Anatomical evidence for cerebellar and basal ganglia involvement in higher cognitive function.

The possibility that neurons in the basal ganglia and cerebellum innervate areas of the cerebral cortex that are involved in cognitive function has been a controversial subject. Here, retrograde transneuronal transport of herpes simplex virus type 1 (HSV1) was used to identify subcortical neurons that project via the thalamus to area 46 of the primate prefrontal cortex. This cortical area is known to be involved in spatial working memory. Many neurons in restricted regions of the dentate nucleus of the cerebellum and in the internal segment of the globus pallidus were labeled by transneuronal transport of virus from area 46. The location of these neurons was different from those labeled after HSV1 transport from motor areas of the cerebral cortex. These observations define an anatomical substrate for the involvement of basal ganglia and cerebellar output in higher cognitive function.

Muscle and movement representations in the primary motor cortex.

What aspects of movement are represented in the primary motor cortex (M1): relatively low-level parameters like muscle force, or more abstract parameters like handpath? To examine this issue, the activity of neurons in M1 was recorded in a monkey trained to perform a task that dissociates three major variables of wrist movement: muscle activity, direction of movement at the wrist joint, and direction of movement in space. A substantial group of neurons in M1 (28 out of 88) displayed changes in activity that were muscle-like. Unexpectedly, an even larger group of neurons in M1 (44 out of 88) displayed changes in activity that were related to the direction of wrist movement in space independent of the pattern of muscle activity that generated the movement. Thus, both "muscles" and "movements" appear to be strongly represented in M1.

Activation of a cerebellar output nucleus during cognitive processing.

Magnetic resonance imaging was used to examine the involvement of the dentate nucleus of the cerebellum in cognitive operations. All seven people examined displayed a large bilateral activation in the dentate during their attempts to solve a pegboard puzzle. The area activated was three to four times greater than that activated during simple movements of the pegs. These results provide support for the concept that the computational power of the cerebellum is applied not only to the control of movement but also to cognitive functions.

Transneuronal transfer of herpes virus from peripheral nerves to cortex and brainstem.

The transneuronal transfer of neurotropic viruses may represent an effective tool for tracing chains of connected neurons because replication of virus in the recipient neurons after transfer amplifies the "tracer signal." Herpes simplex virus type 1 was transferred transneuronally from forelimb and hindlimb nerves of rats to the cortical and brainstem neurons that project to the spinal enlargements to which the nerves receiving injections are connected. This transneuronal transfer of herpes simplex virus type 1 from peripheral nerves has the potential to be used to identify neurons in the brain that are related transsynaptically to different nerves and muscles.

Multiple output channels in the basal ganglia.

The neural circuits that link the basal ganglia with the cerebral cortex are critically involved in the generation and control of voluntary movement. Retrograde transneuronal transport of herpes simplex virus type 1 was used to examine the organization of connections in the cebus monkey between an output nucleus of the basal ganglia, the internal segment of the globus pallidus (GPi), and three cortical areas: the primary motor cortex, the supplementary motor ara, and the ventral premotor area. Spatially separate regions of the GPi were labeled after virus injections into each cortical area. The GPi projects to multiple cortical motor areas, and this pallidal output is organized into discrete channels. This information provides a new anatomical framework for examining the function of the basal ganglia in skeletomotor control.

Direction of action is represented in the ventral premotor cortex.

The ventral premotor area (PMv) is a major source of input to the primary motor cortex (M1). To examine the potential hierarchical processing between these motor areas, we recorded the activity of PMv neurons in a monkey trained to perform wrist movements in different directions with the wrist in three different postures. The task dissociated three major variables of wrist movement: muscle activity, direction of joint movement and direction of movement in space. Many PMv neurons were directionally tuned. Nearly all of these neurons (61/65, 94%) were 'extrinsic-like'; they seemed to encode the direction of movement in space independent of forearm posture. These results are strikingly different from results from M1 of the same animal, and suggest that intracortical processing between PMv and M1 may contribute to a sensorimotor transformation between extrinsic and intrinsic coordinate frames.

The cerebellum: an overview.

Life has been compared to a beautiful tapestry, woven in intricate design of many threads and colors. By means of physics, chemistry, physiology, anatomy, embryology and genetics we unravel this texture, separate its constituent threads and colors, but lose the pattern as a whole. These analytical sciences have enormously increased our knowledge of life's constituent elements and processes, but the pattern of the tapestry is usually neglected or ignored.

Imaging the premotor areas.

