Brain, Vol. 124, No. 1, 176-208,
January 2001
© 2001 Oxford University Press
Cortical innervation of the facial nucleus in the non-human primate
A new interpretation of the effects of stroke and related subtotal brain trauma on the muscles of facial expression
Division of Basic Biomedical Sciences, The University of South Dakota School of Medicine, Vermillion, South Dakota, USA
Correspondence to:
Dr Robert J. Morecraft, Division of Basic Biomedical Sciences, The University of South Dakota School of Medicine, Vermillion, SD 57069, USA E-mail: rmorecra{at}usd.edu
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Abstract |
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The corticobulbar projection to musculotopically defined subsectors of the facial nucleus was studied from the face representation of the primary (M1), supplementary (M2), rostral cingulate (M3), caudal cingulate (M4) and ventral lateral pre- (LPMCv) motor cortices in the rhesus monkey. We also investigated the corticofacial projection from the face/arm transitional region of the dorsal lateral premotor cortex (LPMCd). The corticobulbar projection was defined by injecting anterograde tracers into the face representation of each motor cortex. In the same animals, the musculotopic organization of the facial nucleus was defined by injecting fluorescent retrograde tracers into individual muscles of the upper and lower face. The facial nucleus received input from all face representations. M1 and LPMCv gave rise to the heaviest projection with progressively diminished intensity occurring in the M2, M3, M4 and LPMCd projections, respectively. Injections in all cortical face representations labelled terminals in all nuclear subdivisions (dorsal, intermediate, medial and lateral). However, significant differences occurred in the proportion of labelled boutons found within each functionally characterized subdivision. M1, LPMCv, LPMCd and M4 projected primarily to the contralateral lateral subnucleus, which innervated the perioral musculature. M2 projected bilaterally to the medial subnucleus, which supplied the auricular musculature. M3 projected bilaterally to the dorsal and intermediate subnuclei, which innervated the frontalis and orbicularis oculi muscles, respectively. Our results indicate that the various cortical face representations may mediate different elements of facial expression. Corticofacial afferents from M1, M4, LPMCv and LPMCd innervate primarily the contralateral lower facial muscles. Bilateral innervation of the upper face is supplied by M2 and M3. The widespread origin of these projections indicates selective vulnerability of corticofacial control following subtotal brain injury. The finding that all face representations innervate all nuclear subdivisions, to some degree, suggests that each motor area may participate in motor recovery in the event that one or more of these motor areas are spared following subtotal brain injury. Finally, the fact that a component of the corticofacial projection innervating both upper and lower facial musculature arises from the limbic proisocortices (M3 and M4) and frontal isocortices (M1, M2, LPMCv and LPMCd) suggests a potential anatomical substrate that may contribute to the clinical dissociation of emotional and volitional facial movement.
brainstem; cranial nerves; limbic cortex; motor cortex
BDA = biotinylated dextran amine; DY = diamidino-yellow; FB = fast blue; FD = fluorescein dextran; FR = fluoro ruby; LPMCd = dorsal lateral premotor cortex (or area 6V); LPMCv = ventral lateral premotor cortex (or area 6V); LYD = lucifer yellow dextran; M1 = primary motor cortex; M2 = supplementary motor area (or medial area 6); M3 = rostral cingulate motor cortex (or area 24c); M4 = caudal cingulate motor cortex (or area 23c); MCA = middle cerebral artery; PB = phosphate buffer; PHA-L = Phaseolus vulgaris leucoagglutinin
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Introduction |
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TopAbstractIntroductionMaterial and methodsResultsDiscussionReferences |
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The complex nature of facial expression has greatly influenced the notion that the cerebral cortex plays an important role in mediating facial movement (Duchenne, 1862; Darwin, 1872
; Jackson, 1884; James, 1890; Denny-Brown, 1950; Damasio, 1994). Underscoring this relationship are the many alterations in facial expression that follow subtotal brain injury and occur in psychiatric illness and cranial–cervical dystonia. Despite the critical nature of these disorders, we know little about how the cerebral cortex and facial nucleus interact structurally, and how alterations in this interaction may disrupt our ability to appropriately control the muscles of facial expression. For example, over the past century little has changed in the way we interpret the most common post-stroke condition, in which occlusion of the middle cerebral artery and its branches results in paralysis of the contralateral lower face, but sparing of the upper face. The clinical consequences remain explained by the suggestion that the upper facial musculature receives bilateral innervation from the primary motor cortex (M1) and the lower face only contralateral innervation from M1 (Papez, 1927; Pearson, 1946; Reed and Sheppard, 1976; Brodal, 1981; Wilson-Pauwels et al., 1988; Adams et al., 1997; Kandel et al., 2000). The continued teaching of this tenet is quite surprising, considering the fact that neuronal tracing studies conducted in human and non-human primates do not fully support the classic interpretation (Kuypers, 1958a, b; Jenny and Saper, 1987). For example, it has been shown that parts of the facial nucleus innervating the lower facial musculature receive heavy contralateral input from M1. However, parts of the facial nucleus containing motor neurones innervating the upper facial musculature receive little input from M1. Contributing further to the enigma is evidence indicating that the supplementary motor area (M2 or medial area 6), which contains a face representation, may not innervate the facial nucleus (Jürgens, 1984).
