The organization of cerebellar and basal ganglia outputs to primary motor cortex as revealed by retrograde transneuronal transport of herpes simplex virus type 1

Abstract

We used retrograde transneuronal transport of herpes simplex virus type 1 to map the origin of cerebellar and basal ganglia "projections" to leg, arm, and face areas of the primary motor cortex (M1). Four to five days after virus injections into M1, we observed many densely labeled neurons in localized regions of the output nuclei of the cerebellum and basal ganglia. The largest numbers of these neurons were found in portions of the dentate nucleus and the internal segment of the globus pallidus (GPi). Smaller numbers of labeled neurons were found in portions of the interpositus nucleus and the substantia nigra pars reticulata. The distribution of neuronal labeling varied with the cortical injection site. For example, within the dentate, neurons labeled from leg M1 were located rostrally, those from face M1 caudally, and those from arm M1 at intermediate levels. In each instance, labeled neurons were confined to approximately the dorsal third of the nucleus. Within GPi, neurons labeled from leg M1 were located in dorsal and medial regions, those from face M1 in ventral and lateral regions, and those from arm M1 in intermediate regions. These results demonstrate that M1 is the target of somatotopically organized outputs from both the cerebellum and basal ganglia. Surprisingly, the projections to M1 originate from only 30% of the volume of the dentate and <15% of GPi. Thus, the majority of the outputs from the cerebellum and basal ganglia are directed to cortical areas other than M1.

Fig. 1.

Examples of regions outlined to make area measurements (shaded areas). Two regions were outlined:A, the entire nucleus; and B, the region containing most (>90%) of the labeled neurons.

Fig. 2.

Ventrolateral thalamus of animal Jo 22. Top left, Neuronal cell bodies in VPLo labeled by retrograde transport of WGA-HRP from arm M1 (box B). Also, axonal terminal fields in the reticular nucleus labeled by anterograde transport of WGA-HRP (box A). Top right, An adjacent section stained with cresyl violet. The boxed areas in the top row are shown at higher magnification in the middle and bottom rows. Middle, Reticular nucleus (A, C). Bottom, VPLo (B, D).Arrows in A and C point to the reticular nucleus. Scale bars: top row, 1 mm;middle, bottom row, 100 μm.

Fig. 3.

Ventrolateral thalamus of animal Jo 26.Top, First order neurons in VPLo labeled by retrograde transport of virus from arm M1; survival period of 3 d. Scale bar, 1 mm. Bottom, Higher magnification view of theboxed area at the top. Scale bar, 100 μm. Note that the virus-labeled cells have features typical of thalamocortical neurons.

Fig. 4.

Cerebellar deep nuclei of animal Z10.Top, Dark-field view of the dentate and interpositus nuclei. Scale bar, 1 mm. Bottom left, Higher magnification view of the boxed area at thetop showing second-order neurons labeled by retrograde transneuronal transport of virus from arm M1; survival period of 4.5 d. Scale bar, 500 μm. Bottom right, Higher magnification view of the boxed area at theleft. Scale bar, 50 μm.

Fig. 5.

Basal ganglia of animal Z10. Top, Dark-field view of the globus pallidus and putamen. Scale bar, 1 mm.Bottom left, Higher magnification view of theboxed area at the top showing second-order neurons in GPi labeled by retrograde transneuronal transport of virus from arm M1; survival period of 4.5 d. Scale bar, 500 μm. Bottom right, Higher magnification view of the boxed area at the left. Scale bar, 50 μm. GPe, External segment of GP; i, inner portion of GPi; o, outer portion of GPi;PUT, putamen.

Fig. 6.

Schematic summary of the spatiotemporal patterns of labeling after virus injections into arm M1. Arrowsindicate the direction of transport, retrograde, and retrograde transneuronal.

Fig. 7.

Ventrolateral thalamus of animal Jo 20. Top left, Thalamic neurons in VLo labeled by retrograde transport of virus from arm M1 (box B); survival period of 4.8 d. Also, second-order neurons in the reticular nucleus labeled by retrograde transneuronal transport (box A).Top right, An adjacent section stained with cresyl violet. The boxed areas in the top roware shown at higher magnification in the middle andbottom rows. Middle, Reticular nucleus (A, C). Bottom, VLo (B, D). Arrows in B andD point to a localized site of intense gliosis and neuronal cell loss. Scale bars: top row, 1 mm;middle, bottom row, 100 μm.

