Kenji W. Koyano

Direct Comparison of Spontaneous Functional Connectivity and Effective Connectivity Measured by Intracortical Microstimulation: An fMRI Study in Macaque Monkeys

Correlated spontaneous activity in the resting brain is increasingly recognized as a useful index for inferring underlying functional-anatomic architecture. However, despite efforts for comparison with anatomical connectivity, neuronal origin of intrinsic functional connectivity (inFC) remains unclear. Conceptually, the source of inFC could be decomposed into causal components that reflect the efficacy of synaptic interactions and other components mediated by collective network dynamics (e.g., synchronization). To dissociate these components, it is useful to introduce another connectivity measure such as effective connectivity, which is a quantitative measure of causal interactions. Here, we present a direct comparison of inFC against emEC (effective connectivity probed with electrical microstimulation [EM]) in the somatosensory system of macaque monkeys. Simultaneous EM and functional magnetic resonance imaging revealed strong emEC in several brain regions in a manner consistent with the anatomy of somatosensory system. Direct comparison of inFC and emEC revealed colocalization and overall positive correlation within the stimulated hemisphere. Interestingly, we found characteristic differences between inFC and emEC in their interhemispheric patterns. Our results suggest that intrahemispheric inFC reflects the efficacy of causal interactions, whereas interhemispheric inFC may arise from interactions akin to network-level synchronization that is not captured by emEC.

Unitized representation of paired objects in area 35 of the macaque perirhinal cortex

The perirhinal cortex, which is critical for long‐term stimulus–stimulus associative memory, consists of two cytoarchitectonically distinct subdivisions: area 35 (A35) and area 36 (A36). Previous electrophysiological studies suggested that macaque A36 is involved in both association and retrieval processes during a visual pair‐association task. However, the neuronal properties of macaque A35 have never been examined because A35 is located in a very narrow region, which makes it difficult to systematically record single‐unit activity from there. In the present study, we overcame this technical difficulty for targeting A35 by combining magnetic resonance imaging‐guided in‐vivo localization with postmortem histological localization. This two‐track approach enabled us to record from 181 A35 neurons in two macaque monkeys while they performed a pair‐association task. Among these neurons, 64 showed stimulus‐selective responses during the cue period (cue‐selective neurons), whereas 18 did during the delay period (delay‐selective neurons). As in A36, the responses of cue‐selective neurons in A35 to paired associates were correlated. In both areas, these correlations were stronger in neurons showing delay selectivity than in those without delay selectivity. Notably, delay‐selective neurons in A35 responded similarly to the optimal stimulus and its paired associate, whereas delay‐selective neurons in A36 discriminated between them. However, these neurons in both areas discriminated the primary pair, consisting of the optimal stimulus and its paired associate, from other pairs, indicating that selectivity across pairs was maintained between the two areas. These results suggest that delay‐selective neurons in A35 represent these paired stimuli as a single unitized item rather than two associated items.

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.

Characterization of the Properties of Seven Promoters in the Motor Cortex of Rats and Monkeys After Lentiviral Vector-Mediated Gene Transfer

Lentiviral vectors deliver transgenes efficiently to a wide range of neuronal cell types in the mammalian central nervous system. To drive gene expression, internal promoters are essential; however, the in vivo properties of promoters, such as their cell type specificity and gene expression activity, are not well known, especially in the nonhuman primate brain. Here, the properties of five ubiquitous promoters (murine stem cell virus [MSCV], cytomegalovirus [CMV], CMV early enhancer/chicken β-actin [CAG], human elongation factor-1α [EF-1α], and Rous sarcoma virus [RSV]) and two cell type-specific promoters (rat synapsin I and mouse α-calcium/calmodulin-dependent protein kinase II [CaMKIIα]) in rat and monkey motor cortices in vivo were characterized. Vesicular stomatitis virus G (VSV-G)-pseudotyped lentiviral vectors expressing enhanced green fluorescent protein (EGFP) under the control of the various promoters were prepared and injected into rat and monkey motor cortices. Immunohistochemical analysis revealed that all of the VSV-G-pseudotyped lentiviral vectors had strong endogenous neuronal tropisms in rat and monkey brains. Among the seven promoters, the CMV promoter showed modest expression in glial cells (9.4%) of the rat brain, whereas the five ubiquitous promoters (MSCV, CMV, CAG, EF-1α, and RSV) showed expression in glial cells (7.0-14.7%) in the monkey brain. Cell type-specific synapsin I and CaMKIIα promoters showed excitatory neuron-specific expression in the monkey brain (synapsin I, 99.7%; CaMKIIα, 100.0%), but their specificities for excitatory neurons were significantly lower in the rat brain (synapsin I, 94.6%; CaMKIIα, 93.7%). These findings could be useful in basic and clinical neuroscience research for the design of vectors that efficiently deliver and express transgenes into rat and monkey brains.

