Activity of the Substantia Nigra Pars Reticulata during Saccade Adaptation.

When movements become inaccurate, the resultant error induces motor adaptation to improve accuracy. This error-based motor learning is regarded as a cerebellar function. However, the influence of the other brain areas on adaptation is poorly understood. During saccade adaptation, a type of error-based motor learning, the superior colliculus (SC) sends a postsaccadic error signal to the cerebellum to drive adaptation. Since the SC is directly inhibited by the substantia nigra pars reticulata (SNr), we hypothesized that the SNr might influence saccade adaptation by affecting the SC error signal. In fact, previous studies indicated that the SNr encodes motivation and motivation influences saccade adaptation. In this study, we first established that the SNr projects to the rostral SC, where small error signals are generated, in nonhuman primates. Then, we examined SNr activity while the animal underwent adaptation. SNr neurons paused their activity in association with the error. This pause was shallower and delayed compared with those of no-error trial saccades. The pause at the end of the adaptation was shallower and delayed compared with that at the beginning of the adaptation. The change in the intertrial interval, an indicator of motivation, and adaptation speed had a positive correlation with the changes in the error-related pause. These results suggest that (1) the SNr exhibits a unique activity pattern during the error interval; (2) SNr activity increases during adaptation, consistent with the decrease in SC activity; and (3) motivational decay during the adaptation session might increase SNr activity and influence the adaptation speed.

Cerebro-cerebellar interactions in nonhuman primates examined by optogenetic functional magnetic resonance imaging.

Functional magnetic resonance imaging (fMRI) is a promising approach for the simultaneous and extensive scanning of whole-brain activities. Optogenetics is free from electrical and magnetic artifacts and is an ideal stimulation method for combined use with fMRI. However, the application of optogenetics in nonhuman primates (NHPs) remains limited. Recently, we developed an efficient optogenetic intracortical microstimulation method of the primary motor cortex (M1), which successfully induced forelimb movements in macaque monkeys. Here, we aimed to investigate how optogenetic M1 stimulation causes neural modulation in the local and remote brain regions in anesthetized monkeys using 7-tesla fMRI. We demonstrated that optogenetic stimulation of the M1 forelimb and hindlimb regions successfully evoked robust direct and remote fMRI activities. Prominent remote activities were detected in the anterior and posterior lobes in the contralateral cerebellum, which receive projections polysynaptically from the M1. We further demonstrated that the cerebro-cerebellar projections from these M1 regions were topographically organized, which is concordant with the somatotopic map in the cerebellar cortex previously reported in macaques and humans. The present study significantly enhances optogenetic fMRI in NHPs, resulting in profound understanding of the brain network, thereby accelerating the translation of findings from animal models to humans.

Elimination of the Cortico-Subthalamic Hyperdirect Pathway Induces Motor Hyperactivity in Mice.

The substantia nigra pars reticulata (SNr) is the output station of the basal ganglia and receives cortical inputs by way of the following three basal ganglia pathways: the cortico-subthalamo (STN)-SNr hyperdirect, the cortico-striato-SNr direct, and the cortico-striato-external pallido-STN-SNr indirect pathways. Compared with the classical direct and indirect pathways via the striatum, the functions of the hyperdirect pathway remain to be fully elucidated. Here we used a photodynamic technique to selectively eliminate the cortico-STN projection in male mice and observed neuronal activity and motor behaviors in awake conditions. After cortico-STN elimination, cortically evoked early excitation in the SNr was diminished, while the cortically evoked inhibition and late excitation, which are delivered through the direct and indirect pathways, respectively, were unchanged. In addition, locomotor activity was significantly increased after bilateral cortico-STN elimination, and apomorphine-induced ipsilateral rotations were observed after unilateral cortico-STN elimination, suggesting that cortical activity was increased. These results are compatible with the notion that the cortico-STN-SNr hyperdirect pathway quickly conveys cortical excitation to the output station of the basal ganglia, resets or suppresses the cortical activity related to ongoing movements, and prepares for the forthcoming movement.SIGNIFICANCE STATEMENT The basal ganglia play a pivotal role in the control of voluntary movements, and their malfunctions lead to movement disorders, such as Parkinson's disease and dystonia. Understanding their functions is important to find better treatments for such diseases. Here we used a photodynamic technique to selectively eliminate the projection from the motor cortex to the subthalamic nucleus, the input station of the basal ganglia, and found greatly reduced early excitatory signals from the cortex to the output station of the basal ganglia and motor hyperactivity. These results suggest that the neuronal signals through the cortico-subthalamic hyperdirect pathway reset or suppress ongoing movements and that blockade of this pathway may be beneficial for Parkinson's disease, which is characterized by oversuppression of movements.