Further associated with the mysteries underlying facial deficits following localized brain damage are numerous behavioural and clinical observations that have demonstrated a dissociation between voluntary and emotional facial movements (Darwin, 1872; Jackson, 1884; Wilson, 1924; Monrad-Krohn, 1924, 1939; Allen, 1931; Karnosh, 1945; Podolsky, 1945; Brown, 1967; Brodal, 1981; Duchenne, 1990; Hopf et al., 1992; Damasio, 1994, 1995; Topper et al., 1995; Husain, 1997; Ross and Mathiesen, 1998; Urban et al., 1998; Hopf et al., 2000). The most frequently reported example is `volitional facial paralysis'. This occurs in patients who present with impaired voluntary movement in their lower facial muscles contralateral to a cortical or subcortical lesion but overcome the paralysis when responding to an emotionally provocative or humorous remark. Volitional facial paresis has been associated with lesions involving M1 and the underlying subcortical white matter (Hopf et al., 1992). Similarly, but less commonly encountered, is a condition where emotionally affiliated facial movement is not only spared but excessive. In contrast to this is the inverse condition historically known as `amimia', or more commonly known as `emotional facial paralysis'. This disorder is characterized by an inability to smile on one side of the face; however, voluntary control over the same set of facial muscles is largely unaffected. Emotional facial paresis has been reported in patients with lesions involving the midline cortex, insula, thalamus, striatocapsular region and pons (Laplane et al., 1977; Hopf et al., 1992, 2000; Damasio, 1994; Urban et al., 1998). Collectively, these observations have led to the hypothesis that separate neural systems may mediate emotional and voluntary facial movements. Closely related is the recent suggestion that the anterior cingulate cortex may mediate emotionally associated movements in the upper face and the lateral frontal cortices volitional movements in the lower face (Damasio, 1994). However, specific neural systems that may form an integrated network between voluntary and emotional portions of the cortex and musculotopically defined parts of the facial nucleus have not been localized.
Several lines of research have revealed that there are at least five cortical face representations in the human and non-human primate brain, with each face representation being affiliated with five distinct cortical motor areas. (Fig. 1A and B) (Ferrier, 1886; Horsley and Schäfer, 1888; Penfield and Welch, 1951; Woolsey et al., 1952, 1979; Bates, 1953; Luschei et al., 1971; Muakkassa and Strick, 1979; McGuinness et al., 1980; Sirisko and Sessle, 1983; Luppino et al., 1991; Morecraft and Van Hoesen, 1992; Paus et al., 1993; Tanji, 1994; Godschalk et al., 1995; West and Larson, 1995; Morecraft et al., 1996; Picard and Strick, 1996; Tokuno et al., 1997; Wu et al., 2000; Morecraft and Yeterian, 2001). They include the face representation of the primary motor cortex (M1-areas F1 and F4), ventral lateral-premotor cortex (LPMCv-area 6V), supplementary motor cortex (M2-area 6m), rostral cingulate motor cortex (M3-area 24c) and caudal cingulate motor cortex (M4-area 23c). The fact that there is widespread face representation at the cortical level raises the possibility that each face representation may innervate the facial nucleus, i.e. much like all cortical arm representations, selectively innervate brachial levels of the spinal cord and leg representations the lumbosacral levels (Biber et al., 1978; Murray and Coulter, 1981; Hutchins et al., 1988; Galea and Darian-Smith, 1994; Luppino et al., 1994; Rouiller et al., 1994; Morecraft et al., 1997). It is possible to speculate that all cortical face representations may not only innervate the facial nucleus but also preferentially target subsectors controlling different groups of facial muscles, this premise being based upon the fact that the various subsectors of the primate facial nucleus are musculotopically organized (Fig. 1C) (Jenny and Saper, 1987; Satoda et al., 1987; Porter et al., 1989; Welt and Abbs, 1990; VanderWerf et al., 1998) and the fact that M1 preferentially innervates only the contralateral lateral subnucleus (Jenny and Saper, 1987). Finally, defining the organization of the corticofacial projection from M3 and M4, whose origins are embedded in the proisocortical portion of the `emotive' limbic lobe, may assist in interpreting the clinical observations that have long suggested a dissociation of voluntary and emotional facial movements.
