Neuroanatomical StudyCortical and subcortical connections within the pedunculopontine nucleus of the primate Macaca mulatta determined using probabilistic diffusion tractography
Introduction
The pedunculopontine nucleus (PPN) is important in maintaining posture and controlling locomotion.1, 2, 3 Lesions to the PPN cause akinesia4, 5, 6 and degeneration of the PPN has been observed in late-stage Parkinson’s disease.7 Furthermore, low frequency stimulation8 or disinhibition9 of the PPN can dramatically improve akinesia in monkeys made Parkinsonian with 1-methyl, 4-phenyl, 1,2,3,6-tetrahydropyridine. As a result, neurosurgeons have tested whether low frequency deep brain stimulation of the PPN can improve akinesia in Parkinsonian patients and early results have shown great promise for reducing previously intractable akinesia and gait abnormalities.10, 11, 12 Understanding the anatomical connections of the PPN, and in particular, establishing the organisation of cortical and subcortical connections within the PPN, could help to accurately target critical pathways in deep brain stimulation and could also increase our understanding of the role of the PPN in motor control.
PPN connections have been best characterised using tracing and evoked-potential studies in rodents,13, 14, 15, 16, 17, 18, 19 but these connections differ greatly from those shown in monkey tracing studies.3, 20, 21, 22 Although both the rodent and monkey PPN have strong connections with the subthalamic nucleus (STN), substantia nigra pars compacta and pars reticulata, and the internal and external segments of the globus pallidus,1 connections between the PPN and motor cortical areas have been shown only in primates, whereas projections from the PPN to the deep cerebellar nuclei and extending down to the spinal cord have been shown only in rodents. However, these descending PPN projections probably also exist in primates,1, 3, 23 Projections from the deep cerebellar nuclei to the PPN have been observed in squirrel monkeys.24
Information about the somatotopic organisation of PPN connections is much more limited. Until recently, the organisation of PPN connections within the nucleus has been explored in primates only and had been demonstrated for cerebral cortex connections in only one study,23 which showed that more dorsal regions of the primary motor cortex (M1) connected with more lateral areas of the PPN, while ventral regions connected predominantly with the medial PPN. This same relationship was found within subregions of the M1. For example, dorsal portions of the M1 hindlimb and forelimb regions projected laterally into the PPN whereas ventral portions of these M1 subregions projected to the medial PPN. In animal studies, subcortical structures have not been shown to connect preferentially with a PPN region, although efferents from the internal segment of the globus pallidus project throughout the entire PPN in the squirrel monkey.25
The techniques used in the aforementioned studies – anterograde and retrograde tracers, evoked-potential studies, and immunohistochemistry – have several advantages for studying neuroanatomy, especially when combined. They allow for chemically heterogenous neuronal populations to be differentiated, the tracing origin to be confirmed accurately via tissue sectioning and examination of the injection site location, and the projection direction to be determined. Although these tracing methods provide valuable information about how connections function between different brain structures, they also have disadvantages. First, it is difficult to trace the tracts while still obtaining the cellular details of the traced neurons because the tracer signal tends to fade over long fibre distances.26 Newer methods of tract tracing using neurotropic viruses (e.g. rabies) are better at tracing connections between distant structures as the virus signal is amplified in each infected neuron, but these tracing methods depend largely on the injected virus concentration and come with safety issues.27 Second, it is difficult to detect the connections of several different structures within the same brain when each injection site induces some neuronal damage.28 Third, few tracers work well in fixed tissue, and those that do (e.g. lipophilic carbocyanine dyes) diffuse both very slowly through fixed tissue (over several weeks) and over very limited distances.26 Finally, as these procedures are invasive and require post-mortem histological analysis, they clearly cannot be carried out in vivo in humans.
Recently, diffusion tensor imaging has been used to infer anatomical connections in vivo in the human brain.29, 30 Probabilistic diffusion tractography (PDT) enables tracking of probable fibre pathways between cortical and subcortical grey matter structures in vivo.31, 32, 33, 34, 35 However, unlike histological tracing techniques, probabilistic methods for diffusion tractography36, 37 cannot determine the direction of projections between brain regions, nor whether they are interrupted by synapses.