A bicistronic lentiviral vector-based method for differential transsynaptic tracing of neural circuits

We developed a bicistronic HIV1-derived lentiviral vector system co-expressing green fluorescent protein (AcGFP1) and wheat germ agglutinin (WGA) mediated by picornaviral 2A peptide. This system was first applied to the analysis of the rat cerebellar efferent pathways. When the lentiviral vector was injected into a specific lobule, the local Purkinje cell population (first-order neurons) was efficiently infected and co-expressed both AcGFP1 and WGA protein. In the second-order neurons in the cerebellar and vestibular nuclei, WGA but not AcGFP1 protein was differentially detected, demonstrating that the presence of AcGFP1 protein enables discrimination of first-order neurons from second-order neurons. Furthermore, WGA protein was detected in the contralateral ventrolateral thalamic nucleus (third-order nucleus). This system also successfully labeled rat cortical pathways from the primary somatosensory cortex and monkey cerebellar efferent pathways. Thus, this bicistronic lentiviral vector system is a useful tool for differential transsynaptic tracing of neural pathways originating from local brain regions.

Top-Down Regulation of Laminar Circuit via Inter-Area Signal for Successful Object Memory Recall in Monkey Temporal Cortex.

Memory retrieval in primates is orchestrated by a brain-wide neuronal circuit. To elucidate the operation of this circuit, it is imperative to comprehend neuronal mechanisms of coordination between area-to-area interaction and information processing within individual areas. By simultaneous recording from area 36 (A36) and area TE (TE) of the temporal cortex while monkeys performed a pair-association memory task, we found two distinct inter-area signal flows during memory retrieval: A36 spiking activity exhibited coherence with low-frequency field activity in either the supragranular or infragranular layer of TE. Of these two flows, only signal flow targeting the infragranular layer of TE was further translaminarly coupled with gamma activity in the supragranular layer of TE. Moreover, this coupling was observed when monkeys succeeded in the retrieval of the sought object but not when they failed. The results suggest that local translaminar processing can be recruited via a layer-specific inter-area network for memory retrieval.

fMRI Activity in the Macaque Cerebellum Evoked by Intracortical Microstimulation of the Primary Somatosensory Cortex: Evidence for Polysynaptic Propagation

Simultaneous electrical microstimulation (EM) and functional magnetic resonance imaging (fMRI) is a useful tool for probing connectivity across brain areas in vivo. However, it is not clear whether intracortical EM can evoke blood-oxygenation-level-dependent (BOLD) signal in areas connected polysynaptically to the stimulated site. To test for the presence of the BOLD activity evoked by polysynaptic propagation of the EM signal, we conducted simultaneous fMRI and EM in the primary somatosensory cortex (S1) of macaque monkeys. We in fact observed BOLD activations in the contralateral cerebellum which is connected to the stimulation site (i.e. S1) only through polysynaptic pathways. Furthermore, the magnitude of cerebellar activations was dependent on the current amplitude of the EM, confirming the EM is the cause of the cerebellar activations. These results suggest the importance of considering polysynaptic signal propagation, particularly via pathways including subcortical structures, for correctly interpreting ‘functional connectivity’ as assessed by simultaneous EM and fMRI.