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The occurrence of altered facial expressions that accompany subtotal brain trauma and present in psychiatric illness and cranial–cervical dystonia implies that a strong and highly organized structural linkage occurs between the cerebral cortex and the facial nucleus. The fact that selective muscle groups are adversely affected following localized brain damage indicates that the various cortical face representations may preferentially influence different groups of facial muscles. To advance our understanding of these important neurological issues, we studied the organization of the descending projection from the face representation of five major cortical motor areas to musculotopically defined subsectors of the facial nucleus. In addition, we investigated a potential corticofacial projection from the ventral part of the dorsal lateral premotor cortex (LPMCd), which primarily gives rise to arm movements following stimulation, but occasionally face movements. Our findings provide new insight to the long-standing question of cortical innervation of the upper and lower facial musculature. Together, these observations offer a new and alternative explanation for the clinical deficits that occur following localized cortical damage to the lateral and medial regions of the cerebral hemisphere.
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Material and methods |
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The corticobulbar projection to the facial nucleus was studied in seven adult rhesus monkeys (Macaca mulatta). All protocols received Institutional Animal Care and Use Committee approval and were conducted in accordance with USDA and the Society for Neurosciences guidelines. Each monkey was immobilized with ketamine hydrochloride (10 mg/kg) then anaesthetized intravenously with pentobarbital (2.5–4 mg/kg/h). The head was shaved, scrubbed with betadine and the animal placed into a NIH head holder or Kopf stereotaxic head holder.
Surgery
The lateral surface of the cerebral cortex was exposed as described previously (Morecraft and Van Hoesen, 1992; Morecraft et al., 1992). Bridging cortical veins draining into the superior sagittal sinus were cauterized to expose the medial wall of the cerebral cortex. Intracortical microstimulation was used to localize the arm and face representations in M1, LPMCd, LPMCv and M2. Standard anatomical landmarks were used to localize the face representation in M3 and M4. Graded pressure injections (0.2–0.3 µl per penetration) of anterograde neuronal tracers were made into the various cortical face representations using a Hamilton microsyringe inserted 2–3 mm below the cortical surface under microscopic guidance. To minimize white matter involvement following M2 injections, the superior frontal lobule was gently retracted laterally and the tip of the needle was inserted directly into the intended grey matter lining the vertical wall without involving cortex on the dorsomedial convexity and its underlying white matter. For injections into the lower bank of the cingulate sulcus (e.g. M3 and M4) the arachnoid matter bridging over the cingulate sulcus was removed and the superior frontal lobule was retracted laterally exposing cortex in the lower bank. The needle tip was then inserted directly into the cingulate cortex, avoiding the adjacent frontal cortex (Fig. 2B and C). The location of each stimulation point and injection site was recorded on a JVC videocassette recorder (HR-S9400U, Dage-MTI, Inc., Michigan City, Ind., USA) directly connected to a three-chip colour camera (MTI DC-330, JVC, Aurora, Ill., USA) that was mounted on the photo/video port of the surgical microscope (Stortz M-703F, Olympus, Minneapolis, Minn., USA). These images were metrically calibrated and captured with respect to anatomical landmarks (e.g. sulci) and used for developing an accurate post-mortem reconstruction of the brain surface, the location of each physiological stimulation point and the penetration point of each tract tracer injection. Intramuscular injections of fluorescent retrograde neuronal tracers were made in upper and lower facial muscles in four of the seven monkeys studied by using a microsyringe inserted into a 20 g needle, which was then inserted into the muscle tissue. Each muscle was localized using the criteria of Huber (Huber, 1933).
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Intracortical microstimulation
A monopolar tungsten microelectrode (impedance 1.0–2.0 M
) was attached to a Grass S88 stimulator and a constant current stimulus isolation unit. The system was grounded and the electrode was lowered to a depth of 100 µm and advanced at 500-µm intervals. Cortex was mapped with a train duration of 50–100 ms and a pulse duration of 0.2 ms delivered at 330 Hz. Stimulation points were spaced 0.5–2.5 mm apart to minimize tissue damage. The lowest level of current needed to evoke movement was considered the movement threshold. During all experiments, applied current levels were continuously monitored by measuring the voltage with the use of a digital storage oscilloscope (LG Precision Co., Ltd, Ceritos, Calif., USA) and determining the system resistance. The current values ranged between 3 and 80 µA (Figs 3–6). All movements at threshold were discrete and confined to a specific body part (e.g. upper lip, tongue, wrist, finger).
