Kiyoshi Nakahara

Top-down signal from prefrontal cortex in executive control of memory retrieval

Knowledge or experience is voluntarily recalled from memory by reactivation of the neural representations in the cerebral association cortex. In inferior temporal cortex, which serves as the storehouse of visual long-term memory, activation of mnemonic engrams through electric stimulation results in imagery recall in humans, and neurons can be dynamically activated by the necessity for memory recall in monkeys. Neuropsychological studies and previous split-brain experiments predicted that prefrontal cortex exerts executive control upon inferior temporal cortex in memory retrieval; however, no neuronal correlate of this process has ever been detected. Here we show evidence of the top-down signal from prefrontal cortex. In the absence of bottom-up visual inputs, single inferior temporal neurons were activated by the top-down signal, which conveyed information on semantic categorization imposed by visual stimulus–stimulus association. Behavioural performance was severely impaired with loss of the top-down signal. Control experiments confirmed that the signal was transmitted not through a subcortical but through a fronto-temporal cortical pathway. Thus, feedback projections from prefrontal cortex to the posterior association cortex appear to serve the executive control of voluntary recall.

Transient activation of inferior prefrontal cortex during cognitive set shifting

The Wisconsin Card Sorting Test, which probes the ability to shift attention from one category of stimulus attributes to another (shifting cognitive sets), is the most common paradigm used to detect human frontal lobe pathology. However, the exact relationship of this card test to prefrontal function and the precise anatomical localization of the cognitive shifts involved are controversial. By isolating shift-related signals using the temporal resolution of functional magnetic resonance imaging, we reproducibly found transient activation of the posterior part of the bilateral inferior frontal sulci. This activation was larger as the number of dimensions (relevant stimulus attributes that had to be recognized) were increased. These results suggest that the inferior frontal areas play an essential role in the flexible shifting of cognitive sets.

Glutamate receptors: brain function and signal transduction

Glutamate receptors are important in neural plasticity, neural development and neurodegeneration. N-methyl-d-aspartate (NMDA) receptors and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptors act as glutamate-gated cation channels, whereas metabotropic receptors (mGluRs) modulate the production of second messengers via G proteins. Molecular studies from our and other laboratories indicated that NMDA receptors and mGluRs exist as multiple subunits (NMDAR1 and NMDAR2A-2D) and multiple subtypes (mGluR1-mGluR8). In light of the molecular diversity of glutamate receptors, we explored the function and intracellular signaling mechanisms of different members of glutamate receptors. In the visual system, retinal bipolar cells receive glutamate transmission from photoreceptors and contribute to segregating visual signals into ON and OFF pathways. The molecularly cloned mGluR6 is restrictedly expressed at the postsynaptic site of ON-bipolar cells in both rod and cone systems. Gene targeting of mGluR6 results in a loss of ON responses without changing OFF responses and severely impairs detecting visual contrasts. Since AMPA receptors mediate OFF responses in OFF-bipolar cells, two distinct types of glutamate receptors effectively operate for ON and OFF responses. mGluR1 and mGluR5 are both coupled to inositol triphosphate (IP3)/calcium signal transduction with an identical agonist selectivity. Single-cell intracellular calcium ([Ca2+]i) recordings indicated that glutamate evokes a non-oscillatory and oscillatory [Ca2+]i response in mGluR1-expressing and mGluR5-expressing cells, respectively. This difference results from a single amino acid substitution, aspartate of mGluR1 or threonine of mGluR5, at the G protein-interacting carboxy-terminal domains. Protein kinase C phosphorylation of the threonine of mGluR5 is responsible for inducing [Ca2+]i oscillations in mGluR5-expressing cells and cultured glial cells. Thus, the two closely related mGluR subtypes mediate diverging intracellular signaling in glutamate transmission.

Mammalian hairy and Enhancer of split homolog 1 regulates differentiation of retinal neurons and is essential for eye morphogenesis.

