Masaharu Kinoshita

Genetic dissection of the circuit for hand dexterity in primates

It is generally accepted that the direct connection from the motor cortex to spinal motor neurons is responsible for dexterous hand movements in primates. However, the role of the ‘phylogenetically older’ indirect pathways from the motor cortex to motor neurons, mediated by spinal interneurons, remains elusive. Here we used a novel double-infection technique to interrupt the transmission through the propriospinal neurons (PNs), which act as a relay of the indirect pathway in macaque monkeys (Macaca fuscata and Macaca mulatta). The PNs were double infected by injection of a highly efficient retrograde gene-transfer vector into their target area and subsequent injection of adeno-associated viral vector at the location of cell somata. This method enabled reversible expression of green fluorescent protein (GFP)-tagged tetanus neurotoxin, thereby permitting the selective and temporal blockade of the motor cortex–PN–motor neuron pathway. This treatment impaired reach and grasp movements, revealing a critical role for the PN-mediated pathway in the control of hand dexterity. Anti-GFP immunohistochemistry visualized the cell bodies and axonal trajectories of the blocked PNs, which confirmed their anatomical connection to motor neurons. This pathway-selective and reversible technique for blocking neural transmission does not depend on cell-specific promoters or transgenic techniques, and is a new and powerful tool for functional dissection in system-level neuroscience studies.

Comparative analyses of adeno-associated viral vector serotypes 1, 2, 5, 8 and 9 in marmoset, mouse and macaque cerebral cortex.

Here we investigated the transduction characteristics of adeno-associated viral vector (AAV) serotypes 1, 2, 5, 8 and 9 in the marmoset cerebral cortex. Using three constructs that each has hrGFP under ubiquitous (CMV), or neuron-specific (CaMKII and Synapsin I (SynI)) promoters, we investigated (1) the extent of viral spread, (2) cell type tropism, and (3) neuronal transduction efficiency of each serotype. AAV2 was clearly distinct from other serotypes in small spreading and neuronal tropism. We did not observe significant differences in viral spread among other serotypes. Regarding the cell tropism, AAV1, 5, 8 and 9 exhibited mostly glial expression for CMV construct. However, when the CaMKII construct was tested, cortical neurons were efficiently transduced (>∼70% in layer 3) by all serotypes, suggesting that glial expression obscured neuronal expression for CMV construct. For both SynI and CaMKII constructs, we observed generally high-level expression in large pyramidal cells especially in layer 5, as well as in parvalbumin-positive interneurons. The expression from the CaMKII construct was more uniformly observed in excitatory cells compared with SynI construct. Injection of the same viral preparations in mouse and macaque cortex resulted in essentially the same result with some species-specific differences.

Perceptual filling-in at the scotoma following a monocular retinal lesion in the monkey

Although no visual inputs arise from the blind spot, the same visual attribute there as in the visual field surrounding the blind spot is perceived. Because of this remarkable perceptual filling-in, a hole corresponding to the blind spot is not perceived, even when one eye is closed. Does the same phenomenon occur in the case of a scotoma in which visual inputs are lost postnatally due to a retinal lesion? We report that it did: in the macaque monkey, behavioral evidence for filling-in at a scotoma produced by a laser-induced monocular retinal lesion was obtained. The visual receptive fields of neurons in the primary visual cortex (V1) in and around the representation of the visual field corresponding to the scotoma were also mapped, and no clear difference between the retinotopic organization of this part in V1 and that found in the normal visual field was found. Also, perceptual filling-in was found to occur only two days after the lesion. These findings suggest that the normal visual system possesses a mechanism that yields filling-in when some part of the retina is damaged, and that such a mechanism requires no topographical reorganization in V1.

Surface representation in the visual system

Perception of surface accompanies the impression that a certain area of the visual field is occupied by some quality, such as color, brightness and transparency. This does not mean, however, that information about surface quality must be obtained throughout the area. It has been shown in many situations that our visual system has ability to interpolate information obtained at the border of the surface and to perceive homogeneous surfaces. The most dramatic demonstration of this is the perceptual filling-in at the blind spot. In order to understand the neural representation of surface in the visual system, we conducted a series of experiments using macaque monkeys. First, we examined if neurons in the primary visual cortex (V1) respond when a homogeneous surface is presented on the receptive field. Neurons representing the parafoveal visual field were tested and it was found that about one third of neurons showed significant responses when the cells receptive field was contained in a homogeneous surface. Then we examined neuron activities in the retinotopic representation of the blind spot in V1. Although there is no retinal input in the blind spot, a homogeneous surface is perceived within the blind spot as a result of filling-in. We tested whether neurons in this region were activated when a homogeneous surface was perceived in the blind spot as a result of filling-in. We found some neurons in V1 were activated by stimuli which lead to the filling-in. These results indicate that when a surface area is perceived, neurons are activated throughout the region in V1 topographically corresponding to the perceived surface and not restricted to the region representing the border of the surface.