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Experimental design
Cortical and intramuscular injections
In all monkeys, anterograde tracers were injected into various cortical face representations to study the patterns of terminal labelling in the facial nucleus (Figs 2–6
). Injections and representative volumes were made according to the relative size of each face representation and to minimize arm representation involvement when possible. The anterograde tracers used were 10% biotinylated dextran amine (BDA), 10% lucifer yellow dextran (LYD), 2.5% Phaseolus vulgaris leucoagglutinin (PHA-L), 10% fluorescein dextran (FD) and 10% fluoro ruby (FR). The FD injectate was composed of an equal mixture of 3000 and 10 000 MW volumes, as was the FR injectate. In monkeys SDM 14, 19, 20 and 22, specific facial muscles were injected with the retrograde tracers 4% fast blue (FB) and 4% diamidino-yellow (DY). This enabled us to examine the topographical relationship between corticofacial terminals and the general organization of facial lower motor neurones. The specific injection site parameters in each experiment are described below.
Monkey SDM 9.
FB was injected into the face representation of M1 (four penetrations; total volume 0.8 µl) (see Fig. 1 of Morecraft et al., 1996). In the contralateral hemisphere, BDA was injected into the face area of M3 (two penetrations; total volume 0.4 µl), positioned directly above the genu of the corpus callosum (Fig. 2B).
Monkey SDM 10.
FB was injected into the arm representation of M2 (one penetration; total volume 0.2 µl) and DY was injected into the face representation of M2 (one penetration; total volume 0.2 µl) (see Fig. 3 of Morecraft et al., 1996). On the contralateral hemisphere, BDA was injected into the rostral half of M4 (one penetration; total volume 0.4 µl), positioned at coronal levels involving the arcuate spur to levels extending 2 mm caudal to this landmark.
Monkey SDM 14.
All cortical injections were made into the right hemisphere (Figs 2C and 3A). PHA-L was injected into the face representation of M1 (four penetrations; total volume 0.8 µl) and FD was injected into the physiologically defined face representation of LPMCv (total volume 0.8 µl). On the medial wall of the hemisphere, LYD (total volume 0.6 µl) was injected into the face representation of M2, and BDA was injected into the rostral portion of M3 (lower bank of the cingulate sulcus) corresponding to coronal levels including the genu of the corpus callosum (two penetrations; total volume 0.6 µl) (Fig. 2C). Finally, FR (one penetration; total volume 0.4 µl) was injected into the rostral portion of M4 located at coronal levels including the arcuate spur. In the upper facial musculature located contralateral to the cortical injections, DY was injected into the frontalis/corrugator supercilii complex (three penetrations; 10 µl total volume) and FB was injected into the upper and lower orbital and preseptal portions of the orbicularis oculi (six penetrations; 10 µl total volume) (Fig. 3B). In the lower facial musculature located ipsilateral to the cortical injections, DY was injected into the upper orbicularis oris (upper lip) (three penetrations; 10 µl total volume) and FB was injected into lower orbicularis oris (lower lip) (three penetrations; 10 µl total volume) (Fig. 3B).
Monkey SDM 19.
All cortical injections were made in the right hemisphere (Fig. 4A). LYD was injected into the face representation of M1 (four penetrations; total volume 1.2 µl) and FD was injected into the arm representation of M1 (four penetrations; total volume 0.9 µl). FR was then injected into the arm representation of M2 (two penetrations; total volume 0.6 µl). However, during surgery a large bridging vessel required ligation over the rostral part of M2, yielding the underlying cortex (face area) unresponsive to stimulating current. Therefore, the face area was approximated as being localized immediately anterior to the physiologically defined arm representation and caudal to the pre-SMA (M2 pre-supplementary motor area) which resides in the superior frontal gyrus in a plane above the genu of the corpus callosum (Fig. 1A). BDA was injected into the face area of M2 (two penetrations; total volume 0.6 µl). In the upper facial musculature located contralateral to the cortical injections, DY was injected into the frontalis–corrugator supercilii complex (three penetrations; 10 µl total volume) and FB was injected into the upper and lower orbital and preseptal portions of the orbicularis oculi (six penetrations; 10 µl total volume) (Fig. 4B). In the lower facial musculature located ipsilateral to the cortical injections, DY was injected into the upper orbicularis oris (three penetrations; 10 µl total volume) and FB was injected into the lower orbicularis oris (three penetrations; 10 µl total volume) (Fig. 4B).
Monkey SDM 20.
All cortical injections were made in the right hemisphere. BDA was injected into the face representation of M1 (two penetrations; total volume 1.0 µl) and LYD was injected into the arm representation of M1 (two penetrations; total volume 1.0 µl). FR was injected into cortex lining the lower bank of the cingulate (M3) from a location established at coronal levels, including the genu of the corpus callosum, to a caudal location corresponding to a coronal plane located 2 mm rostral to the genu of the arcuate sulcus (10 penetrations; total volume 10 µl). FB was injected bilaterally into the orbital, preseptal and pretarsal portions of the orbicularis oculi (10 penetrations; total volume 25 µl).
