Diverse thalamic projections to the prefrontal cortex in the rhesus monkey

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    Diverse Thalamic Projections to the Prefrontal Cortex in the Rhesus Monkey

    H. BARBAS, T.H. HASWELL HENION, AND C.R. DERMON Department of Health Sciences, Boston University, and Department of Anatomy and Neurobiology, Boston University School of Medicine, Boston, Massachusetts 02215

    ABSTRACT We studied the sources of thalamic projections to prefrontal areas of nine rhesus monkeys

    with the aid of retrograde tracers (horseradish peroxidase or fluorescent dyes). Our goal was to determine the proportion of labeled neurons contributing to this projection system by the mediodorsal (MD) nucleus compared to those distributed in other thalamic nuclei, and to investigate the relationship of thalamic projections to specific architectonic areas of the prefrontal cortex. We selected areas for study within both the basoventral (areas 11, 12, and ventral 46) and the mediodorsal (areas 32, 14, 46, and 8) prefrontal sectors. This choice was based on our previous studies, which indicate differences in cortical projections to these two distinct architectonic sectors (Barbas, '88; Barbas and Pandya, '89). In addition, for each sector we included areas with different architectonic profiles, which is also relevant to the connec- tional patterns of the prefrontal cortices.

    The results showed that MD included a clear majority (over 80%) of all thalamic neurons directed to some prefrontal cortices (areas 11,46, and 8); it contributed just over half to some others (areas 12 and 321, and less than a third to area 14. Clusters of neurons directed to basoventral and mediodorsal prefrontal areas were largely segregated within MD: the former were found ventrally, the latter dorsally. However, the most striking findings establish a relationship between thalamic origin and laminar definition of the prefrontal target areas. Most thalamic neurons directed to lateral prefrontal cortices, which are characterized by a high degree of laminar definition (areas 46 and 81, originated in the parvicellular and multiform subdivisions of MD, and only a few were found in other nuclei. In contrast, orbital and medial cortices, which have a low degree of laminar differentiation, were targeted by the magnocellular subdivision of MD and by numerous other limbic thalamic nuclei, including the midline and the anterior. Thus topographic specificity in the origin of thalamic projections increased as the laminar definition of the target area increased. Moreover, the rostrocaudal distribution of labeled neurons in MD and the medial pulvinar also differed depending on the degree of the laminar definition of the prefrontal target areas. The rostral parts of MD and the medial pulvinar projected to the eulaminate lateral prefrontal cortices, whereas their caudal parts projected to orbital and medial limbic cortices. Selective destruction of caudal MD is known to disrupt mnemonic processes in both humans and monkeys, suggesting that this thalamic- limbic prefrontal loop may constitute an important pathway for memory.

    Key words: thalamus, prefrontal architecture, orbital cortex, limbic system, mediodorsal nucleus, memory, macaque monkeys, medial cortex, cortical lamination, thalamic evolution

    Classic studies have suggested that the mediodorsal (MD) thalamic nucleus and the prefrontal cortex are closely associated (see Le Gros Clark, '32; Nauta, '71; Reep, '84 for reviews).This view was based on observations that the elaboration of MD is associated with an expansion of the frontal cortex to which it projects (Le Gros Clark, '32; Walker, '36, '38; Von Bonin and Green, '49; Pribram et al., '53; Nauta, '62; Akert, '64; Locke, '69; Tobias, '75; Tanaka, '76). In fact, Rose and Woolsey ('48) suggested that the

    prefrontal cortex should be defined as the region receiving projections from MD. With the advent of new and more sensitive tracing procedures, however, it became apparent that neurons in thalamic nuclei other than MD project to prefrontal areas as well (Carmel, '70; Jones and Leavitt,

    Accepted July 24, 1991. Address reprint requests to H. Barbas, Boston University, 635 Common-

    wealth Ave. #431, Boston, MA 02215.

    o 1991 WILEY-LISS. INC.

  • 66 H. BARBAS ET AL.

    '74; Trojanowski and Jacobson, '74; Bos and Benevento, '75; Kievit and Kuypers, '77; Potter and Nauta, '79; Barbas and Mesulam, '81; Asanuma et al., '85; Baleydier and Mauguiere, '85; Goldman-Rakic and Porrino, '85; Ilinsky et al., '85, '87). The relative density of neurons in MD and in other nuclei directed to prefrontal cortices is, however, not known.