Neural responses in the primary visual cortex of the monkey during perceptual filling-in at the blind spot.

The phenomenon of perceptual filling-in demonstrates that physical stimuli presented on the retina do not necessarily correspond to surface perception, and that our visual system has mechanisms with which to interpolate missing information in order to construct continuous surfaces. Among its various forms, filling-in at the blind spot is one of the most remarkable. To study the neural mechanisms involved in filling-in at the blind spot, we recently conducted a recording experiment aimed at determining whether the neurons in the primary visual cortex (V1) that represent the visual field corresponding to the blind spot are activated when filling-in occurs. We found that neurons located in deep layers of the V1, particularly layer 6, respond to large stimuli that cover the blind spot and induce perceptual filling-in. These neurons tended to have very large receptive fields, which extended out of the blind spot, and preferred relatively large stimuli. We believe that neurons in the V1 region representing the blind spot encode information essential for perceptual filling-in at the blind spot.

Role of Direct vs. Indirect Pathways from the Motor Cortex to Spinal Motoneurons in the Control of Hand Dexterity.

Evolutionally, development of the direct connection from the motor cortex to spinal motoneurons [corticomotoneuronal (CM) pathway] parallels the ability of hand dexterity. Damage to the corticofugal fibers in higher primates resulted in deficit of fractionated digit movements. Based on such observations, it was generally believed that the CM pathway plays a critical role in the control of hand dexterity. On the other hand, a number of “phylogenetically older” indirect pathways from the motor cortex to motoneurons still exist in primates. The indirect pathways are mediated by intercalated neurons such as segmental interneurons (sINs), propriospinal neurons (PNs) reticulospinal neurons (RSNs), or rubrospinal neurons (RuSNs). However, their contribution to hand dexterity remains elusive. Lesion of the brainstem pyramid sparing the transmission through the RuSNs and RSNs, resulted in permanent deficit of fractionated digit movements in macaque monkeys. On the other hand, in our recent study, after lesion of the dorsolateral funiculus (DLF) at the C5 segment, which removed the lateral corticospinal tract (l-CST) including the CM pathway and the transmission through sINs and RuSNs but spared the processing through the PNs and RSNs, fractionated digit movements recovered within several weeks. These results suggest that the PNs can be involved in the recovery of fractionated digit movements, but the RSNs and RuSNs have less capacity in this regard. However, on closer inspection, it was found that the activation pattern of hand and arm muscles considerably changed after the C5 lesion, suggesting limitation of PNs for the compensation of hand dexterity. Altogether, it is suggested that PNs, RSNs RuSNs, and the CM pathway (plus sINs) make a different contribution to the hand dexterity and appearance of motor deficit of the hand dexterity caused by damage to the corticofugal fibers and potential of recovery varies depending on the rostrocaudal level of the lesion.

Optical Imaging of Contextual Interactions in V1 of the Behaving Monkey

Interactions in primary visual cortex (V1) between simple visual elements such as short bar segments are believed to underlie our ability to easily integrate contours and segment surfaces. We used intrinsic signal optical imaging in alert fixating macaques to measure the strength and cortical distribution of V1 interactions among collinear bars. A single short bar stimulus produced a broad-peaked hill of activation (the optical point spread) covering multiple orientation hypercolumns in V1. Flanking the bar stimulus with a pair of identical collinear bars led to a strong nonlinear suppression in the optical signal. This nonlinearity was strongest over the center bar region, with a spatial distribution that cannot be explained by a simple gain control. It was a function of the relative orientation and separation of the bar stimuli in a manner tuned sharply for collinearity, being strongest for immediately adjacent bars lying on a smooth contour. These results suggest intracortical interactions playing a major role in determining V1 activation by smooth extended contours. Our finding that the interaction is primarily suppressive when imaged optically, which presumably reflects the combined inhibitory and excitatory inputs, suggests a complex interplay between these cortical inputs leading to the collinear facilitation seen in the spiking response of V1 neurons. This disjuncture between the facilitation seen in spiking and the suppression in imaging also suggests that cortical representations of complex stimuli involve interactions that need to be studied over extended networks and may be hard to deduce from the responses of individual neurons.

