Abstract
The dysmetria of thought theory holds that the cerebellum is an integral node in the distributed neural circuits subserving cognition. We tested this theory in four rhesus monkeys with bilateral lesions in cerebellar dentate nuclei (DN), targeting ventral sectors linked with cerebral association cortices engaged in cognitive processing. Lesion localization was confirmed by MRI and histology. DN animals and controls were assessed for motor dexterity (Kuypers’ Task), attention, three-choice discrimination, recognition memory (Delayed Non-Matching to Sample task [DNMS]), working memory (Delayed Recognition Span Task [DRST]) and executive function (Conceptual Set Shifting Task [CSST]). Performance by the DN animals was not significantly different than controls on the Kuypers’ task and DNMS, but as a group they were impaired on DRST and CSST. DN monkeys achieved shorter spans on DRST. Their responses on CSST were more perseverative, and larger lesions produced greater deficits. This study provides the first empirical support for anatomical and functional MRI evidence of a motor-cognitive dichotomy in the cerebellar dentate nucleus. It highlights a within-cognition dichotomy in which cerebellum modulates dorsal stream cognitive functions (spatial awareness; dynamic actions of where and how) characterized by executive functions including working memory but is not engaged in ventral stream cognitive processes (static actions of object identification) exemplified by recognition memory.
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Introduction
The traditional notion dating back to the time of Flourens1 that the cerebellum is devoted exclusively to the coordination of movement, has been replaced by a more comprehensive and nuanced understanding of its contribution to multiple aspects of neurological function2,3. This paradigm shift in thinking about the cerebellum was facilitated by studies in rhesus monkey of projections from association and paralimbic cerebral cortices to the basis pontis that constitute the first step in the feedforward limb of the cerebrocerebellar circuits4, complemented by the feedback projections from deep cerebellar nuclei through thalamus to cerebral cortex5,6. The development of functional MRI advanced the understanding of cerebral and cerebellar interconnections in the human brain. Task-based MRI studies show discrete functional topography across and within individuals7,8 and resting state functional connectivity MRI has identified intrinsic connectivity networks in the cerebral hemispheres that are tightly correlated with topographically arranged regions in the cerebellum9,10,11. The identification of the cerebellar cognitive affective syndrome (CCAS) in patients with lesions confined to cerebellum provided the critical clinical underpinning of this new field of cerebellar cognitive neuroscience12,13. The CCAS is characterized by deficits in executive function, visual-spatial processing, linguistic skill including metalinguistics14 and affective dysregulation15. Notably missing from the syndrome is a primary amnestic disorder. On the CCAS/Schmahmann scale derived from patients with cerebellar lesions16, failure of declarative memory including recognition memory and episodic memory is specifically demarcated as a red flag for extracerebellar pathology in locations such as the medial temporal lobe, thalamus, and their interconnecting neural circuits. The description of the CCAS further recognized a double dissociation in topographic arrangement of cerebellar function such that the cerebellar anterior lobe is predominantly motor related, whereas the cerebellar posterior lobe is engaged in cognition and emotional processing12,17. The role of the cerebellum in higher-order function has been extensively replicated in lesion deficit correlation studies in patients, behavioral and anatomical experiments in animal models, and in functional imaging studies in humans using task-based, resting state, and functional connectivity analyses3,18.
The deep cerebellar nuclei are reciprocally connected with topographically precise regions of the cerebellar cortex, and with the exception of the vestibulocerebellum are the only source of efferents from the cerebellum to the spinal cord, brainstem and cerebral hemispheres19. In the case of the dentate nucleus, which has expanded massively through evolution20, anatomical connectivity suggests that the more recently evolved ventral part of the dentate nucleus which is linked with the prefrontal cortex but not with the precentral motor cortex should be engaged in cognitive processing but not in motor control5,6. Task-based activation studies21 and resting state connectivity analyses22provide support for the conclusion that subdivisions of the dentate nucleus contribute to different aspects of neurological function. Clinical confirmation of the role of the deep nuclei in higher-order function is difficult to ascertain with certainty. For reasons largely related to the dual blood supply of the deep cerebellar nuclei from the posterior inferior cerebellar artery and the superior cerebellar artery23, it is exceedingly uncommon to have a stroke confined to the deep cerebellar nuclei, and mass lesions affecting the deep nuclei are promiscuous in the anatomical structures affected by the pathology. We therefore undertook a lesion-deficit behavioral study in the rhesus monkey in which we targeted the ventral dentate nucleus on both sides to test the hypothesis that the deep nuclear lesion would spare motor function and recognition memory, but degrade executive control tasks that rely on prefrontal-cerebellar circuitry. Such a result would provide the first behavioral confirmation of a dissociation of motor versus cognitive outcome for the dentate nuclei as has been demonstrated for the anterior versus posterior lobes of the cerebellar hemispheres.
Results
Lesion localization—MRI
Brain MRI successfully identified coordinates of the planned electrolytic lesions in the ventral parts of the dentate nucleus on each side. Post-operative MRI identified the lesions in the intended ventral regions of the dentate nuclei bilaterally in cases 1 and 2. The lesion sites in cases 3 and 4 were slightly dorsal to the intended target but still involved the dentate nuclei. (Figure 1).
T1-weighted spoiled gradient (SPGR) brain MRI scans in the stereotactic coronal plane in four rhesus monkeys after surgical placement of electrolytic lesions (the focal areas of hypointensity) targeting the ventral part of the dentate nuclei in each cerebellar hemisphere. Rostral planes of section in the four Cases are on the left, and caudal planes are on the right. The right cerebellum is shown on the right. Slice thickness is 1 mm.