    In this study we used a simple quantitative method to address the issue of diversity in the origin of thalamic projections to prefrontal cortices. This was accomplished by noting the relative proportion of retrogradely labeled neu- rons in MD compared to those distributed in other thalamic nuclei after injecting retrograde tracers in specific prefron- tal areas. In addition, we investigated some hitherto unre- solved aspects of the topography of thalamic projections. We noted particularly the distribution of prefrontally di- rected neurons along the rostrocaudal extent of MD and the medial pulvinar, both of which are large nuclei. This information is important because mnemonic deficits have been associated with preferential destruction of the caudal parts of MD in both humans and monkeys (Victor et al., '71; Isseroff et al., '82; Zola-Morgan and Squire, '85).

    Finally, the present study was conducted in the context of our previous findings, which have shown differences in the cortical and some subcortical projections to specific prefron- tal areas (Barbas, '88; Barbas and De Olmos, '90). Those studies established that the degree of laminar definition of individual areas (Barbas and Pandya, '89) is relevant to the pattern of their connections. Therefore, we sought to determine whether the thalamic projections to the same prefrontal cortices varied along these lines. The present study provides evidence consistent with this view.

    METHODS Experiments were conducted on nine rhesus monkeys

    (Macaca mulatta). They were anesthetized with ketamine hydrochloride (10 mg/kg, i.m.) followed by sodium pentobar- bital administered intravenously through a femoral cathe- ter until a surgical level of anesthesia was achieved. Addi- tional anesthetic was administered during surgery as needed. Surgery was performed under aseptic conditions. The monkey's head was firmly positioned in a holder, which left the cranium unobstructed for surgical approach. A bone defect was made, the dura was retracted and the cortex was exposed.

    Horseradish peroxidase (HRP) experiments In eight animals, injections of a solution containing 8%

    HRP conjugated to wheat germ agglutinin (Sigma) were made with a microsyringe (Hamilton, 5 p1) mounted on a microdrive. The needle was lowered to the desired site under microscopic guidance. Small amounts (0.05 ~ 1 ) of the injectate were delivered 1.5 mm below the pial surface at each of two adjacent sites separated by 1-2 mm over a 30-minute period.

    HRP was injected in orbital areas 11 and 12, lateral area 12, and ventral area 46 (cases 1-4). These areas belong to the basoventral prefrontal sector defined on the basis of its architecture and intrinsic connections (Barbas and Pandya, '89). In addition, injections were placed in areas within the mediodorsal prefrontal sector: medial area 32 (case 5), dorsal area 46 (case 71, and dorsal area 8 (cases 8 and 9).

    Following a 40-48-hour survival period, the monkeys were re-anesthetized and perfused through the heart with

    A AN AD AM AV Cau cc Cdc c g Cif Cim c1 Clc CM c s Csl GM GMmc GMpc H IL LD LF LGN Li LO LP MD MDdc MDmc MDmf, mf MDpc ML MO MTT

    arcuate sulcus anterior nuclei anterior dorsal nucleus anterior medial nucleus anterior ventral nucleus caudate corpus callosum central densocellular nucleus cingulate sulcus central inferior nucleus central intermediate nucleus central lateral nucleus central latocellular nucleus centromedian nucleus central superior nucleus central superior lateral nucleus medial geniculate nucleus medial geniculate nucleus, magnocellular sector medial geniculate nucleus, parvicellular sector habenula intralaminar nuclei lateral dorsal nucleus lateral fissure dorsal lateral geniculate nucleus limitans nucleus lateral orbital sulcus lateral posterior nucleus mediodorsal nucleus mediodorsal nucleus, densocellular sector mediodorsal nucleus, magnocellular sector mediodorsal nucleus, multiform sector mediodorsal nucleus, parvicellular sector midline nuclei medial orbital sulcus mamillothalamic tract


    OLF P Pa Pac PAll Pcn Pf Pi PI Pm Po Pro ProM Pt R Re Ro Sf SG SM,Sm ST THI VA VAmc VL VLC VLm VLO



    olfactory cortex principal sulcus paraventricular nucleus paraventricular nucleus, caudal sector limbic periallocortex paracentral nucleus parafascicular nucleus pulvinar nucleus, inferior sector pulvinar nucleus, lateral sector pulvinar nucleus, medial sector pulvinar nucleus, oral sector proisocortex rostral portion of the ventral premotor cortex parataenial nucleus reticular nucleus reuniens nucleus rostral sulcus subfascicular nucleus suprageniculate nucleus stria medullaris superior temporal sulcus habenulo-interpeduncular tract ventral anterior nucleus ventral anterior nucleus, magnocellular sector ventral lateral nuclei ventral lateral caudal nucleus ventral lateral medial nucleus ventral lateral oral nucleus ventral lateral postrema nucleus ventral posterior inferior nucleus ventral posterior lateral caudal nucleus ventral posterior lateral oral nucleus ventral posterior medial nucleus ventral posterior medial nucleus, parvicellular sector area X