Viral vector-mediated selective and reversible blockade of the pathway for visual orienting in mice

Recently, by using a combination of two viral vectors, we developed a technique for pathway-selective and reversible synaptic transmission blockade, and successfully induced a behavioral deficit of dexterous hand movements in macaque monkeys by affecting a population of spinal interneurons. To explore the capacity of this technique to work in other pathways and species, and to obtain fundamental methodological information, we tried to block the crossed tecto-reticular pathway, which is known to control orienting responses to visual targets, in mice. A neuron-specific retrograde gene transfer vector with the gene encoding enhanced tetanus neurotoxin (eTeNT) tagged with enhanced green fluorescent protein (EGFP) under the control of a tetracycline responsive element was injected into the left medial pontine reticular formation. 7–17 days later, an adeno-associated viral vector with a highly efficient Tet-ON sequence, rtTAV16, was injected into the right superior colliculus. 5–9 weeks later, the daily administration of doxycycline (Dox) was initiated. Visual orienting responses toward the left side were impaired 1–4 days after Dox administration. Anti-GFP immunohistochemistry revealed that a number of neurons in the intermediate and deep layers of the right superior colliculus were positively stained, indicating eTeNT expression. After the termination of Dox administration, the anti-GFP staining returned to the baseline level within 28 days. A second round of Dox administration, starting from 28 days after the termination of the first Dox administration, resulted in the reappearance of the behavioral impairment. These findings showed that pathway-selective and reversible blockade of synaptic transmission also causes behavioral effects in rodents, and that the crossed tecto-reticular pathway clearly controls visual orienting behaviors.

Contribution of propriospinal neurons to recovery of hand dexterity after corticospinal tract lesions in monkeys

Significance There are different views about the targets of regenerative therapies to induce functional recovery in patients with motor paralysis following brain and spinal cord injury: whether we should aim at repairing the injured corticospinal tract or at facilitating compensation by other descending motor pathways. To help answer this question, we used double viral vectors to reversibly and selectively block the propriospinal neurons (PNs), one of the major intercalated neurons mediating cortical commands to motoneurons, in monkeys with partial spinal cord injury. We demonstrated causal roles of the PN-mediated pathway in promoting recovery of hand dexterity after the lesion. Thus, targeting the PNs might lead to developing effective treatment to facilitate recovery after spinal cord injury. The direct cortico-motoneuronal connection is believed to be essential for the control of dexterous hand movements, such as precision grip in primates. It was reported, however, that even after lesion of the corticospinal tract (CST) at the C4–C5 segment, precision grip largely recovered within 1–3 mo, suggesting that the recovery depends on transmission through intercalated neurons rostral to the lesion, such as the propriospinal neurons (PNs) in the midcervical segments. To obtain direct evidence for the contribution of PNs to recovery after CST lesion, we applied a pathway-selective and reversible blocking method using double viral vectors to the PNs in six monkeys after CST lesions at C4–C5. In four monkeys that showed nearly full or partial recovery, transient blockade of PN transmission after recovery caused partial impairment of precision grip. In the other two monkeys, CST lesions were made under continuous blockade of PN transmission that outlasted the entire period of postoperative observation (3–4.5 mo). In these monkeys, precision grip recovery was not achieved. These results provide evidence for causal contribution of the PNs to recovery of hand dexterity after CST lesions; PN transmission is necessary for promoting the initial stage recovery; however, their contribution is only partial once the recovery is achieved.

DNA methylation and methyl-binding proteins control differential gene expression in distinct cortical areas of macaque monkey.

Distinct anatomical regions of the neocortex subserve different sensory modalities and neuronal integration functions, but mechanisms for these regional specializations remain elusive. Involvement of epigenetic mechanisms for such specialization through the spatiotemporal regulation of gene expression is an intriguing possibility. Here we examined whether epigenetic mechanisms might play a role in the selective gene expression in the association areas (AAs) and the primary visual cortex (V1) in macaque neocortex. By analyzing the two types of area-selective gene promoters that we previously identified, we found a striking difference of DNA methylation between these promoters, i.e., hypermethylation in AA-selective gene promoters and hypomethylation in V1-selective ones. Methylation levels of promoters of each area-selective gene showed no areal difference, but a specific methyl-binding protein (MBD4) was enriched in the AAs, in correspondence with expression patterns of AA-selective genes. MBD4 expression was mainly observed in neurons. MBD4 specifically bound to and activated the AA-selective genes both in vitro and in vivo. Our results demonstrate that methylation in the promoters and specific methyl-binding proteins play an important role in the area-selective gene expression profiles in the primate neocortex.