Two-photon imaging of neuronal activity in motor cortex of marmosets during upper-limb movement tasks.

Two-photon imaging in behaving animals has revealed neuronal activities related to behavioral and cognitive function at single-cell resolution. However, marmosets have posed a challenge due to limited success in training on motor tasks. Here we report the development of protocols to train head-fixed common marmosets to perform upper-limb movement tasks and simultaneously perform two-photon imaging. After 2-5 months of training sessions, head-fixed marmosets can control a manipulandum to move a cursor to a target on a screen. We conduct two-photon calcium imaging of layer 2/3 neurons in the motor cortex during this motor task performance, and detect task-relevant activity from multiple neurons at cellular and subcellular resolutions. In a two-target reaching task, some neurons show direction-selective activity over the training days. In a short-term force-field adaptation task, some neurons change their activity when the force field is on. Two-photon calcium imaging in behaving marmosets may become a fundamental technique for determining the spatial organization of the cortical dynamics underlying action and cognition.

Development of stereotaxic recording system for awake marmosets (Callithrix jacchus).

The common marmoset has been proposed as a potential alternative to macaque monkey as a primate model for neuroscience and medical research. Here, we have newly developed a stereotaxic neuronal recording system for awake marmosets under the head-fixed condition by modifying that for macaque monkeys. Using this system, we recorded neuronal activity in the cerebral cortex of awake marmosets and successfully identified the primary motor cortex by intracortical microstimulation. Neuronal activities of deep brain structures, such as the basal ganglia, thalamus, and cerebellum, in awake marmosets were also successfully recorded referring to magnetic resonance images. Our system is suitable for functional mapping of the brain, since the large recording chamber allows access to arbitrary regions over almost the entire brain, and the recording electrode can be easily moved stereotaxically from one site to another. In addition, our system is desirable for neuronal recording during task performance to assess motor skills and cognitive function, as the marmoset sits in the marmoset chair and can freely use its hands. Moreover, our system can be used in combination with cutting-edge techniques, such as two-photon imaging and optogenetic manipulation. This recording system will contribute to boosting neuroscience and medical research using marmosets.

Elucidating information processing in primate basal ganglia circuitry: a novel technique for pathway-selective ablation mediated by immunotoxin.

Employing a neuron-specific retrograde gene-transfer vector (NeuRet vector), we have recently developed a novel technique that achieves pathway-selective ablation in the primate brain. This technique is mediated by immunotoxin (IT) and eliminates a neuronal population that constitutes a particular pathway, leaving other pathways intact. By means of this technique, we have made an attempt to remove the hyperdirect pathway selectively from basal ganglia circuitry. The hyperdirect pathway links the motor cortex to the subthalamic nucleus (STN) directly and plays a crucial role in motor control. After electrical stimulation in the motor cortex, triphasic responses consisting of an early excitation, an inhibition, and a late excitation are usually elicited in the internal pallidal segment (GPi). Several pieces of pharmacophysiological evidence imply that the early excitation may be derived from the hyperdirect pathway. In our experiments, the NeuRet vector expressing human interleukin-2 receptor α-subunit was injected into the STN of macaque monkeys. Then, IT injections were performed into the supplementary motor area (SMA). When single neuron activity in the GPi was recorded in response to the SMA stimulation, it was found that the early excitation was significantly reduced with neither the inhibition nor the late excitation affected. The spontaneous firing rate and pattern of GPi neurons remained to be altered. This clearly indicates that IT-mediated tract targeting successfully eliminated the hyperdirect pathway with spontaneous activity of STN neurons unaffected. The electrophysiological findings were histologically confirmed by retrograde and anterograde neuronal labeling. The overall data define that the motor cortically driven early excitation in GPi neurons is conveyed through the hyperdirect pathway. The IT-mediated pathway-selective ablation technique will provide a powerful tool for elucidating information processing in various neural networks.

Immunotoxin-mediated tract targeting in the primate brain: selective elimination of the cortico-subthalamic "hyperdirect" pathway.