Distinct Neuronal Interactions in Anterior Inferotemporal Areas of Macaque Monkeys during Retrieval of Object Association Memory

In macaque monkeys, the anterior inferotemporal cortex, a region crucial for object memory processing, is composed of two adjacent, hierarchically distinct areas, TE and 36, for which different functional roles and neuronal responses in object memory tasks have been characterized. However, it remains unknown how the neuronal interactions differ between these areas during memory retrieval. Here, we conducted simultaneous recordings from multiple single-units in each of these areas while monkeys performed an object association memory task and examined the inter-area differences in neuronal interactions during the delay period. Although memory neurons showing sustained activity for the presented cue stimulus, cue-holding (CH) neurons, interacted with each other in both areas, only those neurons in area 36 interacted with another type of memory neurons coding for the to-be-recalled paired associate (pair-recall neurons) during memory retrieval. Furthermore, pairs of CH neurons in area TE showed functional coupling in response to each individual object during memory retention, whereas the same class of neuron pairs in area 36 exhibited a comparable strength of coupling in response to both associated objects. These results suggest predominant neuronal interactions in area 36 during the mnemonic processing, which may underlie the pivotal role of this brain area in both storage and retrieval of object association memory.

Deeply located granule cells and mitral cells undergo apoptosis after transection of the central connections of the main olfactory bulb in the adult rat

The main olfactory bulb (MOB) is the first relay station of the olfactory system: it receives afferents from sensory neurons and sends efferents to the primary olfactory cortex. The MOB also receives many centrifugal afferents from various regions. Transection of peripheral afferents to the MOB has been reported to induce cell death in granule cells. However, little is known about the effect of transection of these central connections of the MOB in adult rats. Here, we used a unilateral olfactory peduncle transection model in the adult rat to examine neuronal degeneration in the MOB. In the MOB ipsilateral to the surgery, the granule cell layer (GCL) was smaller, and the number of mitral cells was decreased compared with the contralateral MOB at 7 days after surgery. Many degenerating cells were present in both the mitral cell layer (MCL) and GCL in the ipsilateral MOB at 3 days after surgery, although there were no obvious changes in the gross morphology. We also found terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end labeling (TUNEL)-positive cells in the MCL and GCL in the ipsilateral MOB at 3 days after surgery. The majority of the degenerating and TUNEL-positive cells were located in the deep, rather than the superficial, GCL. Immunohistochemistry for activated caspase-9 further supported the occurrence of apoptotic cell death in the mitral and deeply located granule cells. These results indicate that not only axotomized mitral cells, but also deeply located granule cells that were not directly injured, underwent apoptosis after transection of the central connections, and suggest that sensitivities to transection of the central connections differ among granule cells according to their depth in the GCL.

Similarities and differences of functional connectivity as measured by spontaneous correlation of fMRI signals and effective connectivity as measured by simultaneous intracortical microstimulation and fMRI

Hydrogen sulfide (H2S) is a signaling molecule as well as a cytoprotectant in the brain. We have shown that H2S protects neurons from oxidative stress by increasing the levels of glutathione (GSH), a major cellular antioxidant. Here we show that H2S enhances the transport of cysteine to increase GSH production more than cystine transport and to redistribute the localization of GSH to mitochondria. The efficiency of GSH production enhanced by H2S is even greater under oxidative stress by glutamate. H2S reinstated GSH levels in the fetal brain decreased by ischemia/reperfusion in utero. In addition, H2S produced by 3-mercaptopyruvate sulfurtransferase (3MST), along with cysteine aminotransferase (CAT) in mitochondria suppresses oxidative stress in this organelle. Neuro2a cells expressing 3MST along with CAT, showed significant resistance to oxidative stress. The present study shows that H2S protects cells from oxidative stress by two mechanisms. It enhances the production of GSH by enhancing cystine/cysteine transporters. H2S produced in mitochondria also may directly suppress oxidative stress. It provides a new mechanism of neuroprotection from oxidative stress by H2S. Research fund: KAKENHI22590258.