For immunohistochemical detection of ChAT, a polyclonal andmonoclonal antibody were used, both produced against human placen-tal ChAT. The polyclonal antibody (Chemicon International®, CA,USA, AB144p) was raised in goat, and the antiserum affinity purified.Its specificity has been demonstrated (Bruceet al., 1985; Grosmanet al., 1995). The mouse monoclonal antibody (28C4) was generouslysupplied by Dr M. Ma¨der and Dr R. Poethke (Universita¨t Gottingen,Germany), who have characterized it in various immunoassays(Poethkeet al., 1997: table 1).

A polyclonal rabbit antiserum produced against a human VAChTC-terminal peptide (Phoenix Pharmaceuticals®, CA, USA, H-V005)was used as the third cholinergic marker. Its specificity has beendemonstrated previously (Scha¨fer et al., 1995).

The steps listed below were used for immunohistochemical detec-tion of the antigens in serial sections through the thalamus.1 Pretreatment in a solution of 1% H2O2 and 90% methanol in H2Ofor 10 min at room temperature to inhibit endogenous peroxidase.2 Preincubation in blocking sera for 3 h at room temperature. Asolution of 5% bovine serum albumin (BSA) and 0.4% Triton X-100in PB saline (PBS) was employed in the protocols to reveal theChAT. A solution of 10% normal goat serum, 2% BSA and 0.3%Triton X-100 in PBS was used in the VAChT protocol.3 Incubation in the primary antibodies for 60 h at 4 °C. The followingdilutions were employed: 1/100–1/1000 for the polyclonal anti-ChAT;1/500–1/1000 for the monoclonal anti-ChAT; and 1/1000–1/2000 forthe polyclonal anti-VAChT. The anti-ChAT antibodies were in asolution made of 20% normal rabbit serum, 5% BSA and 0.5% TritonX-100 in PBS. The anti-VAChT antiserum was diluted in PBScontaining 1% normal goat serum, 2% BSA and 0.3% Triton X-100.4 Incubation in secondary antisera for 1 h at room temperature. Arabbit antigoat (Chemicon International®, CA, USA, AB221, 1/50)and rabbit antimouse antisera (Chemicon International®, CA, USA,AB240, 1/30) were employed in the polyclonal and monoclonal ChATprotocols, respectively. Both antisera were diluted in a solution of20% normal rabbit serum, 2% BSA and 0.2% Triton X-100. In theVAChT protocol, a goat antirabbit antiserum (Chemicon Inter-national®, CA, USA, AB21, 1/50), diluted in the same solution asthe primary antibody was used.5 Incubation for 1.5 h at room temperature in PAP raised in goat,mouse and rabbit, respectively, for the polyclonal ChAT, monoclonalChAT and polyclonal VAChT protocols (Chemicon International®,CA, USA, PAP10, PAP14, PAP18, 1/600). The PAP was diluted inthe same solutions as the corresponding secondary antisera. In selectedsections incubated with the VAChT antiserum, a standard avidin–biotin–peroxidase method (Vectastain, ABC Standard kit, VectorLabs®, CA, USA) was employed.6 Peroxidase development. In most sections, the sensitive glucose–oxidase–diaminobenzidine–nickel method of Shuet al. (1988) wasused. Selected sections were developed in a PB solution of 0.05%diaminobenzidine and 0.0015% H2O2 under microscope control. Oncemounted and dry, these sections were silver- and gold-intensifiedusing the protocol of Kittet al. (1994), slightly modified by incubationof the sections at room temperature and use of low concentrations ofsilver nitrate (1%) and gold chloride (0.1%).

All steps were carried out under gentle agitation in free floating

© 1998 European Neuroscience Association,European Journal of Neuroscience, 10, 2346–2352

sections, unless otherwise specified. Except for between steps 2 and3, the sections were thoroughly rinsed in PBS between the differentsteps. The sections were finally mounted, dehydrated, defatted andcoverslipped.

Control procedures

All the series of immunohistochemical experiments were run togetherwith positive and negative controls. The positive control protocolswere identical to those of the experimental series, but we reactedtissue that is known to contain cholinergic neuronal bodies: brainstemmotor nuclei, habenula, neostriatum and basal forebrain (Mesulamet al., 1984). In the negative control experiments we substituted theprimary antibodies with sera from the species where they hadbeen raised.

Preadsorption experiments were also run. Before incubation of thetissue with the primary antiserum AB144p, we added 100 mg/mL ofhuman placental ChAT (Sigma®, St Louis, MO, USA, C2898) to thesolution. This preincubation was carried out for 24 h at 4 °C in gentleagitation. The AB144p was used at a concentration of 1/4000 in theseexperiments. This was the maximal dilution of the antiserum yieldingpositive staining of cell bodies. In additional parallel experiments,we replaced the human ChAT with a similar concentration ofbutyrilcholinesterase from human serum (Sigma®, C5386).

