Reorganization of Corticospinal Projections after Prominent Recovery of Finger Dexterity from Partial Spinal Cord Injury in Macaque Monkeys.

We investigated morphologic changes in the corticospinal tract (CST) to understand the mechanism underlying recovery of hand function after lesion of the CST at the C4/C5 border in seven macaque monkeys. All monkeys exhibited prominent recovery of precision grip success ratio within a few months. The trajectories and terminals of CST from the contralesional (n = 4) and ipsilesional (n = 3) hand area of primary motor cortex (M1) were investigated at 5-29 months after the injury using an anterograde neural tracer, biotinylated dextran amine (BDA). Reorganization of the CST was assessed by counting the number of BDA-labeled axons and bouton-like swellings in the gray and white matters. Rostral to the lesion (at C3), the number of axon collaterals of the descending axons from both contralesional and ipsilesional M1 entering the ipsilesional and contralesional gray matter, respectively, were increased. Caudal to the lesion (at C8), axons originating from the contralesional M1, descending in the preserved gray matter around the lesion, and terminating in ipsilesional Laminae VI/VII and IX were observed. In addition, axons and terminals from the ipsilesional M1 increased in the ipsilesional Lamina IX after recrossing the midline, which were not observed in intact monkeys. Conversely, axons originating from the ipsilesional M1 and directed toward the contralesional Lamina VII decreased. These results suggest that multiple reorganizations of the corticospinal projections to spinal segments both rostral and caudal to the lesion originating from bilateral M1 underlie a prominent recovery in long-term after spinal cord injury.

Emergence of an erythroid cell-specific regulatory region in ABO intron 1 attributable to A- or B-antigen expression on erythrocytes in Hominoidea.

A- and B-antigens are present on red blood cells (RBCs) as well as other cells and secretions in Hominoidea including humans and apes such as chimpanzees and gibbons, whereas expression of these antigens on RBCs is subtle in monkeys such as Japanese macaques. Previous studies have indicated that H-antigen expression has not completely developed on RBCs in monkeys. Such antigen expression requires the presence of H-antigen and A- or B-transferase expression in cells of erythroid lineage, although whether or not ABO gene regulation is associated with the difference of A- or B-antigen expression between Hominoidea and monkeys has not been examined. Since it has been suggested that ABO expression on human erythrocytes is dependent upon an erythroid cell-specific regulatory region or the + 5.8-kb site in intron 1, we compared the sequences of ABO intron 1 among non-human primates, and demonstrated the presence of sites orthologous to the + 5.8-kb site in chimpanzees and gibbons, and their absence in Japanese macaques. In addition, luciferase assays revealed that the former orthologues enhanced promoter activity, whereas the corresponding site in the latter did not. These results suggested that the A- or B-antigens on RBCs might be ascribed to emergence of the + 5.8-kb site or the corresponding regions in ABO through genetic evolution.

Diurnal variation in declarative memory and the involvement of SCOP in cognitive functions in nonhuman primates.

Cognitive functions depend on the time of day in various organisms. Previously, we found that 24-h recognition memory performance of nocturnal mice changes diurnally through SCOP protein-dependent regulation. It remains unknown whether diurnal change and SCOP-dependent regulation of memory performance are conserved across species with diurnal/nocturnal habits. We tested whether the memory performance of diurnal Japanese macaques depends on the time of day. The memory association between bitter taste of drinking water and the nozzle color of the water bottle was established during the light period of the day to evaluate of memory performance for macaques. Here we found diurnal variation of declarative memory in Japanese macaques. The middle of the daytime is the most effective time for memory performance during the light period. To assess whether SCOP is involved in declarative memory performance, we interfered with SCOP expression by using lentiviral vector expressing shRNA against Scop in the hippocampus of Japanese macaques. Scop knockdown in the hippocampus abrogated the memory performance in the middle of the daytime. Our results implicate that SCOP in the hippocampus is necessary for the diurnal rhythm of the memory system and that the SCOP-dependent memory regulation system may be conserved in mammals.