Recent imaging studies of motor function provide new insights into the organization of the premotor areas of the frontal lobe. The pre-supplementary motor area and the rostral portion of the dorsal premotor cortex, the 'pre-PMd', are, in many respects, more like prefrontal areas than motor areas. Recent data also suggest the existence of separate functional divisions in the rostral cingulate zone.

Medial wall motor areas and skeletomotor control.

The results of recent studies in primates provide convincing evidence that the cortex on the medial wall of the hemisphere contains multiple areas concerned with the generation and control of body movement. Highlights of these findings include the demonstration that each of these motor areas has substantial direct projections to the spinal cord, somatotopically organized projections to the primary motor cortex, a 'motor' map, revealed by intracortical stimulation, and neuronal activity that precedes trained hand movements.

Cerebellar projections to the prefrontal cortex of the primate.

The cerebellum is known to project via the thalamus to multiple motor areas of the cerebral cortex. In this study, we examined the extent and anatomical organization of cerebellar input to multiple regions of prefrontal cortex. We first used conventional retrograde tracers to map the origin of thalamic projections to five prefrontal regions: medial area 9 (9m), lateral area 9 (9l), dorsal area 46 (46d), ventral area 46, and lateral area 12. Only areas 46d, 9m, and 9l received substantial input from thalamic regions included within the zone of termination of cerebellar efferents. This suggested that these cortical areas were the target of cerebellar output. We tested this possibility using retrograde transneuronal transport of the McIntyre-B strain of herpes simplex virus type 1 from areas of prefrontal cortex. Neurons labeled by retrograde transneuronal transport of virus were found in the dentate nucleus only after injections into areas 46d, 9m, and 9l. The precise location of labeled neurons in the dentate varied with the prefrontal area injected. In addition, the dentate neurons labeled after virus injections into prefrontal areas were located in regions spatially separate from those labeled after virus injections into motor areas of the cerebral cortex. Our observations indicate that the cerebellum influences several areas of prefrontal cortex via the thalamus. Furthermore, separate output channels exist in the dentate to influence motor and cognitive operations. These results provide an anatomical substrate for the cerebellum to be involved in cognitive functions such as planning, working memory, and rule-based learning.

The inferior parietal lobule is the target of output from the superior colliculus, hippocampus, and cerebellum.

The inferior parietal lobule (IPL) is a functionally and anatomically heterogeneous region that is concerned with multiple aspects of sensory processing and sensorimotor integration. Although considerable information is available about the corticocortical connections to the IPL, much less is known about the origin and importance of subcortical inputs to this cortical region. To examine this issue, we used retrograde transneuronal transport of the McIntyre-B strain of herpes simplex virus type 1 (HSV1) to identify the second-order neurons in subcortical nuclei that project to the IPL. Four monkeys (Cebus apella) received injections of HSV1 into three different subregions of the IPL. Injections into a portion of the lateral intraparietal area labeled second-order neurons primarily in the superficial (visual) layers of the superior colliculus. Injections of HSV1 into a portion of area 7a labeled many second-order neurons in the CA1 region of the hippocampus. In contrast, virus injections within a portion of area 7b labeled second-order neurons in posterior regions of the dentate nucleus of the cerebellum. These observations have some important functional implications. The IPL is known to be involved in oculomotor and attentional mechanisms, the establishment of maps of extrapersonal space, and the adaptive recalibration of eye-hand coordination. Our findings suggest that these functions are subserved by distinct subcortical systems from the superior colliculus, hippocampus, and cerebellum. Furthermore, the finding that each system appears to target a separate subregion of the IPL provides an anatomical substrate for understanding the functional heterogeneity of the IPL.

Cerebellar output: motor and cognitive channels.

The input to the cerebellum has long been known to originate from widespread regions of the cerebral cortex including the frontal, parietal and temporal lobes. The output of the cerebellum, however, was thought to project mainly to the primary motor cortex. Recent anatomical observations have challenged this view. It is now apparent that cerebellar output goes to multiple cortical areas, including not only the primary motor cortex, but also areas of premotor and prefrontal cortex. In fact, there is growing evidence that each of the areas of cerebral cortex that project to the cerebellum is also the target of cerebellar output. The cerebellar output to individual cortical areas originates from distinct clusters of neurons in the deep nuclei which we have termed `output channels'. The individual output channels to the cortical areas we have examined display little or no overlap. Physiological recordings in awake trained primates indicate that neurons in different output channels appear to be involved in distinct aspects of behavior, and in both motor and cognitive functions. These observations indicate that the cerebellar influence on the cerebral cortex is more extensive than previously recognized.