We have used PDT to trace connections of the PPN in healthy humans.34, 35 These results show that PPN connections in humans are similar to those traced in lower primates, including descending connections with the spinal cord. In addition, using PDT, the connections of the human STN and the organisation of these connections within the STN matched what was previously observed in monkeys and rodents.34, 35 However, the organisation of PPN connections differed from what has been described in monkeys. For example, Matsumura et al. reported that the medial PPN connected with the ventral M1 whereas the lateral PPN was connected with the dorsal M1,23 but this did not seem to be the case in humans. Instead, connections with the motor cortex overlapped greatly in the lateral and dorsal PPN. In addition, although no segregation of PPN connections with subcortical regions has been observed in animals, PPN regions connected with subcortical brain structures were significantly segregated from each other.34, 35 These differing results illustrate the importance of verifying the existence of PPN connections across different species using the same methodological techniques.
In this study, we examine the connections of the PPN and the organisation of these connections within the PPN in a post-mortem, fixed rhesus monkey brain using PDT. To demonstrate the use of PDT as an accurate anatomical tracing technique, we compare our results to those of previous anatomical tracer and evoked-potential studies. We chose to use a fixed brain for our analyses so we could determine whether our PDT results were directly comparable to results from other anatomical tracing studies that are generally done in fresh or fixed post-mortem brains. We also compare the organisation of rhesus monkey PPN connections with those we obtained previously using PDT in vivo in humans34, 35 to determine differences in the role of the PPN across species.
Section snippets
Brain extraction and fixation
One male rhesus monkey (Macaca mulatta, 9 years old) was used in this experiment. The studies were conducted under project and personal licences issued by the British Home Office and in accordance with the British Animal Scientific Procedures Act (1986). The animal was deeply anesthetised with a lethal dose of sodium pentobarbitone. Immediately after cardiac arrest, the animal was perfused through the heart with 90% normal saline and 10% formalin. The brain was removed whole and kept in 10%
Connections of the PPN
The PPN had connections with the cerebral cortex and basal ganglia but not with the cerebellum or spinal cord (Table 1, Fig. 4, Fig. 5). The cortical regions that showed connections with the right PPN were the dorsal premotor cortex, supplementary motor area, and with all M1 regions (Fig. 4, Fig. 5), although only the dorsal premotor cortex, M1 wrist and hand, and M1 orofacial regions showed connections with the left PPN as well (Fig. 5). Subcortical connections of both the left and right PPN
Discussion
We have shown that PDT can be used to probe the anatomical connections of the primate in a fixed post-mortem rhesus monkey brain. The PPN exhibited connections with the supplementary motor cortex, dorsal premotor cortex, and all M1 regions, although connections with the dorsal premotor cortex and the M1 wrist, hand, and orofacial regions were most robust. The PPN was also connected with the thalamus, globus pallidus, substantia nigra, and STN. No connections between the PPN and the cerebellum
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Cited by (26)
Gait control by the frontal lobe
2023, Handbook of Clinical NeurologyNon-motor connections of the pedunculopontine nucleus of the rat and human brain
2022, Neuroscience LettersA Causal Role for the Pedunculopontine Nucleus in Human Instrumental Learning
2021, Current BiologyCitation Excerpt :The next question is how reward-related information reaches the PPN in the first place. There are plenty of possibilities, given the richness of afferent inputs to the PPN established in non-human primates,68,74–76 coming from both the basal ganglia (such as the globus pallidus and subthalamic nucleus) and cortical areas (such as the ventromedial prefrontal and anterior cingulate cortex). Another remaining question is whether the PPN reward signals that we observed here in the human brain are indeed conveyed to dopamine neurons.
Structure and function of the mesencephalic locomotor region in normal and parkinsonian primates
2019, Current Opinion in Physiology