Origin of Multisynaptic Corticospinal Pathway to Forelimb Segments in Macaques and Its Reorganization After Spinal Cord Injury.

Removal of the monosynaptic corticospinal pathway (CSP) terminating within the forelimb segments severely impairs manual dexterity. Functional recovery from the monosynaptic CSP lesion can be achieved through the remaining multisynaptic CSP toward the forelimb segments. In the present study, we applied retrograde transsynaptic labeling with rabies virus to a monkey model of spinal cord injury. By injecting the virus into the spinal forelimb segments immediately after the monosynaptic CSP lesion, we showed that the contralateral primary motor cortex (M1), especially its caudal and bank region (so-called "new" M1), was the principal origin of the CSP linking the motor cortex to the spinal forelimb segments disynaptically (disynaptic CSP). This forms a striking contrast to the architecture of the monosynaptic CSP that involves extensively other motor-related areas, together with M1. Next, the rabies injections were made at the recovery period of 3 months after the monosynaptic CSP lesion. The second-order labeled neurons were located in the ipsilateral as well as in the contralateral "new" M1. This indicates that the disynaptic CSP input from the ipsilateral "new" M1 is recruited during the motor recovery from the monosynaptic CSP lesion. Our results suggest that the disynaptic CSP is reorganized to connect the ipsilateral "new" M1 to the forelimb motoneurons for functional compensation after the monosynaptic CSP lesion.

[Species Differences in the Brain between Human and Non-Human Primates].

Cognitive abilities of humans are superior to that of non-human primates (NHP). Differences in genes, gene expression, cellular composition and response, neural circuit, and brain volume between humans and NHP underlying cognitive abilities are discussed, with a focus on cortical expansion, prefrontal expansion, language-related areas, and the adaptation of longer life spans in humans.

Neuronal and microglial localization of secreted phosphoprotein 1 (osteopontin) in intact and damaged motor cortex of macaques.

We previously reported that mRNA encoding secreted phosphoprotein 1 (SPP1), also known as osteopontin, is preferentially expressed in large neurons in layer V of the macaque motor cortex, most of which are presumed to be corticospinal tract neurons. As a first step to elucidating the cellular function of SPP1 in macaque neurons, we examined the localization of SPP1 in the primary motor cortex (M1) of the macaque by using immunohistochemistry. SPP1 immunoreactivity was found to be localized in the cell bodies of neurons, but not outside the cells, indicating that SPP1 was not secreted from these neurons. The results of electron microscope analysis and double-labeling analysis with marker proteins suggested that SPP1 was localized in the mitochondria of neurons. The distributions of SPP1 in the neurons corresponded to those of integrin αV, a putative receptor for SPP1. The distribution of SPP1 was also investigated in macaques whose M1 had been lesioned. We found that SPP1 was secreted by proliferated microglia in the lesioned area. Double-labeling analysis indicated that SPP1 immunoreactivity in the microglia was colocalized with CD44, another putative receptor for SPP1. Success rates in the small-object-retrieval task were positively correlated with SPP1 immunoreactivity in the neurons in the perilesional area. SPP1 has multiple roles in the macaque motor cortex, and it may be a key protein during recovery of hand movement after brain damage.

Human-specific features of spatial gene expression and regulation in eight brain regions.

Molecular maps of the human brain alone do not inform us of the features unique to humans. Yet, the identification of these features is important for understanding both the evolution and nature of human cognition. Here, we approached this question by analyzing gene expression and H3K27ac chromatin modification data collected in eight brain regions of humans, chimpanzees, gorillas, a gibbon, and macaques. An analysis of spatial transcriptome trajectories across eight brain regions in four primate species revealed 1851 genes showing human-specific transcriptome differences in one or multiple brain regions, in contrast to 240 chimpanzee-specific differences. More than half of these human-specific differences represented elevated expression of genes enriched in neuronal and astrocytic markers in the human hippocampus, whereas the rest were enriched in microglial markers and displayed human-specific expression in several frontal cortical regions and the cerebellum. An analysis of the predicted regulatory interactions driving these differences revealed the role of transcription factors in species-specific transcriptome changes, and epigenetic modifications were linked to spatial expression differences conserved across species.