Lesion localization—histology
In three control animals we used Image J on serial histological sections to define and outline the boundaries of the dentate nuclei to measure their volumes. We used the same method for the four DN monkeys, measuring the volume of the remaining intact dentate nuclei in both right and left hemispheres. In cases 1 and 2 there was extensive destruction of the ventral lateral part of the dentate nuclei as intended. In cases 3 and 4 the lesions were more laterally situated in the dentate nuclei, and the lesion volumes were smaller than in cases 1 and 2, but nevertheless were markedly reduced compared to the control animals. (Table 1; Figure 2).
Representative histological sections in the coronal plane in the rhesus monkey brains in Cases 1 through 4, stained for Nissl substance with thionin. The electrolytic lesions are the focal areas of necrosis located in the ventral parts of the DN in Cases 1 and 2, and in more dorsal parts of the DN in cases 3 and 4. Bar = 1 mm. D, dentate nucleus; F, fastigial nucleus; IA, interpositus anterior nucleus; IP, interpositus posterior nucleus. The right cerebellum is shown on the right.
Manual dexterity task
We measured the latency to retrieve the pellets from the wells in the Kuypers’ task before and after lesion placement in cases 2 and 4. There was no difference between the pre-lesion and post-lesion mean and standard deviations of latencies. Case 2, pre-surgery 2.0 +/- 1.0 (N = 58), post-surgery 1.8 +/- 1.0 (N = 60), P = 0.37. Case 4, pre-surgery 2.5 +/- 2.2 (N = 57), post-surgery 2.5 +/- 1.7 (N = 50), P = 0.99.
Attention task
The attention task requiring the animals to point to an object on the screen was accomplished without error in the four DN animals and four controls, and therefore group performance was comparable (P < 0.001).
Three-choice discrimination task
Each of the DN and control animals performed without error on each of the 10 attempts, and therefore group performance was comparable (P < 0.001).
Delayed non-matching to sample (DNMS)
On the DNMS basic task the four DN animals acquired criterion in an average of 316.0 trials (SD 229.6) and committed an average of 93.5 errors (SD 67.5), compared to the control cohort that took an average 222.5 trials (SD 53.2) and committed an average of 52.25 errors (SD 12.3). On the 120 s delay condition of the DNMS task the DN group performed with 83% accuracy (SD 4.0%), and the control animals performed with 85% accuracy (SD 4.0%). For the 600 s delay condition, DN group achieved 80% (SD 6.0%) accuracy and the controls 80% (SD 6.0%). Mixed-model regression analyses revealed no significant interaction between group and delay [X2(2) = 0.538, P = 0.76], and a marginally significant effect of delay across all animals, both lesioned and controls, [X2(1) = 3.866, P = 0.049]. When we compared lesioned with control monkeys using two-sample t-tests, for Acquisition Trials, Acquisition Errors, T120, and T600, the P-values were 0.48, 0.31, 0.56, 0.87, respectively. Table 2. There was no significant correlation between percent dentate remaining and any of the six behavioral outcomes – Acquisition Trials and Errors, T120 errors and percent errors, and T600 errors and percent errors. Table 3.
Delayed recognition span task (DRST)
The Spatial DSRT task was substantially harder than the Object DSRT task for all monkeys (means spans 2.9 and 4.3, respectively), and significantly more difficulty for DN-lesioned monkeys than for controls (Group x Task interaction, P = 0.006). In a mixed-model regression with the experimental outcome (span) as response, and monkey ID as a random effect, Group, Task, and the Group x Task interaction were all statistically significant, with P-values 0.001, < 0.001, and 0.05, respectively. We tested the significance of the difference between lesion and control for the Object task, the Spatial task, and the difference of these two tasks. Adjusting for the three hypothesis tests, the corresponding P-values were < 0.001, 0.016 and 0.037, respectively. Table 3 AND Figure 3B.
Performance of the four controls animals (white bars) and four DN animals (gray bars) on behavioral tasks. (A) Object and Spatial Delayed Recognition Span Tasks (DRST). Lesioned animals were significantly impaired compared to controls. Mean span on the y-axis. Overall group difference P =.001; Object P <.001; Spatial P =.016. (B) Conceptual Set Shifting Task (CSST). Percent errors committed by the DN animals were significantly greater than controls on Tri (P <.001), Red-Tri, and Blue-Tri switches (P <.001 and P =.001, respectively). Mean perseverative errors on the y-axis, conditions on the x-axis. (C) Conceptual Set Shifting Task (CSST). Mean perseverative errors committed by the DN animals were significantly greater than the control animals. The overall P-value for Group (main effect plus the significant interaction) was P <.001. Mean perseverative errors are on the y-axis, conditions on the x-axis. *, P <.05; **, P <.005; ***, P <.001.
Conceptual set shifting task (or CSST)
For perseverative and non-perseverative errors combined, the error rates in lesioned monkey were greater than controls for all trial types (P < 0.001). Perseverative error rates were recorded for three experiment types: Blue, Star, and Tri. In a mixed-model regression with perseverative error as response and monkey ID as a random effect, both the main effect of Group (P = 0.02) and the Group x Type interaction (P = 0.02) were statistically significant. The overall P-value for Group (i.e. for the main effect plus the significant interaction) was P < 0.001. We tested the significance of the difference between lesion and control for each of Blue, Star, and Tri, and the switches between Tri-Blue, Star-Blue, and Tri-Star. We determined P-values, adjusting for the six hypotheses tested. The difference of the Tri-Blue switch between groups was significant (P = 0.004), and the difference of Tri-Star between groups trended towards significance (P = 0.098). Table 4, Figure 3C, and 3D.