Fig. 8.

Cerebellar cortex of animal Jo 21.Top, Third-order neurons labeled in cerebellar cortex by retrograde transneuronal transport of virus from arm M1; survival period of 7.0 d. The Purkinje cell layer runs horizontally through the approximate middle of the micrograph (see bottom). Note the virus-labeled neuron in the Purkinje cell layer (left) and cluster of labeled somata and processes in the outer molecular and inner granular layers (right).Bottom, An adjacent section stained with cresyl violet. Note the glial proliferation in the outer molecular layer (right). Scale bar, 100 μm.

Fig. 9.

Examples of typical HSV1 injection sites in M1. The survival periods were 4–5 d. Scale bar, 1 mm.

Fig. 10.

Location of virus injections in arm M1 of animal Jo 19. A, Lateral view of the left hemisphere of theCebus monkey. The enclosed area indicates the region of cortex enlarged in B. B, The region of M1 that was mapped by intracortical stimulation. Sites from which movements were evoked are marked with a letterdesignating the response observed (inset).Uppercase letters indicate sites where thresholds for movement were ≤10 μA. Lowercase letters indicate sites where thresholds were 11–35 μA. Circlesindicate where the microsyringe needle entered the surface of the cortex for virus injection. Squares indicate the point of entry for injections into the sulcus. The central sulcus has been opened to reveal its anterior bank. C, Coronal sections through the virus injection sites. The AP level of each section is indicated by the numbered arrows in B. The shaded areas in B andC indicate the spread of virus. ArSi, Inferior limb of the arcuate sulcus; ArSs, superior limb of the arcuate sulcus; C, caudal; CS, central sulcus; D, dorsal; L, lateral;M, medial.

Fig. 11.

Relative locations of virus injection sites within arm M1 for three different animals (Z10, Jo 19, and Jo 20).ArSi, Inferior limb of the arcuate sulcus;ArSs, superior limb of the arcuate sulcus;CS, central sulcus; M, medial;PS, principal sulcus; R, rostral.

Fig. 12.

Location of virus injections in leg M1 of animal Jo 17. See Figure 10 for details. The dashed lines inC represent areas of tissue loss.

Fig. 13.

Location of virus injections in face M1 of animal Jo 18. See Figure 10 for details.

Fig. 14.

Rostrocaudal distributions of labeled neurons in three animals with virus injections into arm M1. Top, Histograms of labeled neurons in dentate. Bottom, Histograms of labeled neurons in GPi. The height of the columns indicates the number of labeled neurons observed in each tissue section.

Fig. 15.

Histograms of the distribution of labeled neurons in dentate after virus injections into leg (Jo 17, top), arm (Jo 19, middle), or face (Jo 18,bottom) M1. The numbers highlighted on the abscissae correspond to the sections illustrated in Figure16.

Fig. 16.

Plots of labeled cells in the cerebellar deep nuclei. Each dot represents the position of a neuron labeled by transneuronal transport of virus from leg (Jo 17,left), arm (Jo 19, middle), or face (Jo 18, right) M1. D, Dorsal;M, medial; ND, dentate nucleus;NIA, anterior interpositus nucleus; NIP, posterior interpositus nucleus; NF, fastigial nucleus.

Fig. 17.

Plots of labeled cells in the globus pallidus. Each dot represents the position of a neuron labeled by transneuronal transport of virus from leg (Jo 17, left), arm (Jo 19, middle), or face (Jo 18,right) M1. The section numbers correspond to those highlighted on the abscissae of Figure 18. D, Dorsal;GPe, external segment of GP;GPi_i, inner portion of the internal segment of GP; GPi_o, outer portion of the internal segment of GP; M, medial.

Fig. 18.

Histograms of the distribution of labeled neurons in GPi after virus injections into leg (Jo 17, top), arm (Jo 19, middle), or face (Jo 18, bottom) M1. The height of the light and dark stippling indicates the proportion of labeled cells observed in the inner portion of GPi and all of GPi (inset).