Data analysis

The sections were examined under a Zeiss Axioskop light microscope.The thalamic nuclei were identified by reference to the adjacentsections stained for cell bodies, myelin or AChE activity. In order toascertain the types of ChAT-immunopositive neurons (projectionneurons and/or interneurons), we estimated their size in two monkeys,one of which had not received tracer injections in the cortex. Neuronswere sampled from the Pcn nucleus of six regularly spaced sections,immunostained with the AB144p antibody, from each monkey (62neurons from one monkey and 42 from the other). By using an 1003oil immersion objective and camera lucida, the boundary of theperikaryon of these cells was traced on the plane of the nucleus. Thesomatic areas were measured using a digitizing tablet.

Results and discussion

Control experiments

Examination of the positive control tissue revealed that all threeantibodies labelled well-established cholinergic neuronal groups in thebrainstem (motor nuclei), diencephalon (habenula) or telencephalon(nucleus basalis, caudate and putamen). The labelling characteristicsof the immunopositive neurons of the caudate nucleus and putamenwere particularly useful for comparison with the immunopositiveneurons in the thalamus, since both populations were present in thesame tissue sections and thus had undergone identical experimentalconditions (see below).

No immunolabelling was present in the thalamus when the primaryantibodies were omitted in the protocol (Fig. 1). In the preadsorptioncontrol experiments, no immunolabelling was evident in the tissue.However, preincubation with non-specific human protein instead ofChAT antigen resulted in positive immunolabelling.

Immunolabelling in the paracentral nucleus

ChAT- and VAChT-immunopositive neuronal bodies and axons wereconsistently present in the Pcn nucleus of the thalamus in all animalswhose sections had been incubated with the polyclonal and monoclonal

2348 B. Rico and C. Cavada

FIG. 1. Immunostaining with ChAT antibodies in the left hemithalamus (A,D) and absence of reaction-product in the right hemithalamus, used as control (C,F).(A) Reacted with the polyclonal antiserum (ChATp), (D) reacted with the monoclonal antibody (ChATm). The hemithalamus of (C) and (F) was reacted inparallel to that of (A) and (D), but the primary antibodies were substituted with non-specific sera. (B) and (E) show sections, respectively adjacent to (A/C) and(D/F), that were histochemically treated to reveal AChE activity, which helps identify the thalamic nuclei. The regions shown in (A/C) and (D/F) are indicatedby the insets in (B) and (E), and corresponding blood vessels are marked with asterisks and stars. The borders of the Pcn nucleus in (A), (C), (D) and (F) areindicated with arrowheads. Calibration bars are common for (A) and (C), and (D) and (F). AD, anterodorsal nucleus; AM, anteromedial nucleus; AV, anteroventralnucleus; MD, mediodorsal nucleus; Pcn, paracentral nucleus; VA, ventral anterior nucleus.



FIG. 2. Immunostaining with ChAT polyclonal antiserum (A,B,D). (C) An adjacent section processed to reveal AChE activity. The midline is to the right.Asterisks indicate corresponding blood vessels in (A), (B) and (C). Note that the ChAT-immunoreactive cell bodies in (B) are limited to the dorsal region ofthe nucleus (inset). This region is shown in (A) at higher power. (D) A representation of a sector of the Pcn nucleus shown in (A) is presented next to a pictureof an adjacent section stained with cresyl violet (E). The immunostained neurons of (D) are similar in size to the largest cells of the Pcn nucleus in (E). Thisimpression was confirmed after measurement of the somatic areas of the immunostained neurons. Note the morphological detail of the neurons immunostainedwith the ChAT antibody (A,D), and also the presence of immunostained neuropil throughout the Pcn nucleus (A,B,D). The calibration bar is common for (D)and (E). MD, mediodorsal nucleus; Pcn, paracentral nucleus; VP, ventral posterior nucleus.

ChAT antibodies, or the VAChT antiserum. This was the case in bothgroups of monkeys, those that were experimentally naive and thosethat had received cortical injections of different tracers. In the latter,we discarded the possibility that the presence of immunoreactive cellbodies might be a consequence of the unilateral cortical HRPinjections, because the immunolabelling showed similar characteristicsin the right and left Pcn nuclei. In addition, no immunoreactive cellbodies were observed in thalamic nuclei known to project heavilyinto the injected regions, e.g. the mediodorsal or medial pulvinar.