Lesion analysis
In the DN animals, dentate nucleus residual volume was significant for both perseverative and non-perseverative errors on the CSST, P = 0.02 for both analyses, based on likelihood ratio chi-square tests. Smaller residual dentate nucleus volumes corresponded to a larger number of errors. Table 5
Discussion
We tested the hypothesis that there is dissociation of motor versus cognitive processing in the dentate nucleus of the cerebellum of rhesus monkey by placing electrolytic lesions bilaterally in the dentate nuclei, targeting the ventral dentate linked with prefrontal and posterior parietal association areas. We then tested the animals on tasks of motor control, recognition memory, working memory, and mental flexibility. Our results demonstrate preservation of fine motor control and recognition memory, but impairment on tests of working memory and mental flexibility. Table 6
Appendicular dysmetria with inaccurate reaching, degraded fine motor control, and clumsiness of hand movements are hallmarks of the cerebellar motor syndrome. The Kuypers’ motor task24assesses upper extremity dysmetria and impaired dexterity, and the finding that this task is preserved in monkeys with DN-lesions provides the behavioral confirmation that the ventral dentate nucleus is not necessary for upper extremity motor control. This is consistent with observations from a previous ablation study in monkey25, and in male Long Evans rats in which lateral (dentate) nucleus lesions do not impair motor function but reduce hedonic and purposive motivation26.
The DNMS, a benchmark test of rule learning and recognition memory in the non-human primate27 was not impaired in the DN animals. These functions are degraded by lesions of medial temporal cortex, hippocampal and parahippocampal and perirhinal formations, and related structures engaged in learning and recall28,29. This finding was consistent with our hypothesis that recognition memory should be spared, based on neuroanatomical tract tracing studies in monkey and clinical observations in patients with cerebellar lesions, as discussed below.
In contrast, the DN animals demonstrated significant impairment on the DRST, a test of working memory27 that examines the ability to remember an increasing array of stimuli28. In monkeys the DRST recruits the hippocampus for the acquisition and retention of new information28, and it also relies on the prefrontal cortex, as shown by impaired performance in young monkeys with lesions of the dorsolateral prefrontal cortex27. Further, spatial span tasks in humans, similar to the DRST, activate the prefrontal cortex and are impaired in patients with large frontal lobe lesions, particularly the right dorsolateral prefrontal cortex, reflecting the role of the dorsolateral prefrontal cortex for the strategic and goal-based component of working memory30. Here we show for the first time that the DRST is impaired in monkeys with lesions in the DN.
The Wisconsin Card Sorting Test (WCST) in humans assesses abstraction, concept formation and cognitive set shifting that localize to the prefrontal cortex31,32,33. The CSST is a monkey version of the WCST that taps the executive – working memory component of prefrontal cortex function34,35. Perseveration on the CSST task, characterized by the erroneous repetition of a previously rewarded choice, is a hallmark dysfunction of damaged executive control systems subserved by the prefrontal cortex. Similar to performance on the DRST, here we show for the first time that the monkeys with lesions in the DN are impaired on the CSST. Further, at the level of the individual animals, the degree of impairment on the CSST correlated with the size of the DN lesions, providing proof of principal for the causal relationship of the DN lesion with the impaired performance on the mental flexibility tasks.
We thus demonstrate in the rhesus monkey a dissociation of behavioral consequences following focal dentate nucleus lesions: preservation of motor dexterity and recognition memory, but degradation of mental dexterity needed for cognitive control of set switching and intellectual flexibility.
Cerebrocerebellar circuity underlying these behavioral observations
Our findings provide a behavioral correlate of the anatomical, connectional, and functional topography identified within the cerebellum and dentate nucleus. Evolutionary considerations have led to the understanding that the lateral cerebellar hemispheres, particularly the expansion of lobule VI and of crus I and crus II of lobule VII, together with the ventral part of the dentate nucleus have expanded through mosaic evolution in concert with the cerebral association areas and constitute the anatomical representation of the cognitive cerebellum2,3,6,7,12,17,20,36,37. This stands in contrast to the cerebellar anterior lobe, notably lobules III through V and adjacent regions of lobule VI that constitute the primary somatosensory cerebellum together with lobule VIII that contains the secondary sensorimotor cerebellar representation3, a hypothesis that has extensive support from anatomical and physiological studies in animal models. Vermis, lobule IX and the fastigial nucleus in particular have been proposed as the cerebellar representation of limbic circuitry2,38,39 critical for affective regulation and neuropsychiatric phenomena in the clinical setting23,38. Linkage of the fastigial nucleus with the ventral tegmental area40, the origin of the mesolimbic dopaminergic system, provides further support for this contention.
The prefrontal cortex (PFC) is strongly interconnected with the cerebellum, and particularly the cognitive regions in the cerebellar posterior lobe2,3,36, as well as with the more recently evolved ventral sectors of the dentate nucleus5,20. Within the dentate nucleus the use of trans-synaptic anterograde and retrograde viral studies demonstrated a dichotomy of motor versus cognitive connectivity6. The dorsal dentate nucleus, together with the interpositus nuclei, is linked with sensorimotor regions of the cerebral cortex, and engaged in tight reciprocal relationships with afferents from spinal cord and with regions of the inferior olivary nuclei that participate in sensorimotor control19. In contrast, the ventral dentate is devoid of connections with inferior olivary nuclei that receive input from spinal cord19,41, is linked with the D2 zone of the cerebellar hemisphere that includes lobules VI and VII19devoted to cognitive control7,8,9,10, and projects to thalamic nuclei6 that are reciprocally interconnected with prefrontal and other association areas rather than sensorimotor cortices42,43. This pattern of anatomical connectivity to spinal cord, brainstem, and cerebral hemisphere regions in tract tracing studies is complemented by functional imaging studies of the dentate nucleus in human. The mediolateral topography of the corticonuclear connections within the cerebellum in the animal model44 is confirmed in the human with diffusion tractography, showing regions of the dentate nucleus that are linked with the motor anterior lobe, and a topographic arrangement of dentate connections with different regions of the cognitive posterior lobe45 Task-based functional MRI21 and resting state functional connectivity MRI extend this dentate nucleus relationship to the cerebral cortex, showing three major divisions within the dentate nucleus, a default-mode, salience-motor, and visual cerebral cortical network22.