Monkey SDM 22.
All cortical injections were made in the right hemisphere (Fig. 5A). On the lateral wall, FR was injected into the face representation of M1 (four penetrations; total volume 1.2 µl), and BDA was injected into the face representation of LPMCv (four penetrations; total volume 1.2 µl). FR was injected into the face area of M2 (four penetrations; total volume 0.4 µl). PHA-L was injected into the face area of M3 (two penetrations; total volume 0.5 µl) and LYD was injected into the face area of M4 (one penetration; total volume 0.4 µl). In the upper facial musculature contralateral to the cortical injections, FB was injected into the upper and lower orbital, preseptal and pretarsal portions of the orbicularis oculi (seven penetrations; 10 µl total volume) and DY was injected into the orbito-auricularis muscle (two penetrations; 10 µl total volume) (Fig. 5B). In the lower facial musculature located ipsilateral to the cortical injections, FB was injected into the upper orbicularis oris (three penetrations; 10 µl total volume) and DY was injected into the lower orbicularis oris (three penetrations; 10 µl total volume) (Fig. 5B).
Monkey SDM 23.
FD was injected into the ventral part of the arm area of LPMCd as defined using microstimulation (three penetrations; total volume 1.0 µl) (Fig. 6).
Tissue processing
Following a survival period of 21–30 days each monkey was deeply anaesthetized with Nembutal and perfused with 300–500 ml of 0.9% saline solution followed by 2 l of 4% paraformaldehyde in 0.1 M phosphate buffer (PB), then 1 l of 10% sucrose in 0.1 M PB. The perfusion was completed by an infusion of a 10% then 30% solution of sucrose in 0.1 M PB. The brain was removed, placed in 30% sucrose in 0.1 M PB and stored for 2–4 days at 4°C. Post-mortem photographs and electronic images were taken of the dorsal, ventral, lateral and medial surfaces of the cerebral cortex, brainstem and spinal cord. Each injected facial muscle in monkeys SDM 14, 19, 20 and 22 as well as the adjacent non-injected muscles were dissected and placed in 30% sucrose in 0.1 M PB, stored at 4°C.
The cortex was frozen then sectioned coronally (monkeys SDM 9, 10, 20 and 22) or horizontally (monkeys SDM 14, 19 and 23) on an American Optical sliding microtome (AO 860) at a thickness of 50 µm in cycles of 10. The brainstem was frozen with dry ice and cut transversely (perpendicular to its long axis) at a thickness of 50 µm in cycles of five. In monkey 5, brainstem serial sections were cut with a vibratome at a thickness of 200–300 µm. Finally, each muscle was frozen sectioned and examined for tracer localization (Vander Werf et al., 1996, 1998).
The first section of each cortical and brainstem series was mounted on subbed slides, dried overnight, coverslipped with DPX mounting medium (Aldrich Chemicals, Milwaukee, Wis., USA) and used for fluorescent analysis (Morecraft and Van Hoesen, 1992, 1993, 1998; Morecraft et al., 1993) (Fig. 7). After analysis, each fluorescent section was stained for Nissl substance using thionin. The second section in each series was processed for BDA using the avidin–biotin method (Fig. 8). Following successive rinses in PB, the sections were incubated in 0.05% DAB (diaminobenzidine tetrachloride) and 1% nickel ammonium sulphate for 10 min. Then, 0.009% hydrogen peroxide was added and the tissue incubated for another 10–15 min, rinsed in PB, serially dehydrated, mounted on subbed slides and coverslipped using Permount (Fisher Chemicals, Denver, Co., USA). The BDA injection site, axons and cells stained by nickel-enhanced DAB appeared blue-black upon visualization.