The immunoreactive cell bodies were only observed in the dorsalregion along the antero-posterior extent of the Pcn nucleus. Thisregional specificity was a consistent feature of the three antibodiesemployed here (Figs 1A,D, 2A–C and 3A–C). In general, the intensity


of the immunostaining with either the ChAT or VAChT antibodieswas somewhat lower in the neurons of the Pcn nucleus than in thoseof the neostriatum or nucleus basalis.

The morphological features of the immunopositive neurons weredifferent with each antibody tested. The polyclonal ChAT antiserumyielded the best immunostaining of cell bodies (Figs 1A and 2A,D);these appeared as large polygonal neurons with distinct dendriticprofiles (Fig. 2D). The monoclonal ChAT antibody provided lessmorphological detail than the polyclonal ChAT antiserum. Nonethe-less, even when the lowest concentrations of antibody were used, theimmunopositive cell bodies of the Pcn nucleus invariably stand outagainst a remarkably pale background (Fig. 1D). VAChT-immunoposi-tive neurons were more difficult to identify than ChAT-immunopositive


FIG. 3. Immunostaining with VAChT antiserum in the thalamus (A,B) and caudate nucleus (D). (C) Corresponds to a section adjacent to (A) and (B) processedto reveal AChE activity. The midline is to the right. The inset in (C) corresponds to the region shown in (B). In turn, the inset in (B) is shown at a higherpower in (A). Cell bodies immunoreacted with anti-VAChT show less morphological detail than those immunostained with anti-ChAT and are more difficult toidentify [arrows in (B), compare with Fig. 2A]. However, the neuropil appears more heavily stained after VAChT immunostaining than after ChAT immunostaining(compare with Fig. 2A,D). The morphology of the VAChT-immunoreactive neurons was similar in the Pcn nucleus (A,B) and caudate nucleus of the samesection (D), which was used as a positive control. Calibration bars are common for (A) and (D). Cd: caudate nucleus; MD: mediodorsal nucleus; Pcn: paracentralnucleus; VL: ventral lateral nucleus.

ones (Fig. 3A,B). Unlike the uniform perikaryal distribution ofthe ChAT immunostaining, VAChT immunostaining was markedlygranular, and hardly any reaction product was observed in dendrites.These features were essentially similar to those of the VAChT-immunostained cells in the caudate nucleus (Fig. 3D), and othersreported in the literature (Scha¨fer et al., 1995; Gilmoret al., 1996;Ichikawaet al., 1997).

The above evidence is the first indication that the primate thalamusholds a group of cholinergic neurons. The obvious question is whythese Pcn ChAT-immunoreactive neurons went undetected in earlierstudies. Indeed, previous reports on the cholinergic innervation of theprimate thalamus using immunohistochemistry do not mention thepresence of ChAT-positive neurons (Heckerset al., 1992; Schwartz& Mrzljak, 1993). Likewise, earlier research aimed at revealing thecholinergic cell groups of the monkey brain has also failed to detectChAT-immunopositive neurons in the thalamus (Hedreenet al., 1983;Mesulamet al., 1984). We believe that differences in the specificityand sensitivity of the immunohistochemical procedures employedmost likely account for the above discrepancies. In our study we haveused two new ChAT antibodies, one an affinity-purified polyclonalantibody and the other monoclonal, and have routinely intensified thefinal histochemical reactions. It is possible that the antibodies weemployed were highly sensitive and thus able to recognize lowerconcentrations of the enzyme and/or different epitopes than the earlierones. Indeed, the lower staining intensity of the Pcn neurons incomparison to that of the neostriatum or nucleus basalis neuronssuggests that the thalamic neurons contain less ChAT. The fact thatdorsal Pcn neurons are not only immunopositive for ChAT, but alsofor the new cholinergic marker VAChT, gives further support to the


notion that this is a cholinergic cell group. It is pertinent to recallthat other cholinergic cell groups in the primate brain have also beendifficult to identify. These groups include hypothalamic and cerebellarpopulations, which were not revealed in the original studies on theprimate cholinergic systems and have come to light more recently(Tagoet al., 1987; De Lacalleet al., 1993).

In addition to the immunoreactive cell bodies, we observed a denseplexus of immunoreactive axons extending throughout the entiremacaque Pcn nucleus. This plexus was most evident after VAChTimmunostaining (Fig. 3A,B), but the polyclonal ChAT antiserum alsogave good fibre staining (Figs 1A and 2A,B,D). The coexistence inthe Pcn nucleus of a dense mesh of immunoreactive fibres and cellsomata raises the possibility of some translocation of the proteinscontained in the fibres as a result of inadequate tissue processing.However, this possibility is improbable here because the immunoreac-tive fibres are present throughout the Pcn nucleus, while the immuno-reactive somata are consistently confined to its dorsal region. Thehuman Pcn nucleus has also been shown to be densely innervated byChAT-immunoreactive axons (Heckerset al., 1992).