Relevance for clinical neuroscience
The CCAS is characterized by deficits in executive function, visual-spatial processing, linguistic skill, and affect regulation, and whereas the executive control of recall can be degraded, episodic and recognition memory are typically spared12. The syndrome results from lesions of the cerebellar posterior lobe, underscores the clinical relevance of the cerebellar contribution to nonmotor function, and has been regarded as third cornerstone of clinical ataxiology along with the cerebellar motor syndrome and the cerebellar vestibular syndrome46. These clinical manifestations are consistent with the dysmetria of thought theory that holds that the cerebellum regulates emotional and cognitive processing in a manner analogous to the disorder of motor control2,47. The postulated mechanism is damage to the universal cerebellar transform23,36, the underlying computation unique to the paracrystalline architecture characteristic of the cerebellar cortex48. According to this theory, the universal cerebellar transform is applied to different streams of information processing according to the precise topographic arrangement of the cerebellar connections with extracerebellar structures. These theoretical formulations receive support from functional imaging studies revealing topographic arrangement in the resting state connectivity networks that link cerebral and cerebellar hemispheres9,10, from the topographic maps of cognitive representations throughout the cerebellum7,8, and from the behaviorally distinct phenotypes of anterior versus posterior lobe lesions in patients with focal cerebellar damage3,17.
Based on anatomical and functional imaging studies, the dentate nuclei also appear to be organized according to distinct motor and cognitive domains. For reasons likely related to the rarity of the experiments of nature damaging the cerebellar deep nuclei in isolation, to our knowledge there are no clinical reports describing a dichotomy of motor versus cognitive deficits following dentate nucleus lesions, and this study in the monkey provides empirical evidence for this behavioral relationship.
These findings have relevance for the clinical neurology and neuropsychiatry of disease or damage to the deep cerebellar nuclei. This is particularly true for deep lesions such as occur in posterior fossa tumors in childhood, the resection of which is frequently followed by the CCAS and the cerebellar mutism syndrome49, cerebellar hemispheric infarctions that include the deep nuclei, and brain hemorrhage and other disruptions that damage the cerebellar nuclei producing neurological dysfunction in the domains of motor control, intellect, emotion and social cognition18,50,51.
Implications of a dichotomy within cerebellum of cognitive domains
We report that DN lesions degraded the executive functions of working memory (DRST) and cognitive flexibility (CSST) traditionally regarded as prefrontal cortical functions, but spared the DNMS task of recognition memory known to rely on medial temporal systems including the hippocampus28. We note, however, the earlier observation that animals tested after induction of large lesions of the dorsolateral and dorsomedial prefrontal cortex (Brodmann areas 46, 8, 9, 10, 12, 6) were impaired on both the acquisition (rule learning) and performance (recognition memory) conditions of the DNMS task52. In contrast, when the DNMS acquisition task was performed before frontal lobe damage, DNMS retention was impaired when lesions were in the ventromedial or orbital prefrontal cortex, but spared when lesions were in the dorsal prefrontal cortex53. The details of lesion timing and the differential nature and degree of the contribution of the medial and inferior temporal cortices vs the prefrontal lobe to recognition memory have been discussed previously52, but they raise an intriguing question relevant to this cerebellar focused study. If lesions of prefrontal areas play a role in learning and recognition memory, why do lesions of the cerebellar DN that convey cerebellar efferents to prefrontal cortices degrade the DRST and CSST but spare the DNMS task?
A possible anatomical explanation is that the cerebrocerebellar circuits disrupted by the DN lesions did not include the prefrontal areas involved in recognition memory as identified in monkey and human, particularly, the left anterior prefrontal cortex area 10 and left inferior frontal gyrus and opercular cortices (areas 45/47)54, but rather involved prefrontal circuits engaged in working memory, namely the dorsolateral and mid-dorsolateral prefrontal cortex, particularly areas 9 and 4655,56. Our present data do not allow us to resolve this possibility.
A more conceptual account of DNMS sparing relates to the putative mechanisms of information processing unique to PFC vs cerebellar circuitry3,47 even if the same PFC areas were included in the neural circuits disrupted by the DN lesions. This is exemplified by the finding in human fMRI studies that focal PFC and cerebellar regions activated by the same cognitive paradigm contribute differentially to the functional outcome. In a word stem completion task with MANY vs FEW possible correct answers, a double dissociation was identified, with left middle frontal gyrus (Brodmann areas 9/10) activated by the MANY condition reflecting response selection, and right cerebellar lobules VI and Crus I (“posterior quadrangular lobule and superior semilunar lobule”) activated in the FEW condition reflecting the search for responses57. This finding in healthy individuals received support from the observation that patients with cerebellar disorders were more impaired on the FEW condition relative to the MANY condition7.
The postulated distinct operations subserved by the PFC vs the cerebellum have relevance for the DRST-CSST/DNMS dichotomy following the DN lesions. We view these differences within the overarching framework of the dorsal vs ventral streams of cognitive processing: the dorsal stream emanating from visual areas of the parietal lobe, involved in spatial processing and guiding actions – the where/how pathway mediating sensorimotor transformations for visually guided actions; and the ventral stream leading from the occipital lobe to the temporal lobe engaged in object recognition, form representation and feature identification – the what pathway58,59. This sets up the dichotomy relevant here, of a dorsal stream for action, and a ventral stream for perception, with the caveat that the two streams do interact at the level of cortical connections60.