    -("- o/ *. 25 - - - -._ ~ _ _ _ _ _ _ ~ - ---/ - - 2 4 *.A-

    5 mm 9 C

    Fig. 1. Composite of injection sites shown on the medial (A), lateral (B), and basal (C) surfaces of the cerebral hemisphere. The injection sites are superimposed on an architectonic map of the prefrontal cortex (Barbas and Pandya, '89). Mediodorsal areas appear above and basoven- tral below the heavy dashed line (B). Within the mediodorsal sector, stepwise increases in laminar definition are observed in a direction from the medial periallocortex (PAll) to proisocortical areas 24, 25, and 32, and then through medial areas 14, 10, and 9, to dorsal areas 9, 10, and rostral 46, and finally through caudal areas 46 and 8. Within the basoventral sector, gradual increases in the number of layers and their delineation are observed from the orbital PAll to the proisocortex (Pro), orbital area 25, area 13, then to orbital areas 12, 14, and 11, and then through ventrolateral areas 10, 12, rostral 46, and finally to caudal areas 46 and 8. Small dashed lines demarcate architectonic areas; large dashed lines demarcate sulci. Large numbers designate architectonic areas; small numbers refer to cases. Injection sites in mediodorsal prefrontal cortices are shown by the striped area; in basoventral sites they are shown in black.

    saline followed by 2 liters of fixative (1.25% glutaraldehyde, 1% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4). The fixative was followed by perfusion with 2 liters of cold (4C) phosphate buffer (0.1 M, pH 7.4).

    The brain was then removed from the skull, photo- graphed, placed in glycerol phosphate buffer (10% glycerol and 2% DMSO in 0.1 M phosphate buffer at pH 7.4) for 1 day and in 20% glycerol phosphate buffer for another 2 days. The brain was frozen in -75C isopentane, trans- ferred to a freezing microtome, and cut in the coronal plane at 40 pm in 10 series. One series of sections was treated to visualize HRP (Mesulam et al., '80). The tissue was mounted, dried, and counterstained with neutral red. Adja- cent series of sections were stained for Nissl bodies, myelin, acetylcholinesterase, or cytochrome oxidase to aid in delin- eating architectonic borders (Geneser-Jensen and Blacks- tad, '71; Gallyas, '79; Wong-Riley, '79).

    Fluorescent tracer experiment With the surgical procedures outlined above, one animal

    received an injection of the fluorescent tracer fast blue (3%, 0.4 pl) in medial area 14 (case 6). After a survival period of 10 days, the animal was deeply anesthetized and perfused with 6% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4). The brain then was placed in a solution of 6% paraformaldehyde with glycerol and 2% DMSO for 3 days, frozen, and cut (as described above). Sections were mounted on subbed slides.

    Data analysis Brain sections were viewed microscopically under bright-

    and darkfield illumination for HRP cases, or fluorescence illumination for case 6. Outlines of brain sections, the location of retrogradely labeled neurons ipsilateral to the injection site, and the site of blood vessels used as land- marks were transferred from the slides onto paper by using an X-Y recorder (Hewlett Packard, 7044B), which was electronically coupled to the stage of the microscope. In later experiments data were transferred from the slides onto paper by means of a digital plotter (Hewlett Packard, 7475A) coupled to the stage of the microscope (as described above) and to a computer (Compaq 386). In this system the analog signals are converted to digital signals via an analog-to-digital converter (Data Translation). Software developed in this laboratory allowed each labeled neuron to be recorded only once. Both procedures allow accurate topographic presentation of labeled neurons within the thalamus.

    All sections in one series that contained labeled neurons in the thalamus were charted. In initial experiments labeled neurons (represented as dots on the charted sections) were counted by using a grid inserted in the microscope eyepiece. When the data were stored in a computer, labeled neurons were counted by outlining the area of interest (e.g., one nucleus) by moving the X and Y axes of the stage of the microscope. The number of labeled neurons within the enclosed area was calculated by means of an algorithm written for this purpose. A designated region was then subdivided automatically along its dorsoventral or mediolat- eral extent into five sectors and the number of labeled neurons per sector recorded. In initial experiments this operation was conducted manually. Labeled neurons in each dorsoventral, mediolateral, or rostrocaudal sector of a given nucleus were counted from serial sections as de-

  • 68 H. BARBAS ET AL.

    Fig. 2. Left: Brightfield photomicrographs o f coronal sections from rostra1 (A) through caudal (E) sectors of the thalamus showing the location of the various nuclei and their cytoarchitecture. Right: Brightfield photomicrographs of matched sections stained for myelin are shown in B, D, and F. (Frozen cut tissue, cresyl violet stain (A,C,E) and Gallyas myelin stain (B,D,F). Scale bar = 1 mm.


    scribed previously (Barbas and De...


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