Using a neuron-specific retrograde gene-transfer vector (NeuRet vector), we established immunotoxin (IT)-mediated tract targeting in the primate brain that allows ablation of a neuronal population constituting a particular pathway. Here, we attempted selective removal of the cortico-subthalamic "hyperdirect" pathway. In conjunction with the direct and indirect pathways, the hyperdirect pathway plays a crucial role in motor information processing in the basal ganglia. This pathway links the motor-related areas of the frontal lobe directly to the subthalamic nucleus (STN) without relay at the striatum. After electrical stimulation in the motor-related areas such as the supplementary motor area (SMA), triphasic responses consisting of an early excitation, an inhibition, and a late excitation are usually detected in the internal segment of the globus pallidus (GPi). Several lines of pharmacophysiological evidence suggest that the early excitation may be derived from the hyperdirect pathway. In the present study, the NeuRet vector expressing human interleukin-2 receptor α-subunit was injected into the STN of macaque monkeys. Then, IT injections were made into the SMA. In these monkeys, single-neuron activity in the GPi was recorded in response to the SMA stimulation. We found that the early excitation was largely reduced, with neither the inhibition nor the late excitation affected. The spontaneous firing rate and pattern of GPi neurons remained unchanged. This indicates that IT-mediated tract targeting successfully eliminated the hyperdirect pathway selectively from the basal ganglia circuitry without affecting spontaneous activity of STN neurons. The electrophysiological finding was confirmed with anatomical data obtained from retrograde and anterograde neural tracings. The present results define that the cortically-driven early excitation in GPi neurons is mediated by the hyperdirect pathway. The IT-mediated tract targeting technique will provide us with novel strategies for elucidating various neural network functions.

Increased number of new neurons in the olfactory bulb and hippocampus of adult non-human primates after focal ischemia.

Adult neurogenesis is modulated by growth factors, physical conditions, and other alterations in the physical microenvironment. We studied the effects of focal ischemia on neurogenesis in the subventricular zone (SVZ), olfactory bulb (OB), and hippocampal dentate gyrus (DG) (known to be persistent neurogenic regions) in the adult non-human primate, the cynomolgus monkey. Three monkeys underwent middle cerebral artery occlusion-induced focal ischemia and were given multiple BrdU injections during the first 2 weeks after ischemia. Twenty-eight days later, the animals were perfused. The number of new neurons (3182 +/- 408/mm3) in the ipsilateral DG of ischemic monkeys was 4.7-fold that in the DG of non-operated monkeys. The number of new neurons (9176 +/- 2295/mm3) in the ipsilateral olfactory bulb of ischemic monkeys was 18.0-fold that in normal olfactory bulb. These observations suggest an increase in the number of new OB neurons, as well as new DG neurons, after focal ischemia in a primate. This substantial increase in new neurons after focal ischemia could result from the enhancement of cell proliferation rather than a change in the rate of cell commitment. Of the three monkeys subjected to ischemia, only one animal possessed a unique progenitor cell type at the most anterior aspect of the ipsilateral SVZ. Within this region, a short migration (approximately 500 microm) of doublecortin-expressing immature neuronal progenitor cells was observed.

Successful retrograde transport of fluorescent latex nanospheres in the cerebral cortex of the macaque monkey.

Retrograde axonal transport of latex nanospheres offers a means of delivering chemical agents to a targeted region of the central nervous system (CNS). In this study we performed microinjections of latex nanospheres into the cerebral cortex of cynomolgus monkeys and observed successful retrograde labeling of neurons in the contralateral region. Our data indicate the successful use of this delivery system, reported in studies using other animals, may also be achievable with primates as well.

Nonrenewal of neurons in the cerebral neocortex of adult macaque monkeys.

The concept that, after developmental periods, neocortical neurons become numerically stable and are normally nonrenewable has been challenged by a report of continuous neurogenesis in the association areas of the cerebral cortex in the adult Macaque monkey. Therefore, we have reexamined this issue in two different Macaque species using the thymidine analog bromodeoxyuridine (BrdU) as an indicator of DNA replication during cell division. We found several BrdU+/NeuN+ (neuronal nuclei) double-labeled cells, but cortical neurons, distinguished readily by their size and cytological and immunohistochemical properties, were not BrdU positive. We examined in detail the frontal cortex, where it is claimed that the largest daily addition of neurons has been made, but did not see migratory streams or any sign of addition of new neurons. Thus, we concluded that, in the normal condition, cortical neurons of adult primates, similar to other mammalian species, are neither supplemented nor renewable.