Corticopontine projections, the first link in the feedforward limb of the cerebrocerebellar loops, are derived heavily from dorsal stream areas in parietal, occipitotemporal and superior temporal lobe regions, but are either absent or sparse from ventral stream regions in the ventral occipital and temporal lobes61,62,63,64. These observations led us to suggest that within the visual realm, the corticopontocerebellar system is more concerned with visual spatial related functions and parameters of visual motion than with functions related to visual object identification or discrimination63. This dichotomy extends to the PFC64where pontine projections are derived from the dorsolateral and medial prefrontal convexities; that is, from areas 8Ad, 8B, 9 (lateral and medial), 10, 9/46d, 9/46v, and 32 important for the spatial attributes of memory, as well as executive functions such as initiative, planning, execution, and verification of willed actions and thoughts65,66,67,68,69, and from area 45B thought to be homologous with the language area of humans70. In contrast, and in agreement with others71,72,73, we did not detect pontine input in monkey from the ventrolateral and orbitofrontal cortices including areas 11, 47/12, 14, and 9/46v4,74 that are interconnected with ventral stream areas engaged in object identification, and feature detection, discrimination and recognition.
Thus, we hypothesize the cerebellum is concerned with dynamic action, but not static information processing. The transition from movement to thought36, reflected in the dysmetria of thought theory2,75, would apply to dynamic processing, whether motor control, cognitive operation/mental manipulation or regulation of emotion according to context. In this sense, the active mental manipulation required for working memory inherent in the DRST, and the active switching reflecting the dynamism of mental flexibility from one perception/understanding (blue) to another (red) in the CSST are dorsal stream processes requiring active engagement with the environment. Amending and respecting the terminology of Hughlings Jackson, cerebellum is as critical for the dynamic process of movement of an idea as it is for the movement of a limb2,76,77.
In contrast, the recognition of the unique features of an object that discriminates it from another object that may or may not have been seen before, is a feature detection task that is a static/fact acquisition distinct from the mental manipulation requiring the coordination of the cerebellar system. This would account for the sparing of performance on the DNMS, whether or not there may be a frontal lobe contribution, because, so the hypothesis holds, recognition memory is not in the purview of the cerebellar computation.
In sum, the idea that the universal cerebellar transform is to maintain behavior around a homeostatic baseline, automatically, without conscious awareness, informed by implicit learning, and performed according to context15applies to the coordination of dynamic action, awareness of self in relation to the internal and external environment, and the interpretation, manipulation, analysis and synthesis of complex stimuli, whether cognitive or motor. Cerebellar lesions cause incoordination of willed motor acts, but they do not cause motor weakness. This notion explains why, in the cognitive domain, cerebellum is involved in metalinguistics14, agrammatism and dynamic aphasia78 – the how of language, not the what of a word meaning as in semantic dementia. It is not facts that cerebellum cares about – it is the monitored, controlled, context-appropriate, coordinated use of facts. The dorsal stream is the purview of cerebellum, not the ventral stream – where and how, not what: DRST and CSST, not DNMS. The findings of a within-cognition dorsal–ventral stream dichotomy provides a hypothesis-driven invitation to experimental paradigms to further explore the nature of cerebellar cognition and elucidate its underlying mechanisms in the experimental animal and in the human condition in health and disease.
Limitations
There was a relatively small number of monkeys in this study, although this number is consistent with existing literature on lesion-deficit behavioral studies in animal models. The DN lesions in two animals were more restricted than intended, but we were able to capitalize on this observation to demonstrate a significant relationship between residual DN volume and CSST performance in the four DN animals.
We note that there was some encroachment on the dorsal part of the DN in cases 3 and 4, but the pre- and post- lesion testing in case 4 showed no impairment on the Kuyper’s motor task. In the absence of physiological testing, it is not possible to know whether lesion involving the dorsal part of the DN in this case involved subregions linked to M1 arm, or to M1 leg, M1 face, and premotor or SMA regions6,79,80. To the extent that there was some encroachment on the dorsal DN in Case 4, it is possible that the Kuyper’s task may have been unaffected because the upper extremity M1-DN circuitry was spared. It is also possible that the dorsal DN to M1 connectivity is less concerned with motor control tasks exemplified by the Kuyper’s paradigm than the interpositus nucleus that provides powerful timing signals important in coordinating limb movements81. The behavioral correlate of the dorsal dentate to M1 connection remains to be shown.
Conclusions
Damage to the ventral part of the dentate nucleus of monkey linked with association areas of the cerebral cortex spares manual motor dexterity and recognition memory, but degrades tasks of working memory and mental dexterity. This lesion-deficit behavioral study provides empirical support for anatomical and functional imaging evidence of a motor-cognitive dichotomy in the cerebellar dentate nucleus. It also highlights a within-cognition dichotomy of cerebellar modulation of dorsal stream functions characterized by working memory and mental flexibility but sparing of ventral stream processes exemplified by recognition memory.
Methods
Subjects
Four experimentally naïve male rhesus monkeys ranging from 5–6 years of age were used for lesion placement and behavioral testing. Each of the four animals received bilateral electrolytic lesions of the dentate nuclei (the DN cohort). Data from a cohort of eight age-matched naïve monkeys were used to formulate a standard for performance on the Attention Task, Three-Choice Discrimination Task, and DNMS task. Four additional age-matched naïve monkeys were used as healthy controls for the DRST Spatial and Object Tasks and the CSST tasks. Another three non-lesioned control animals were used for histological analysis. While on study, monkeys were individually housed in colony rooms in the Boston University Animal Science Center where they were in constant auditory and visual range of other monkeys. This facility is fully AAALAC accredited, and research was conducted in accordance with the guidelines of the National Institutes of Health and the Institute of Laboratory Animal Resources Guide. for the Care and Use of Laboratory Animals. All procedures were approved by the Boston University. Institutional Animal Care and Use Committee. Diet consisted of Lab Diet Monkey Chow (#5038—Lab- Diet Inc., St. Louis, MO) supplemented by fruit and vegetables with feeding taking place once per day, immediately following behavioral testing. Water was available continuously. The monkeys were housed under a 12-h light/dark cycle with cycle changes occurring in a graded fashion over the course of an hour. They were checked daily by trained observers for health and well-being and were given a medical exam every 6 months by a Clinical Veterinarian in the Boston University Animal Science Center.