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Monkeys SDM 14, 19 and 22 received injections of multiple anterograde tracers (e.g. BDA, LYD, PHA-L, FR and FD). In these experiments, BDA was processed alone in a complete series of tissue sections as indicated above, mounted on glass slides and coverslipped. In additional serial sets of unprocessed tissue sections through the cortex, brainstem and spinal cord, double labelling immunohistochemistry was performed (Fig. 8F
). In all of the double labelling experiments, BDA was reacted first according to the above protocol with the exception that BDA was stained brown using DAB in the absence of nickel ammonium sulphate (Fig. 8B and C). After reacting for BDA, the same tissue sections were then incubated in avidin–biotin blocking reagent (Vector SP-2001), rinsed in 10% normal goat serum for 2 h then incubated overnight, with the appropriate biotinylated antibody directed against a second neuronal tract tracer (e.g. biotinylated anti-LYD). The tissue was then incubated in a solution of avidin–biotinylated peroxidase complex, rinsed in buffer and stained blue using the Vector SG peroxidase substrate kit (Vector SK-4700). Thus, BDA was colourized brown and the second tracer (e.g. LYD) was stained blue in the same tissue sections (Fig. 8A and F). All sections were rinsed, dehydrated, mounted and coverslipped on glass slides. This process was conducted on another set of serial sections staining BDA brown with DAB, then another anterograde tracer blue (e.g. PHA-L), again using the appropriate antibody and blue reaction process to visualize the second tracer (e.g. biotinylated anti-PHA-L). FR and FD were also processed immunohistochemically in the same manner (e.g. BDA brown then FR blue). In these series, FR and FD labelling was verified by comparing the locations of immunochemically localized reaction products to fluorescent labelling in the non-reacted fluorescent series. The specific antibodies used for each anterograde tracer were biotinylated anti-Lucifer Yellow rabbit IgG fraction, biotinylated anti-PHA-L rabbit IgG fraction, biotinylated anti-FR rabbit IgG fraction and biotinylated anti-FD rabbit IgG fraction (all from Vector Laboratories, Burlingame, Calif., USA). The remaining cortical tissue sections were stained for myelin using the gold chloride method (Schmued, 1990) and cytochrome oxidase for evaluating histochemical boundaries of the frontal motor areas (Matelli et al., 1985).
Data analysis
Fluorescent material was studied using epi-fluorescent illumination and immunohistochemically processed material was examined under bright-field illumination. Data from all tissue sections were collected with the use of a conventional Hewlett Packard X-Y plotter (HP-7045B, Hewlett Packard, Palo Alto, Calif., USA) that was attached to the x–y axes of a microscope stage (Olympus BX60, Minneapolis, Mass., USA). The outline of the tissue section, anatomical landmarks such as blood vessels and ventricles, the white and grey matter interfaces, periphery of the injection sites, and transported tracing substances were plotted in each tissue section. The criterion of Condé was used to assess the effectiveness of the fluorescent injection sites (Condé, 1987). Accordingly, zones 0–II were interpreted as corresponding to the effective uptake site. For immunohistochemically developed material, the effective injection site was defined by the heavily stained area that surrounds the point of tracer administration and extends to the edge of the deposit. The edge of the injection site corresponding to the region where the dense precipitate diminishes, neurones and labelled axons are distinguishable, and the intensity of fibre staining is similar to fibre staining intensity occurring in local, primary projection targets (Fig. 2). Cortical sections were spaced at intervals of 500 µm. Nissl-, myelin- and cytochrome-stained sections were used for plotting architectonic boundaries directly onto the chartings. Brainstem tissue sections were analysed at intervals of 250 µm. Fluorescent labelling (retrogradely labelled motor neurones with FB and DY and corticofacial terminals labelled with FD and FR) and immunohistochemically developed sections (BDA, LYD, PHA-L, FD and FR) were also plotted using the x–y plotter. To study in greater detail facial nucleus labelling patterns in immunohistochemically processed sections, axon varicosities in the nucleus were charted a second time, using maximum range conditions of the x–y recorder or with a drawing tube. White and grey matter borders were added to the brainstem chartings using Nissl-stained sections. In monkey SDM 20, vibratomed sections through the brainstem were charted for FR terminals. FR labelling in the facial nucleus was verified in this case using a Bio-Rad 1024 confocal microscope (Bio-Rad Laboratories, Hercules, Calif., USA).
The terminal projection from representative cases of each motor area (M1, LPMCv, LPMCd, M2, M3 and M4) was quantified within the facial nucleus using immunohistochemically processed material (Fig. 9). To accomplish this, the total number of labelled boutons was counted in every immunohistochemically reacted serial section through the ipsilateral and contralateral facial nucleus (e.g. at 250 µm intervals through a rostrocaudal distance of 1.7–2.2 mm). Terminal boutons were further categorized by ipsilateral and contralateral nuclear subdivision (medial, lateral, dorsal and intermediate). Terminal boutons, or axon varocosities, represent putative synaptic contacts between a projection axon and local neurones (Wouterlood and Groenewegen, 1985). The total number of boutons in each subdivision of each side was divided by the total number of boutons counted in the entire case. The ratio was converted to a percentage and expressed by subdivision in each side.
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The cortex was reconstructed using surface drawings developed from metrically calibrated photographs and video images taken of the brain prior to tissue sectioning (Figs 3–6
). Nissl-, myelin- and cytochrome-oxidase-stained tissue sections were used to delineate architectonic areas of the frontal lobe and cingulate gyrus. A physiological map of the cortical surface illustrating the locations of the stimulation points, electrolytic lesions, sulci and vessels was constructed from images taken during surgery of each penetration point and surrounding landmark. The stimulation map was superimposed on the injection site map (Figs 3–6). The brainstem sections were reconstructed by stacking each serial section in a caudal to rostral sequence depicting terminal labelling in all cases and motor neuronal labelling in selected cases (Figs 10–15).