Comprehensive analysis of area-specific and time-dependent changes in gene expression in the motor cortex of macaque monkeys during recovery from spinal cord injury.

The present study aimed to assess the molecular bases of cortical compensatory mechanisms following spinal cord injury in primates. To accomplish this, comprehensive changes in gene expression were investigated in the bilateral primary motor cortex (M1), dorsal premotor cortex (PMd), and ventral premotor cortex (PMv) after a unilateral lesion of the lateral corticospinal tract (l-CST). At 2 weeks after the lesion, a large number of genes exhibited altered expression levels in the contralesional M1, which is directly linked to the lesioned l-CST. Gene ontology and network analyses indicated that these changes in gene expression are involved in the atrophy and plasticity changes observed in neurons. Orchestrated gene expression changes were present when behavioral recovery was attained 3 months after the lesion, particularly among the bilateral premotor areas, and a large number of these genes are involved in plasticity. Moreover, several genes abundantly expressed in M1 of intact monkeys were upregulated in both the PMd and PMv after the l-CST lesion. These area-specific and time-dependent changes in gene expression may underlie the molecular mechanisms of functional recovery following a lesion of the l-CST.

Co-Opted Megasatellite DNA Drives Evolution of Secondary Night Vision in Azara's Owl Monkey.

Owl monkeys (genus Aotus) are the only taxon in simian primates that consists of nocturnal or otherwise cathemeral species. Their night vision is superior to that of other monkeys, apes, and humans but not as good as that of typical nocturnal mammals. This incomplete night vision has been used to conclude that these monkeys only secondarily adapted to a nocturnal lifestyle, or to their cathemeral lifestyle that involves high night-time activity. It is known that the rod cells of many nocturnal mammals possess a unique nuclear architecture in which heterochromatin is centrally located. This "inverted nuclear architecture", in contrast with "conventional nuclear architecture", provides elevated night vision by passing light efficiently to the outer segments of photoreceptors. Owl monkey rod cells exhibit an intermediate chromatin distribution, which may provide them with less efficient night vision than other nocturnal mammals. Recently, we identified three megasatellite DNAs in the genome of Azara's owl monkey (Aotus azarae). In the present study, we show that one of the three megasatellite DNAs, OwlRep, serves as the primary component of the heterochromatin block located in the central space of the rod nucleus in A. azarae. This satellite DNA is likely to have emerged in the Aotus lineage after its divergence from those of other platyrrhini taxa and underwent a rapid expansion in the genome. Our results indicate that the heterochromatin core in the A. azarae rod nucleus was newly formed in A. azarae or its recent ancestor, and supports the hypothesis that A. azarae, and with all probability other Aotus species, secondarily acquired night vision.

Evolutionary acquisition of promoter-associated non-coding RNA (pancRNA) repertoires diversifies species-dependent gene activation mechanisms in mammals.

BACKGROUND: Recent transcriptome analyses have shown that long non-coding RNAs (ncRNAs) play extensive roles in transcriptional regulation. In particular, we have reported that promoter-associated ncRNAs (pancRNAs) activate the partner gene expression via local epigenetic changes. RESULTS: Here, we identify thousands of genes under pancRNA-mediated transcriptional activation in five mammalian species in common. In the mouse, 1) pancRNA-partnered genes confined their expression pattern to certain tissues compared to pancRNA-lacking genes, 2) expression of pancRNAs was significantly correlated with the enrichment of active chromatin marks, H3K4 trimethylation and H3K27 acetylation, at the promoter regions of the partner genes, 3) H3K4me1 marked the pancRNA-partnered genes regardless of their expression level, and 4) C- or G-skewed motifs were exclusively overrepresented between-200 and-1 bp relative to the transcription start sites of the pancRNA-partnered genes. More importantly, the comparative transcriptome analysis among five different mammalian species using a total of 25 counterpart tissues showed that the overall pancRNA expression profile exhibited extremely high species-specificity compared to that of total mRNA, suggesting that interspecies difference in pancRNA repertoires might lead to the diversification of mRNA expression profiles. CONCLUSIONS: The present study raises the interesting possibility that the gain and/or loss of gene-activation-associated pancRNA repertoires, caused by formation or disruption of the genomic GC-skewed structure in the course of evolution, finely shape the tissue-specific pattern of gene expression according to a given species.