Brain imaging
Brain MRI scans were used to identify the coordinates of the dentate nuclei using a stereotactic head frame. MR image acquisition was performed in the MRI Research Scanner at the MRI Division, Department of Radiology, Brigham and Women’s Hospital, a 1.5 Tesla General Electric SIGNA System (GE Medical Systems, Milwaukee, WI). The high-resolution MRI data acquisition protocol included a T2-weighted fast spin echo sequence that covered the entire cranium to screen for unsuspected pathology, and a T1-weighted spoiled gradient (SPGR) 3-D scan sequence for fast imaging acquisition and high contrast. The animals were anaesthetized throughout (Ketamine, 10 mg/kg, IM), the head stabilized in a standard stereotactic plane using a specially designed stereotactic apparatus constructed from plexiglass and brass, to avoid interfering with image acquisition, after Saunders et al.82 This device stabilized the head, provided reproducible orientation of the head for surgical purposes, and the radiopaque ear bars provided a stereotaxic landmark from which locations of the dentate nuclei were measured. Post-operative MRI was performed to confirm lesion size and location before the motor and cognitive testing.
SPGR scans in the coronal plane were used to identify the locations of the dentate nuclei within the deep white matter of each cerebellar hemisphere. Using the GE computer, measurements were taken to define the vertical (superior-posterior) distance of the dentate nucleus from the cerebral cortical surface as well as from the tentorium. Horizontal (medial–lateral) measurements were taken from the midline (defined as the plane between the two cerebral hemispheres) to the location corresponding to the center of the dentate nucleus according to the atlas of Szabo and Cowan83. The anterior–posterior location of the dentate was determined by measuring the anterior–posterior distance from the radiopaque ear bars. The final locations of the dentate nuclei, in stereotaxic space, were calculated from the zero measurements reading on the stereotaxic apparatus.
Electrolytic lesion placement
Animals were sedated with ketamine hydrochloride (10 mg/kg IM), anesthetized with intravenous sodium pentobarbital (9.75 mg/kg) and an endotracheal tube was inserted. The animal was placed in the stereotaxic headframe taking care to ensure that it was oriented with the same zero measurements from the time of MRI scanning. A midline scalp incision was made, both temporalis muscles retracted laterally, and the skull surface was cleared of fascia. A craniotomy was performed, the dura was retracted medially to expose the hemisphere and the interhemispheric fissure, mannitol was introduced intravenously, midline vessels cauterized, and the splenium of the corpus callosum was located and the distance from its most caudal aspect to the ear bars ascertained. This distance was compared to the measurements from the MRI to confirm placement in the stereotaxic space. A small incision was made in the tentorium cerebelli at the point at which the needle was to penetrate the dura to ensure the unimpeded and direct passage of the needle into the cerebellum. The stereotactic instrument was used to place a conducting electrode that delivered electric current of 2.0 mAmp for 1.5 min each, in three locations spaced 1.5 mm apart from anterior to posterior in the dentate nucleus bilaterally. After the lesions were placed, the dura was stitched, the temporalis muscles reapproximated, the scalp closed, the suture site covered with antibiotic ointment to prevent infection, and the animals monitored postoperatively in a pediatric incubator for 5 h until recovery.
Perfusion procedure
At the conclusion of the experiment, the four DN and three control monkeys were deeply anesthetized with sodium pentobarbitol (0.15 cc/kg) and euthanized by exsanguination during transcardial perfusion with 4% paraformaldehyde. The brain was blocked in situ in the coronal stereotactic plane. The tissue was cryoprotected by storage in 10% glycerol in 0.1 M phosphate buffer at –20 °C overnight, transferred to a solution of 20% glycerol in 0.1 M phosphate buffer and returned to the freezer. After one week in the 20% glycerol solution, the brain was flash frozen at −75 °C (per Rosene et al.)84 and stored at –80 °C.
Histological processing
The cerebellar block was cut on a sliding microtome into series of 60-micron thick sections, from posterior to anterior, and mounted onto gelatin subbed, glass slides. Section numbers were noted to facilitate reconstruction of the cerebellum, including intact and damaged tissue. The sections were stained with a thionin solution and coverslipped. The sections were examined microscopically to identify areas of gliosis and neuron loss, with or without tissue preservation in these areas. The lesion was reconstructed onto images of these coronal sections using a Nikon Eclipse E800 microscope (Nikon THP, Tokyo, Japan) with a camera attached (Diagnostic Instruments, Inc., Sterling, MI), and a computer running Adobe Photoshop 5.0 (Adobe Systems Incorporated, San Jose, CA). Image J software85 was used to analyze the sections quantitatively to determine the volume of remaining intact dentate nucleus (mm3) in the DN animals.
Neurobehavioral assessments
Preoperative behavioral testing
A simple test of fine motor control developed by Lawrence and Kuypers24 was used to compare preoperative and postoperative motoric function. All behavioral testing was performed in a Wisconsin General Testing Apparatus (WGTA) in a dark room with white noise to mask extraneous sound. The experimenter sat behind a one-way screen facing a 25 cm × 70 cm testing tray containing 15 numbered wells of diminishing sizes. Figure 4. The monkey sat behind a wooden barrier. A raisin was placed in one of the wells and the experimenter’s screen was closed. The animal’s barrier was lifted, and the time taken by the animal to extract the raisin from the well was measured and recorded. Subsequent raisins were placed in wells following a predetermined list of 30 trials. The animal was tested again the following day using a second list of 30 trials.
The Kuypers’ 25 cm × 70 cm testing tray containing food pellets (M&M candies here) in fifteen numbered wells of diminishing sizes presenting varying levels of difficulty. The food pellets were placed in the different wells following a predetermined list over 30 trials.