Mammalian hairy and Enhancer of split homolog 1 (HES1), a basic helix-loop-helix factor gene, is expressed in retinal progenitor cells, and its expression decreases as differentiation proceeds. Retinal progenitor cells infected with HES1-transducing retrovirus did not differentiate into mature retinal cells, suggesting that persistent expression of HES1 blocks retinal development. In contrast, in the retina of HES1-null mutant mice, differentiation was accelerated, and rod and horizontal cells appeared prematurely and formed abnormal rosette-like structures. Lens and cornea development was also severely disturbed. Furthermore, in the mutant retina, bipolar cells extensively died, and finally disappeared. These studies provide evidence that HES1 regulates differentiation of retinal neurons and is essential for eye morphogenesis.

Functional magnetic resonance imaging of macaque monkeys performing visually guided saccade tasks: comparison of cortical eye fields with humans.

The frontal and parietal eye fields serve as functional landmarks of the primate brain, although their correspondences between humans and macaque monkeys remain unclear. We conducted fMRI at 4.7 T in monkeys performing visually-guided saccade tasks and compared brain activations with those in humans using identical paradigms. Among multiple parietal activations, the dorsal lateral intraparietal area in monkeys and an area in the posterior superior parietal lobule in humans exhibited the highest selectivity to saccade directions. In the frontal cortex, the selectivity was highest at the junction of the precentral and superior frontal sulci in humans and in the frontal eye field (FEF) in monkeys. BOLD activation peaks were also found in premotor areas (BA6) in monkeys, which suggests that the apparent discrepancy in location between putative human FEF (BA6, suggested by imaging studies) and monkey FEF (BA8, identified by microstimulation studies) partly arose from methodological differences.

Local and retrograde gene transfer into primate neuronal pathways via adeno-associated virus serotype 8 and 9.

Viral vector-mediated gene transfer has become increasingly valuable for primate brain research, in particular for application of genetic methods (e.g. optogenetics) to study neuronal circuit functions. Neuronal cell tropisms and infection patterns are viable options for obtaining viral vector-mediated transgene delivery that is selective for particular neuronal pathways. For example, several types of viral vectors can infect axon terminals (retrograde infections), which enables targeted transgene delivery to neurons that directly project to a particular viral injection region. Although recent studies in rodents have demonstrated that adeno-associated virus serotype 8 (AAV8) and 9 (AAV9) efficiently transduce neurons, the tropisms and infection patterns remain poorly understood in primate brains. Here, we constructed recombinant AAV8 or AAV9, which expressed an enhanced green fluorescent protein (EGFP) gene driven by a ubiquitous promoter (AAV8-EGFP and AAV9-EGFP, respectively), and stereotaxically injected it into several brain regions in marmosets and macaque monkeys. Immunohistochemical analyses revealed almost exclusive colocalization of EGFP fluorescence via AAV9-mediated gene transfer with a neuron-specific marker, indicating endogenous neuronal tropism of AAV9, which was consistent with our previous results utilizing AAV8. Injections of either AAV8-EGFP or AAV9-EGFP into the marmoset striatum resulted in EGFP expression in local striatal neurons as a result of local infection, as well as expression in dopaminergic neurons of the substantia nigra via retrograde transport along nigrostriatal axonal projections. Retrograde infections were also observed in the frontal cortex and thalamus, which are known to have direct projections to the striatum. These local and retrograde gene transfers were further demonstrated in the geniculocortical pathway of the marmoset visual system. These findings indicate promising capabilities of AAV8 and AAV9 to deliver molecular tools into a range of primate neural systems in pathway-specific manners through their neuronal tropisms and infection patterns.

Intrasulcal electrocorticography in macaque monkeys with minimally invasive neurosurgical protocols.