Physiological demonstration of multiple representation in the forelimb region of the cat motor cortex.

Earlier studies in primates have demonstrated a double representation of the distal forelimb in area 4. In this study intracortical stimulation was used to map the representation of the forelimb in area 4 of the cat. Maps of individual animals revealed two spatially separate representations for the distal forelimb in area 4. Two "digit zones," regions in which threshold stimulation evoked contractions limited to digit musculature, were seen in all animals. Although the absolute location of the two digit zones varied among animals, the zones were always separated by a field in which more proximal musculature was represented. In some experiments EMG activity was monitored from selected forelimb muscles in order to determine the muscles represented in the two zones. Activity of the same digit muscle could be evoked by stimulation in each digit zone. The analysis demonstrated that some digit muscles were represented in both the digit zones. Thus, this study demonstrates that multiple representation of the distal forelimb in area 4 is not an isolated, species-specific phenomenon, but is likely to be a generalized pattern of motor cortex organization.

Anatomical demonstration of multiple representation in the forelimb region of the cat motor cortex.

Retrograde transport of HRP was employed to examine the pattern of callosal connections in the forelimb region of area 4 gamma in the cat. According to the conventional view, areas of the motor cortex which contain the representation of distal body parts neither send nor receive callosal fibers. If this is true, then an absence of callosal connections would define the sites of distal forelimb representation. Following multiple injections of HRP into the contralateral motor cortex, many labeled neurons were found in the forelimb region of area 4 gamma. However, within this region, two spatially separate areas were found where labeled neurons were either absent or present in very low density ("callosal holes"). The anatomically defined callosal holes corresponded in size, shape, and location to the physiologically defined digit zones. To provide direct evidence for this correspondence, retrograde HRP transport was combined with intracortical stimulation in the same animal. Small lesions placed in physiologically identified digit zones were located within the anatomically defined callosal holes. Thus, a double representation of the distal forelimb can be defined in area 4 gamma of the cat motor cortex using both anatomical and physiological methods.

Cerebellar connections with the motor cortex and the arcuate premotor area: an analysis employing retrograde transneuronal transport of WGA-HRP.

We have employed transneuronal transport to examine the anatomical relationships between the deep cerebellar nuclei and 2 cortical motor areas: the primary motor cortex and the arcuate premotor area (APA). In the same animals, we have also examined the patterns of labeling in the thalamus and the red nucleus to provide evidence for the potential routes of transneuronal transport to the cerebellum. When the appropriate technical procedures were employed, cortical injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) resulted in transneuronal labeling within portions of the contralateral deep cerebellar nuclei. Injections into the primary motor cortex labeled neurons in the dentate and in the 2 subdivisions of the interpositus. Injections into the APA labeled neurons in the dentate and in only the posterior subdivision of the interpositus. In most instances, dentate neurons were more intensely labeled following the cortical injections than interpositus neurons. The transneuronal labeling observed in the dentate nucleus was topographically organized. The dentate region that was labeled following injections into the "arm area" of the APA was caudal and ventral to the dentate region that was labeled following injections into the "arm area" of the primary motor cortex. This observation provides evidence for two "arm areas" in the dentate: one anatomically related to the APA, and the other related to the primary motor cortex. More than one route of transport may be responsible for the labeling of cerebellar neurons. We propose that the labeling observed in the dentate nucleus reflects the pattern of connections in the cerebellothalamocortical pathways that link the dentate with the cerebral cortex. Thus, our observations support the concept proposed by Schell and Strick (J. Neurosci. 4:539-560, '84)--that the cortical targets of the dentate nucleus include both the primary motor cortex and the APA.

Novel proteoglycan epitope expressed in functionally discrete patterns in primate cortical and subcortical regions.

The molecular diversity of neuronal subpopulations was examined with a new monoclonal antibody, 8B3, that recognizes a condroitin sulfate proteoglycan expressed in anatomically discrete domains of central nervous system regions. In the neocortex, interneurons display 8B3 immunoreactivity in a rostrocaudal gradient, with a distinctive staining pattern that distinguishes known cytoarchitectonic and functional boundaries. The distribution pattern of 8B3 immunoreactivity in subcortical structures is very restricted. In the striatum, 8B3 stains spiny stellate neurons clearly defining a compartment that may correspond to the matrix. Gradients of immunoreactivity are detected in the putamen, globus pallidus, and deep cerebellar nuclei, where the most dense areas of 8B3 immunoreactivity corresponds to zones of polysynaptic projections to association prefrontal cortex. In contrast, the sensorimotor domains express lower levels of immunoreactivity. Only the projection neurons of the ventrolateral nucleus and the GABAergic neurons of the reticular nucleus express significant 8B3 immunoreactivity in the thalamus. In the spinal cord, 8B3 immunoreactivity is primarily associated with a subpopulation of motor neurons in the ventral horn and neurons in Clarke's nucleus. The complex distribution pattern reflects novel aspects of the functional organization of cortical and subcortical systems in the CNS of the primate brain and represents a potentially useful tool to assess subpopulations of neurons and brain areas as putative targets in human disease.