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Cytoarchitectonic organization of the facial nucleus
The facial nucleus can be subdivided into several distinct cellular groups or subnuclei (Papez, 1927
; Courville, 1966; Jenny and Saper, 1987; Satoda et al., 1987; Baker et al., 1994). In this report, the organization of the monkey facial nucleus was determined according to the criteria of Jenny and Saper (Jenny and Saper, 1987) using Nissl-stained sections to facilitate comparison of our work with previously published results on the corticofacial projection. Thus, the nucleus was subdivided into intermediate, dorsal, medial and lateral subsectors (Fig. 1C, right). Furthermore, patterns of motor neurone labelling following injection of retrograde tracers in the facial muscles and organization of corticofacial terminals following injections into cortex correlated well with the basic designation of intermediate, dorsal, medial and lateral subsectors (Fig. 1C, left). Cell sparse bands, packing, somal diameter and staining intensity were considered for parcellation.
Definition and characterization of cortical face areas
The face area of M1 is located rostral to the ventral extent of the central sulcus and on average has a low physiological threshold for excitability (Fig. 1A and B) (Weinrich and Wise, 1982; Huang et al., 1988; Huntley and Jones, 1991; Murray and Sessle, 1992; Preuss et al., 1996) (Fig. 1B). The caudal portion of the M1 face area corresponds to area 4 in Nissl-stained sections (Brodmann, 1909; Barbas and Pandya, 1987; Preuss and Goldman-Rakic, 1991) and area F1 in cytochrome oxidase preparations (Matelli et al., 1985, 1991). The rostral portion corresponds to the caudal portion of area 6V in Nissl-stained sections (Barbas and Pandya, 1987; Preuss and Goldman-Rakic, 1991) and area F4 in cytochrome-oxidase-stained sections (Matelli et al., 1985, 1991). Immediately anterior and adjacent to the face representation of M1 is the face area of the ventral lateral premotor cortex (LPMCv) (Fig. 1B). This cortex corresponds to the rostral portion of area 6V (Barbas and Pandya, 1987; Preuss and Goldman-Rakic, 1991) as well as area F5 (Matelli et al., 1985, 1991). Physiologically, the face representation of LPMCv has a higher threshold of excitability in unanaesthetized monkeys when compared with the caudal portion of M1 (Rizzolatti et al., 1988; Preuss et al., 1996). Electrophysiological evidence indicates the arm and face representations overlap in the dorsal portion of LPMCv (Gentilucci et al., 1988; Rizzolatti et al., 1988; Godschalk et al., 1995; Preuss et al., 1996). Within this transitional cortex, arm movements are heavily represented, but face movements are occasionally evoked. Similarly, the ventral part of LPMCd is primarily related to arm movements and less so to face movements (Godschalk et al., 1995; Preuss et al., 1996). Histologically, the ventral part of LPMCd corresponds to area 6D in Nissl preparations and area F2 in cytochrome oxidase preparations. This overlap in somatotopy was taken into consideration in our experimental design as we investigated the corticofacial projection from the ventral part of LPMCv as well as the corticofacial projection from the ventral portion of LPMCd. The face representation of M2 lies within the rostral portion of medial area 6 (Mitz and Wise, 1987; McGuire et al., 1991; Preuss and Goldman-Rakic, 1991; Godschalk et al., 1995; Morecraft et al., 1996). This same location corresponds to area F3 using cytochrome oxidase staining (Matelli et al., 1985, 1991; Luppino et al., 1993). The face representation of M2 is relatively smaller than the lateral face areas and has, on average, a higher threshold of movement excitation when compared with the average threshold values of cortex residing in the caudal portion of M1 (Mitz and Wise, 1987; Luppino et al., 1991; Huntley and Jones, 1991). The face area of M2 lies 1–3 mm rostral to a coronal plane established through the genu of the arcuate sulcus (Mitz and Wise, 1987; Matsuzaka and Tanji, 1996; Morecraft et al., 1996). Rostral to area F3 is area F6, which corresponds to the pre-SMA (Matelli et al., 1985; Luppino et al., 1991). The pre-SMA is related primarily to complex arm movements (Alexander and Crutcher, 1990; Matsuzaka and Tanji, 1996) and is much less excitable than adjacent area F3 (Luppino et al., 1991). The cingulate motor cortex lines the depths of the cingulate sulcus and is formed by the rostral and caudal cingulate motor cortices (Biber et al., 1978; Muakkassa and Strick, 1979; Morecraft and Van Hoesen, 1988, 1992; Hutchins et al., 1988; Dum and Strick, 1991; Devinsky et al., 1995; Nimchinsky et al., 1996). The rostral cingulate motor cortex (M3) corresponds cytoarchitecturally to area 24c (Vogt et al., 1987; Dum and Strick, 1991; Morecraft