Connectional and functional considerations

The size of the ChAT-immunopositive Pcn neurons suggests that theyare projection neurons rather than intrinsic neurons. Their meansurface area was 195.21µm2 (SD: 62.94). The monkey intralaminarnuclei, including the Pcn, have been shown to hold two neuronalpopulations that can be differentiated on the basis of their content ingamma-aminobutyric acid (GABA) and cell body size (Huntet al.,1991). GABAergic neurons, which are considered to be intrinsicneurons (Bentivoglioet al., 1991), have somatic areas of 65–100µm2,


whereas non-GABAergic neurons, considered to be projectionneurons, have somatic areas of 150–230µm2. The size range of theChAT-immunoreactive Pcn neurons falls within that of the non-GABAergic population, and therefore supports the hypothesis thatthe axons of these neurons project outside the thalamus.

This brings up the question of the origin of the dense cholinergicplexus in the Pcn nucleus. Although it is possible that the axons ofthe Pcn neurons emit collaterals within the nucleus, it seems morelikely that the dense cholinergic plexus of the Pcn nucleus has anextrathalamic origin. The primate thalamus is known to receive inputfrom the upper brainstem cholinergic groups (Steriadeet al., 1988)and the nucleus basalis (Hreibet al., 1988). Whether the Pcncholinergic plexus of the macaque monkey originates in one or theother, or both, is not known. Indirect evidence indicates that thenucleus basalis projects to the Pcn nucleus: Heckerset al. (1992)demonstrated that the human Pcn nucleus holds a rich fibre plexusthat is immunoreactive to ChAT and nerve growth factor receptor.Because both antigens colocalized in the neurons of the primatenucleus basalis, and the nerve growth factor receptor is absent in theupper brainstem cholinergic neurons, they concluded that the nucleusbasalis contributes to the cholinergic innervation of the Pcn. Doublelabelling studies employing tract-tracing and immunohistochemistryare needed to verify this proposal, and also to elucidate if upperbrainstem cholinergic neurons supply the macaque Pcn nucleus withcholinergic input. This is functionally relevant because neurophysio-logical research propounds that the cholinergic innervation of therostral intralaminar nuclei plays a key role in arousal mechanisms(Steriade & McCarley, 1990).

Identification of the targets of the cholinergic Pcn neurons alsorequires double labelling studies. Published data on the connectionsof the Pcn nucleus indicate that its dorsal region projects to the dorsalstriatum (Parentet al., 1983) and selected cortical areas. These includeprincipally visual association regions in the inferior temporal cortex(Websteret al., 1993), occipital cortex and posterior parietal cortex(Tiggeset al., 1982; Schmahmann & Pandya, 1990). In addition, thefrontal eye field (Barbas & Mesulam, 1981; Huertaet al., 1986),presupplementary motor area and supplementary eye field (Matelli &Luppino, 1996) receive dorsal Pcn input. If the cholinergic neuronsof the Pcn nucleus contribute to these projections, the innervatedareas may have a supplementary source of ACh distinct from thatarising in the basal forebrain (Mesulamet al., 1983). Likewise,striatal ACh may, in part, have an extrastriatal origin in the Pcnthalamic nucleus.

The dorsal Pcn nucleus holds visually responsive and eye-positionneurons (Schlag-Rey & Schlag, 1984; Schlag & Schlag-Rey, 1984).In light of their physiological characteristics, they have been attributeda role in the control of gaze and eye movement initiation. It isrelevant to note the significant afferent input to the Pcn nucleus thatarises in the superior colliculus and pretectum (Beneventoet al.,1977; Partlowet al., 1977). Since the role attributed to corticalcholinergic innervation in attentional mechanisms is also critical(Everitt & Robbins, 1997), we speculate that the cholinergic cellgroup of the Pcn thalamic nucleus is involved in visual attention andoculomotor control.

Acknowledgements

We thank Dr M. Mader and Dr R. Poethke for their generous donation of theChAT monoclonal antibody; and G. de la Fuente, P. Romero and R. Sa´nchezLozano for technical assistance. This work was supported by DGICYT PM92-0040 and PM95-0028.


Abbreviations

ACh acetylcholineAChE acetylcholinesteraseBSA bovine serum albuminChAT choline acetyltransferaseGABA gamma-aminobutyric acidHRP horseradish peroxidasePAP peroxidase-antiperoxidasePcn paracentral nucleusPB phosphate bufferPBS phosphate-buffered salineVAChT vesicular acetylcholine transporter

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A population of cholinergic neurons is present in the macaque monkey thalamus

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