Interconnections between the prefrontal cortex and the premotor areas in the frontal lobe.

We examined interconnections between a portion of the prefrontal cortex and the premotor areas in the frontal lobe to provide insights into the routes by which the prefrontal cortex gains access to the primary motor cortex and the central control of movement. We placed multiple injections of one retrograde tracer in the arm area of the primary motor cortex to define the premotor areas in the frontal lobe. Then, in the same animal, we placed multiple injections of another retrograde tracer in and around the principal sulcus (Walker's area 46). This double labeling strategy enabled us to determine which premotor areas are interconnected with the prefrontal cortex. There are three major results of this study. First, we found that five of the six premotor areas in the frontal lobe are interconnected with the dorsolateral prefrontal cortex. Second, the major site for interactions between the prefrontal cortex and the premotor areas is the ventral premotor area. Third, the prefrontal cortex is interconnected with only a portion of the arm representation in three premotor areas (supplementary motor area, the caudal cingulate motor area on the ventral bank of the cingulate sulcus, and the dorsal premotor area), whereas it is interconnected with the entire arm representation in the ventral premotor area and the rostral cingulate motor area. These observations indicate that the output of the prefrontal cortex targets specific premotor areas and even subregions within individual premotor areas.

Motor areas on the medial wall of the hemisphere.

The primary motor cortex (M1) receives input from four premotor areas on the medial wall of the hemisphere: the supplementary motor area (SMA) and three cingulate motor areas located on the banks of the cingulate sulcus (CMAr, CMAd and CMAv). All four premotor areas have maps of the body containing distinct proximal and distal representations of the arm. Surprisingly, the size of the distal representation is comparable to or larger than the size of the proximal representation in each area. Thus, contrary to some previous hypotheses, the anatomical substrate exists for the premotor areas on the medial wall to be involved in the control of distal, as well as proximal arm movements. Each of the premotor areas on the medial wall also has substantial direct projections to the spinal cord. Corticospinal axons from these premotor areas terminate in the intermediate zone of the spinal cord. Some corticospinal axons from SMA, CMAd, and CMAv terminate around motoneurons. In this respect, these motor areas are like M1 and appear to have direct connections with spinal motoneurons, particularly those innervating muscles of the fingers and wrist. These results suggest that the premotor areas on the medial wall are an important source of descending commands for the generation and control of movement. In recent experiments we examined the pattern of functional activation in the premotor areas on the medial wall during the performance of sequences of pointing movements. The patterns of activation were then compared with the body maps revealed by our anatomical studies. Overall, our initial results indicate that the attributes of motor control are unequally represented across the premotor areas. For example, one of the areas on the medial wall, the CMAd, was strongly and selectively activated during the performance of highly practised, remembered sequences of movement. Further insights into the function of the premotor areas are likely to come from examining their participation in a broad range of behavioural paradigms. These initial results support our hypothesis that each premotor area makes some unique contribution to the planning, initiation and/or execution of movement.

Rabies as a transneuronal tracer of circuits in the central nervous system.

The ability of selected neurotropic viruses to move transneuronally in the central nervous system makes them particularly well suited for use as tracers in experimental neuroanatomy. Recently, techniques have been developed for using rabies virus as a transneuronal tracer. Several features of rabies infection make the virus particularly useful for this purpose. We examined transneuronal transport of rabies in the central nervous system of primates after intracortical and intramuscular injections. Rabies was transported in a time-dependent manner to infect synaptically-connected chains of neurons. Transport occurred exclusively in the retrograde direction. At the survival times we used, rabies infection was restricted to neurons and did not cause cell lysis. There are several methodological and safety issues that must be considered when designing studies that use rabies as a transneuronal tracer. When appropriate protocols and laboratory practices have been established, transneuronal transport of rabies can be a safe and efficient tool for revealing the organization of multi-synaptic circuits in the central nervous system.