and Van Hoesen, 1992; Morecraft et al., 1996). The face area of M3 is located in the rostral portion of area 24c, in coronal levels that include the genu of the corpus callosum (Muakkassa and Strick, 1979; Morecraft and Van Hoesen, 1992; Morecraft et al., 1996; Tokuno et al., 1997). The genu and its anterior limit were visualized during surgical exposure to assist in localizing the M3 face area. The caudal cingulate motor area (M4) corresponds cytoarchitecturally to area 23c (Vogt et al., 1987; Hutchins et al., 1988; Dum and Strick, 1991; Morecraft and Van Hoesen, 1992; Morecraft et al., 1996). The face representation of M4 is located in the rostral part of area 23c (Morecraft et al., 1996; Tokuno et al., 1997). This general area gives rise to face movements using intracortical microstimulation in the unanaesthetized monkey (Luppino et al., 1991; Godchalk et al., 1995). Anatomically, this location corresponds to coronal levels that include the anterior commissure medially and spur of the arcuate sulcus laterally (Morecraft and Van Hoesen, 1992; Morecraft et al., 1996; Nimchinsky et al., 1996; Tokuno et al., 1997). If the spur is absent, the location of the M4 face area was approximated 1.0–2.0 mm caudal to the genu of the arcuate sulcus.
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Results |
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Retrograde labelling of facial motor neurones
Labelled neurones were found in the facial nucleus in all monkeys receiving injections of FB and DY into the facial muscles (Figs 3–6
). All injections of retrograde tracer placed into the frontalis–corrugator muscle complex labelled motor neurones in the dorsal subnucleus (Figs 10, 13 and 14, right). Occasionally, a few labelled cells appeared in the medial edge of the intermediate subnucleus. Labelled neurones occurred in the dorsal region of the medial subnucleus following injections in the orbito-auricularis muscle (Figs 11 and 15, right). Injections in the orbicularis oculi muscle consistently labelled neurones that were confined to the intermediate subnucleus (Figs 10, 11 and 13–15, right). Injections in upper orbicularis oris muscle labelled motor neurones in the dorsolateral sector of the lateral subnucleus (Figs 10, 11 and 13–15, left) and injections in the lower orbicularis oris muscle labelled neurones in the ventrolateral sector of the lateral subnucleus (Figs 10, 11 and 13–15, left).
Location of cortical injection sites
In all cases presented in this report, the injection sites were evaluated for somatotopic affiliation on the basis of multiple criteria. First, the location and peripheral extent of the injection sites made in M1, M2, LPMCv and LPMCd were reconstructed in direct relation to all electrophysiological stimulation points. Secondly, matching Nissl and cytochrome oxidase sections were used to evaluate histological boundaries in each motor area. The face areas of M1, LPMCv and LPMCd correlated to the ventral extent of each respective histological region. The face areas of M2, M3 and M4 correlated to the rostral extent of each respective histological region. Thirdly, corticocortical interconnections amongst the various injection sites in the same animal were evaluated for topographical reciprocity. For example, cortical arm representations are selectively interconnected, as are cortical face representations (Pandya and Vignolo, 1971; Muakkassa and Strick, 1979; Morecraft and Van Hoesen, 1988, 1992; Luppino et al., 1994; Nimchinsky et al., 1996; Tokuno et al., 1997). Fourthly, descending projections from each injection site to the facial nucleus and spinal cord were analysed. Injection site involvement of cortical face areas was supported by the presence of projections to the facial nucleus (Kuypers, 1958a, b; Jenny and Saper, 1987; Morecraft et al., 1996). Injection site involvement of cortical arm areas was supported by the presence of direct projections to the brachial spinal cord (Biber et al., 1978; Hutchins et al., 1988; Dum and Strick, 1991; Galea and Darian-Smith, 1994; Rouiller et al., 1994; Luppino et al., 1994; Morecraft et al., 1997).
Monkey SDM 9.
The BDA injection site in M3 was confined to the face representation as determined by FB tracer transport from M1 to the BDA injection site in M3 (Fig. 2B) [also see Morecraft et al. (1996), Fig. 1]. This was verified by the presence of BDA labelling in the facial nucleus [see Morecraft et al. (1996), Fig. 4]. No BDA labelling was found in the spinal cord, indicating no BDA involvement of the M3 arm representation.
Monkey SDM 10.
The BDA injection site involved the arm and face representations of M4 as determined by the corticocortical projection patterns and the fact that BDA labelling was found in both the facial nucleus and spinal cord (see Morecraft et al., 1996, Figs 3 and 4