Electrocorticography (ECoG), multichannel brain-surface recording and stimulation with probe electrode arrays, has become a potent methodology not only for clinical neurosurgery but also for basic neuroscience using animal models. The highly evolved primates brain has deep cerebral sulci, and both gyral and intrasulcal cortical regions have been implicated in important functional processes. However, direct experimental access is typically limited to gyral regions, since placing probes into sulci is difficult without damaging the surrounding tissues. Here we describe a novel methodology for intrasulcal ECoG in macaque monkeys. We designed and fabricated ultra-thin flexible probes for macaques with micro-electro-mechanical systems technology. We developed minimally invasive operative protocols to implant the probes by introducing cutting-edge devices for human neurosurgery. To evaluate the feasibility of intrasulcal ECoG, we conducted electrophysiological recording and stimulation experiments. First, we inserted parts of the Parylene-C-based probe into the superior temporal sulcus to compare visually evoked ECoG responses from the ventral bank of the sulcus with those from the surface of the inferior temporal cortex. Analyses of power spectral density and signal-to-noise ratio revealed that the quality of the ECoG signal was comparable inside and outside of the sulcus. Histological examination revealed no obvious physical damage in the implanted areas. Second, we placed a modified silicone ECoG probe into the central sulcus and also on the surface of the precentral gyrus for stimulation. Thresholds for muscle twitching were significantly lower during intrasulcal stimulation compared to gyral stimulation. These results demonstrate the feasibility of intrasulcal ECoG in macaques. The novel methodology proposed here opens up a new frontier in neuroscience research, enabling the direct measurement and manipulation of electrical activity in the whole brain.

MRI-based localization of electrophysiological recording sites within the cerebral cortex at single-voxel accuracy.

The localization of microelectrode recording sites in the layers of primate cerebral cortex permits the analysis of relationships between recorded neuronal activities and underlying anatomical connections. We present a magnetic resonance imaging method for precise in vivo localization of cortical recording sites. In this method, the susceptibility-induced effect thickens the appearance of the microelectrode and enhances the detectability of the microelectrode tip, which usually occupies less than a few percent of the volume of an image voxel. In a phantom study, the optimized susceptibility-induced effect allowed tip detection with single-voxel accuracy (in-plane resolution, 50 μm). We applied this method to recording microelectrodes inserted into the brains of macaque monkeys, and localized the microelectrode tip at an in-plane resolution of 150 μm within the cortex of 2–3 mm in thickness. Subsequent histological analyses validated the single-voxel accuracy of the in vivo tip localization. This method opens up a way to investigate information flow during cognitive processes in the brain.

Exploring the neural basis of cognition: multi-modal links between human fMRI and macaque neurophysiology.

Although functional magnetic resonance imaging (fMRI) with sophisticated behavioral paradigms has enabled the investigation of increasingly higher-level cognitive functions in humans, these studies seem to lose touch with neurophysiological studies in macaque monkeys. The application of fMRI and other MRI-based techniques to macaque brains allows studies in the two species to be linked. fMRI in human and macaque subjects using equivalent cognitive tasks enables direct comparisons of the functional brain architecture, even for high-level cognitive functions. Combinations of functional or structural MRI and microelectrode techniques provide ways to explore functional brain networks at multiple spatiotemporal scales. These approaches would illuminate the neurophysiological underpinnings of human cognitive functions by integrating human functional neuroimaging with macaque single-unit recordings.

A rapid presentation event-related functional magnetic resonance imaging study of response inhibition in macaque monkeys

Rapid presentation event-related functional magnetic resonance imaging was applied to macaque monkeys performing a symmetrically rewarded go/no-go task, to investigate neural correlate of response inhibition. Sensorimotor activation related to the task performance was observed predominantly in the hemisphere contralateral to the response forelimb. Furthermore, no-go dominant activation possibly related to response inhibition, was observed in the ventral prefrontal cortex, in accordance with previous electrophysiological studies. These results show the feasibility of rapid presentation event-related functional magnetic resonance imaging in behaving monkeys.