Increased expression of the growth-associated protein-43 gene after primary motor cortex lesion in macaque monkeys.

We recently showed that changes of brain activity in the ipsilesional ventral premotor cortex (PMv) and perilesional primary motor cortex (M1) of macaque monkeys were responsible for recovery of manual dexterity after lesioning M1. To investigate whether axonal remodeling is associated with M1 lesion-induced changes in brain activity, we assessed gene expression of growth-associated protein-43 (GAP-43) in motor and premotor cortices. Increased expression was observed in the PMv during the period just after recovery and in the perilesional M1 during the plateau phase of recovery. Time-dependent and brain region-specific remodeling may play a role in functional recovery after lesioning M1.

Temporal plasticity involved in recovery from manual dexterity deficit after motor cortex lesion in macaque monkeys.

The question of how intensive motor training restores motor function after brain damage or stroke remains unresolved. Here we show that the ipsilesional ventral premotor cortex (PMv) and perilesional primary motor cortex (M1) of rhesus macaque monkeys are involved in the recovery of manual dexterity after a lesion of M1. A focal lesion of the hand digit area in M1 was made by means of ibotenic acid injection. This lesion initially caused flaccid paralysis in the contralateral hand but was followed by functional recovery of hand movements, including precision grip, during the course of daily postlesion motor training. Brain imaging of regional cerebral blood flow by means of H2 (15)O-positron emission tomography revealed enhanced activity of the PMv during the early postrecovery period and increased functional connectivity within M1 during the late postrecovery period. The causal role of these areas in motor recovery was confirmed by means of pharmacological inactivation by muscimol during the different recovery periods. These findings indicate that, in both the remaining primary motor and premotor cortical areas, time-dependent plastic changes in neural activity and connectivity are involved in functional recovery from the motor deficit caused by the M1 lesion. Therefore, it is likely that the PMv, an area distant from the core of the lesion, plays an important role during the early postrecovery period, whereas the perilesional M1 contributes to functional recovery especially during the late postrecovery period.

Sporadic premature aging in a Japanese monkey: a primate model for progeria.

In our institute, we have recently found a child Japanese monkey who is characterized by deep wrinkles of the skin and cataract of bilateral eyes. Numbers of analyses were performed to identify symptoms representing different aspects of aging. In this monkey, the cell cycle of fibroblasts at early passage was significantly extended as compared to a normal control. Moreover, both the appearance of senescent cells and the deficiency in DNA repair were observed. Also, pathological examination showed that this monkey has poikiloderma with superficial telangiectasia, and biochemical assay confirmed that levels of HbA1c and urinary hyaluronan were higher than those of other (child, adult, and aged) monkey groups. Of particular interest was that our MRI analysis revealed expansion of the cerebral sulci and lateral ventricles probably due to shrinkage of the cerebral cortex and the hippocampus. In addition, the conduction velocity of a peripheral sensory but not motor nerve was lower than in adult and child monkeys, and as low as in aged monkeys. However, we could not detect any individual-unique mutations of known genes responsible for major progeroid syndromes. The present results indicate that the monkey suffers from a kind of progeria that is not necessarily typical to human progeroid syndromes.

Age-related decrease of the phosphorus content in the ligamentum capitis femoris of monkeys.