Following the fine motor task, the animal was pretrained in the WGTA to displace objects that cover the testing wells to retrieve the reward placed in the well. Throughout testing, raisins, M&M’s, or small pieces of apple were used as rewards. The testing tray contained a single row of three wells, 5 cm in diameter, spaced 15 cm from the center of one well to the center of the neighboring well. A gray square plaque (10 cm × 10 cm) was placed in between the middle and right wells and the center well was baited with an M&M candy. The tray was then presented to the animal, allowing the reward to be obtained. On subsequent trials the gray plaque was gradually moved closer to the well, eventually covering the well, in part and then totally. Once the animal learned to displace the plaque to retrieve the reward, 20 pseudorandom trials were performed using the right and left wells. This task was performed daily until a criterion of 20 consecutive successful trials, determined by a timely displacement of the plaque and retrieval of the reward.
For the computer screen component of the testing, the monkey was seated in the WGTA in a dark room with white noise to mask extraneous sound. The monkey had access to a computer screen placed in front of it. An image of an apple appeared on the screen. The monkey touched the apple and received a food reward for each apple touched. The stimulus was presented on 20 consecutive trials, and the monkey continued its trial days until all 20 apples were touched in the session.
No further preoperative behavioral testing was performed.
Postoperative behavioral testing
All postoperative testing for this study was performed in the WGTA. The procedure was like the preoperative testing, using the Kuypers’ task with the monkey seated in the WFTA. The monkeys were tested on 2 consecutive days, 2 weeks following surgery, for a total of 20 trials per day.
Delayed non-matching to sample (DNMS)
After completing pretraining the animal’s performance was assessed on the DNMS task, which tests rule learning, object discrimination, and object memory. For the DNMS task, the testing tray contained a single row of three wells, 5 cm in diameter, spaced 15 cm center to center. First, a sample object was presented over the baited central well and the animal was required to displace the object to obtain the reward. Subsequently the door blocking the animal’s view of the board was lowered and, after a delay of ten seconds, the recognition trial was conducted with the sample object appearing over one of the lateral wells and a novel object appearing over the other lateral well. The single reward was concealed underneath the novel object. To obtain the reward, the animal had to recognize the now familiar original sample object and displace the unfamiliar object. After completing the trial, the animal’s barrier was closed and, following a 20-s delay, a different sample object was placed in the central location, followed by another recognition trial using another novel object. The position of the two objects on recognition trials was assigned in a pseudo-random predetermined order using a non-correctional procedure. Twenty trials per day were administered, five days per week. DNMS acquisition trials continued until the animal reached a learning criterion of 90 correct responses in 100 consecutive trials. Objects were drawn from a pool of 800 objects separated into groups of 80 objects. No group was used twice within three days.
DNMS delays
Upon reaching learning criterion on the basic task, the memory demand of the task was enhanced by increasing the delay between the presentation of the sample object and the recognition trial. Two delays were used in this study: 120 s (T120) and 600 s (T600). Each delay condition was administered for ten consecutive days, ten trials per day, for a total of 100 trials. The 120-s delay was performed first, followed by the 600-s delay block. The 20-s inter-trial interval between sample object presentation and recognition was maintained for both delay conditions.
Delayed recognition span test (DRST)
The DRST is a working memory task that requires the subject to identify, trial-by-trial, the new stimulus within an increasing array of serially presented stimuli. The task is administered using different classes of stimulus material (e.g., spatial, object or color) to help characterize recognition memory deficits across several stimulus domains. In the present study, the DRST task was administered using the spatial and object conditions.
DRST—spatial
In the spatial condition, fifteen identical plain brown discs, 6 cm in diameter, were used as stimuli. A reward was placed in one of the eighteen wells and covered with one of the brown discs according to a standard predetermined order. The screen was raised, and the monkey was allowed to displace the disc to obtain the reward. The screen was lowered, the first disc was returned to its original position (now over an unbaited well) and a second well was baited and covered with a disc. After a ten-second delay, the screen was raised, and the monkey was required to identify and displace the new disc in order to obtain the reward. Each successive correct response was followed by the addition of a new disc until the monkey made an error (i.e., it chose one of the previously selected discs). With the occurrence of the first error, the trial was terminated and the number of discs on the test tray minus one constituted the recognition span score for that trial (i.e. number of correct responses). Ten trials were presented each day, seven or eight of which were unique sequences of stimuli and two or three of which were the same single sequence that was repeated intermittently throughout the spatial condition. This continued for ten consecutive days for a total of 100 trials – 75 unique and 25 repeated.
DRST—object
The object condition of this task was administered in a similar fashion to the spatial condition. However, the stimuli for all but the repeated trials were drawn from the previously mentioned pool of 800 toy objects. Within each trial, the position of the previously correct stimuli was changed in a pseudorandom fashion so that the animal was forced to identify the new stimulus without the aid of spatial cues.
Tasks evaluated using the computer monitor
Attention task
The monkeys were seated in the WGTA facing a computer screen. They were required to touch the images of apples that appeared on the computer screen as in the pretraining task, but the time intervals between the presentation were randomly generated and varied unpredictably.
Three-choice discrimination task
The animals were then tested on the Three-Choice Discrimination task. Three stimuli were presented on the screen – an orange cross, a brown star, and a pink square. The task was to learn to choose one of three stimuli on the computer screen. The monkey was rewarded with a food pellet for successfully touching the correct stimulus, e.g., the pink square. The animal reached criterion when the correct stimulus was touched ten consecutive times.
Conceptual set shifting task (CSST)
The CSST34 was developed as a test of executive function in the monkey, based on the Wisconsin Card Sorting Test (WCST) that taps executive function in humans. Figure 5. The CSST, like the WCST, is sensitive to damage to the prefrontal cortex. We chose to administer this test in this study because of the strong prefrontal cortex component of the cerebrocerebellar loops, and because of the executive deficits in the CCAS. In the CSST, like the WCST, the monkey was required to choose one feature of a stimulus, such as color, while ignoring other features, such as shape. The animal was provided a food reward each time it chose the correct stimulus feature, until a criterion of ten correct consecutive choices was reached. The stimulus dimension was then changed from the color to shape, and the animal was required to shift set, and recognize that a new sorting principle had been established. After the learning of the initial sorting principle (the red color), there were a total of three consecutive shifts, from Red objects regardless of shape, to Triangles regardless of color, to Blue objects regardless of shape, to Stars regardless of color. The monkey was exposed to 80 trials per day. The outcome variable for the CSST was errors to criterion for each of the four conditions. The nature of the error was recorded, i.e., perseverative error in which the monkey chose a stimulus based on the previously rewarded concept condition, versus a non-perseverative error.
Schematic of the Conceptual Set Shifting Task (CSST)34. The monkey is required to choose one feature of a stimulus, such as color, while ignoring other features, such as shape. When the animal reaches criterion, ten correct consecutive choices, the stimulus dimension is changed from color to shape, and the animal is required to shift set, recognizing the new sorting principle.
Analysis
Each of the behavioral measures in this study was compared between experimental animals and a cohort of normal young monkeys using either mixed-model regression or t-tests P-values were reported to avoid the false rejection of near-significant findings. The 0.05 significance level was used. Post-hoc tests were adjusted for multiple comparisons. The tables and formulae provided by Cohen86 were not used to evaluate the power of the sample size used in the present study because there have been no preliminary studies performed by our group, nor had any similar studies been reported in the literature. However, existing literature of non-human primate lesion studies describes group sizes that range from a low of three monkeys per group to a maximum of eight per group.
To test for effect of the DN lesion on motor performance measured by the Kuypers’ task, latency data were obtained on two monkeys (Cases 2 and 4). Each monkey was tested in two sessions, on separate days, before surgery, and on two additional test days after surgery. The change in latency between pre- and post-surgery sessions was investigated separately for each monkey, using mixed-model regression, with latency as response, period (pre- vs. post-surgery) as a fixed effect, and day as a random effect. Hand use data for the task was also available. For Case 2, we included hand used to retrieve the food pellet and its interaction with period as additional fixed effects. Case 4 used the right hand almost exclusively, to the extent that hand choice could not be included in the regression.
All data analyses were performed using R version 4.2.387. Mixed-model regression was employed for the analyses of the DNMS, DRST, and CSST using the R libraries"lme4"88and"lmerTest"89, adjusting for multiple comparisons using the approach implemented in the"multcomp"library90. For all mixed model regression analyses, hypotheses were tested using likelihood-ratio chi square tests.
On the DNMS the mixed-model regression was used to analyze the effect of group and delay across all conditions. We used two-sample t-tests with Welch’s approximation for degrees of freedom to compare performance of the DN-lesioned and controls animals across each condition.
The DRST analysis used error rate as response, Group (Control vs. Lesion), Type (Spatial vs. Object), Rep ([R] Repeated vs NR [Novel]), and their interaction as fixed effects, and monkey as a random effect.
The CSST analysis error rate as response and Group (Lesion vs. Control) and Type (Red, Tri, Blue, Star), and Perseverative (Y, N) and their interaction as fixed effects, and monkey as a random effect.
We investigated the association between residual dentate nucleus volume in the four lesioned animals and both perseverative and non-perseverative errors on the CSST using mixed-model Poisson regression analyses, with error count (perseverative or non-perseverative) as response. Monkey was modeled as a random effect, and the fixed effects were test type (Blue, Star, Tri) and dentate volume. Hypotheses were tested using analysis of deviance chi-square tests.
Data availability
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
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Acknowledgements
Jason MacMore, Amy S. Hurwitz and Charlene C. DeMong contributed to the acquisition of data in this study. Anna Burt assisted with the incorporation of the statistical data in the graphs and tables. The authors wish to honor and express their gratitude to their mentor Professor Deepak Pandya (1932-2020), the great pioneer of cerebral cortical connectional anatomy, who was steadfast in his support for this line of inquiry into the role of the cerebellum in systems and clinical neuroscience.
This work was first presented in 2004 in plenary session at the Annual Meeting of the American Neurological Association, and at the Annual Meeting of the Society for Neuroscience: Schmahmann JD, Killiany RJ, Moore TL, DeMong C, MacMore JP, Rosene DL, Moss MB. Cerebellar dentate nucleus lesions impair mental flexibility but not motor function in monkeys.
Funding
National Institutes of Mental Health,RO1 MH067980,McDonnell-Pew Program in Cognitive Neuroscience,Aaron J. Berman Neurosurgical Research Fund,Birmingham Foundation
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JDS conceived of the project. JDS, RDK, TLM, MBM, DLR designed the study. JDS, RDK and DLR collected the data aided by the research assistants on the study. MV performed the statistical analyses together with JDS. JDS and DLR wrote the manuscript. All authors discussed the results, contributed to, reviewed and authorized the final manuscript. RDK participated in the project until his untimely passing in 2024.
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This study was approved by the Institutional Animal Care and Use Committee (IACUC) of the Massachusetts General Hospital and of Boston University School of Medicine, and all surgical procedures were performed under aseptic conditions on anesthetized animals in accordance with the NIH Guide for the Care and Use of Laboratory Animals. This study is reported in accordance with ARRIVE guidelines. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
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Schmahmann, J.D., Killiany, R.D., Vangel, M. et al. Cerebellar dentate nucleus lesions in monkey reveal both a motor-cognitive dichotomy and within-cognition dichotomy of dorsal vs ventral stream processing. Sci Rep 15, 32563 (2025). https://doi.org/10.1038/s41598-025-17998-9
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DOI: https://doi.org/10.1038/s41598-025-17998-9