To elucidate compositional changes of the ligament with aging, the authors investigated age-related changes of elements in the ligamentum capitis femoris (LCF) of monkeys with a wide range of ages by direct chemical analysis. Used rhesus and Japanese monkeys consisted of 9 males and 22 females, ranging in age from newborn to 31 years (average age = 10.4 ± 10.9 years). After incineration with nitric acid and perchloric acid, element contents were determined by inductively coupled plasma-atomic emission spectrometry. It was found that the P content decreased significantly in the LCFs of monkeys with aging, but other six element contents, Ca, S, Mg, Zn, Fe, and Na, did not change significantly with aging. Assuming that the P content indicated the active cell density and the S content indicated the protein amount, an age-related change of the mass ratio of P/S was examined in the LCFs. The mass ratio of P/S decreased significantly in the LCFs in childhood. Regarding the relationships among elements, significant direct correlations were found among the Ca, P, S, and Mg contents in the LCFs. It was suggested that the active cell density of the connective tissue cells might decrease significantly in the LCF in childhood.

Differential expression of secreted phosphoprotein 1 in the motor cortex among primate species and during postnatal development and functional recovery.

We previously reported that secreted phosphoprotein 1 (SPP1) mRNA is expressed in neurons whose axons form the corticospinal tract (CST) of the rhesus macaque, but not in the corresponding neurons of the marmoset and rat. This suggests that SPP1 expression is involved in the functional or structural specialization of highly developed corticospinal systems in certain primate species. To further examine this hypothesis, we evaluated the expression of SPP1 mRNA in the motor cortex from three viewpoints: species differences, postnatal development, and functional/structural changes of the CST after a lesion of the lateral CST (l-CST) at the mid-cervical level. The density of SPP1-positive neurons in layer V of the primary motor cortex (M1) was much greater in species with highly developed corticospinal systems (i.e., rhesus macaque, capuchin monkey, and humans) than in those with less developed corticospinal systems (i.e., squirrel monkey, marmoset, and rat). SPP1-positive neurons in the macaque monkey M1 increased logarithmically in layer V during postnatal development, following a time course consistent with the increase in conduction velocity of the CST. After an l-CST lesion, SPP1-positive neurons increased in layer V of the ventral premotor cortex, in which compensatory changes in CST function/structure may occur, which positively correlated with the extent of finger dexterity recovery. These results further support the concept that the expression of SPP1 may reflect functional or structural specialization of highly developed corticospinal systems in certain primate species.

Effects of early versus late rehabilitative training on manual dexterity after corticospinal tract lesion in macaque monkeys.

Dexterous hand movements can be restored with motor rehabilitative training after a lesion of the lateral corticospinal tract (l-CST) in macaque monkeys. To maximize effectiveness, the optimal time to commence such rehabilitative training must be determined. We conducted behavioral analyses and compared the recovery of dexterous hand movements between monkeys in which hand motor training was initiated immediately after the l-CST lesion (early-trained monkeys) and those in which training was initiated 1 mo after the lesion (late-trained monkeys). The performance of dexterous hand movements was evaluated by food retrieval tasks. In early-trained monkeys, performance evaluated by the success rate in a vertical slit task (retrieval of a small piece of food through a narrow vertical slit) recovered to the level of intact monkeys during the first 1-2 mo after the lesion. In late-trained monkeys, the task success rate averaged ∼30% even after 3 mo of rehabilitative training. We also evaluated hand performance with the Klüver board task, in which monkeys retrieved small spherical food pellets from cylindrical wells. Although the success rate of the Klüver board task did not differ between early- and late-trained monkeys, kinematic movement analysis showed that there was a difference between the groups: late-trained monkeys with an improved success rate frequently used alternate movement strategies that were different from those used before the lesion. These results suggest that early rehabilitative training after a spinal cord lesion positively influences subsequent functional recovery.

Functional annotation of genes differentially expressed between primary motor and prefrontal association cortices of macaque brain.

DNA microarray-based genome-wide transcriptional profiling and gene network analyses were used to characterize the molecular underpinnings of the neocortical organization in rhesus macaque, with particular focus on the differences in the functional annotation of genes in the primary motor cortex (M1) and the prefrontal association cortex (area 46 of Brodmann). Functional annotation of the differentially expressed genes showed that the list of genes selectively expressed in M1 was enriched with genes involved in oligodendrocyte function, and energy consumption. The annotation appears to have successfully extracted the characteristics of the molecular structure of M1.

Ultrasound safety with midfrequency transcranial sonothrombolysis: preliminary study on normal macaca monkey brain.

We investigated the safety of transcranial-targeting midfrequency (0.1 to 1 MHz) ultrasonic thrombolysis for acute ischemic stroke. We applied a new therapeutic and imaging transducer to healthy Macaca monkey brains via sonication of the ipsilateral middle cerebral artery through an acoustic temporal window. Young adult cynomolgus monkeys (Macaca fascicularis) were assigned to a group without sonication (control), a group maintained for 1 d after sonication (C1) and a group maintained for 7 d after sonication (C7; n = 3 for each). Two elder rhesus monkeys (Macaca mulatta) were ultrasonicated under transvenous injection of the recombinant tissue plasminogen activator alteplase (0.9 mg/kg), and maintained for 7 d (R). An automatic switching circuit alternately operated a therapeutic ultrasound beam (T-beam) generator for thrombolysis (frequency = 490 kHz; intensity = 0.72 W/cm(2)) and a diagnostic color-flow imaging ultrasound beam (D-beam; frequency = 2.5 MHz; intensity = 0.20 W/cm(2)). A 15-min protocol, comprising four repeats of a sequence of 120-s T-beam activation followed by 30-s D-beam activation and then 5-min T-beam deactivation together with D-beam activation, was repeated four times over 60 min. After confirmation of neurologic deficits, the brains were removed and investigated histologically and immunohistochemically. Three skull samples were subjected to 494-kHz continuous waveform ultrasound, the transcranial intensity was measured and the mechanical index was calculated. None of the monkeys showed neurologic deficits after ultrasonication. The transskull ultrasound intensity rate was 48 ± 12%. The intracranial mechanical index value was 0.15. The novel system did not cause tissue damage in the primate brain and no cavitation effect was detected intracranially.

Response of ERß and aromatase expression in the monkey hippocampal formation to ovariectomy and menopause.

Changes in the expression of estrogen-related substances in monkeys' brains at the menopausal transition, when estrogen deficit starts to occur, have not yet been examined thoroughly. In the present study, we immunohistochemically investigated the expression levels of estrogen receptor beta (ERß) and aromatase (local estrogen synthesizing enzyme) in the hippocampal formation of premenopausal, menopausal, and ovariectomized premenopausal monkeys. In all monkeys tested, ERß immunoreactivity was observed in interneurons located in the subiculum and the Ammon's horn, and most of these ERß-immunoreactive neurons coexpressed a GABAergic neuron marker, parvalbumin. In the menopausal monkeys who exhibited a decline in estrogen concentration, hippocampal ERß was highly upregulated, while aromatase expression was not markedly changed. By contrast, aromatase in the ovariectomized monkeys was significantly upregulated, while ERß expression was not changed. In the brains of ovariectomized and menopausal monkeys, depletion of ovary-derived estrogen brought about different reactions which may be attributed to the senescence of brain aging.

Distribution and pharmacological characterization of primate NK-2 tachykinin receptor in the central nervous system of the rhesus monkey.

Tachykinin NK-2 receptor, a cognate receptor for neurokinin A, expressed in the brain has been suggested as a new target for the treatment of psychiatric disorders. In rodents, treatment with NK-2 receptor agonists causes anxiogenic effects, while NK-2 receptor antagonists show anxiolytic and antidepressant-like effects. However, information about the distribution and functions of NK-2 receptors in the central nervous system (CNS) in primates is still lacking. Here, we examined the distribution and pharmacological profile of NK-2 receptors in the rhesus monkey (Macaca mulatta) to clarify the molecular basis of NK-2-mediated tachykininergic functions in the primate CNS. NK-2 receptors cloned from the rhesus monkey brain showed similar pharmacological properties to those of human NK-2 receptors. Substantial expression levels of NK-2 mRNA were observed in all the brain regions examined, including areas pertinent to the emotional networks such as the prefrontal cortex, cingulate cortex and amygdala. These findings suggest that NK-2 receptors may play important roles in the pathophysiology of psychiatric disorders.