Automated and parallelized spike collision tests to identify spike signal projections.

The spike collision test is a highly reliable technique to identify the axonal projection of a neuron recorded electrophysiologically for investigating functional spike information among brain areas. It is potentially applicable to more neuronal projections by combining multi-channel recording with optogenetic stimulation. Yet, it remains inefficient and laborious because an experimenter must visually select spikes in every channel and manually repeat spike collision tests for each neuron serially. Here, we automated spike collision tests for all channels in parallel (Multi-Linc analysis) in a multi-channel real-time processing system. The rat cortical neurons identified with this technique displayed physiological spike features consistent with excitatory projection neurons. Their antidromic spikes were similar in shape but slightly larger in amplitude compared with spontaneous spikes. In addition, we demonstrated simultaneous identification of reciprocal or bifurcating projections among cortical areas. Thus, our Multi-Linc analysis will be a powerful approach to elucidate interareal spike communication.

Inhibitory neurons exhibit high controlling ability in the cortical microconnectome.

The brain is a network system in which excitatory and inhibitory neurons keep activity balanced in the highly non-random connectivity pattern of the microconnectome. It is well known that the relative percentage of inhibitory neurons is much smaller than excitatory neurons in the cortex. So, in general, how inhibitory neurons can keep the balance with the surrounding excitatory neurons is an important question. There is much accumulated knowledge about this fundamental question. This study quantitatively evaluated the relatively higher functional contribution of inhibitory neurons in terms of not only properties of individual neurons, such as firing rate, but also in terms of topological mechanisms and controlling ability on other excitatory neurons. We combined simultaneous electrical recording (~2.5 hours) of ~1000 neurons in vitro, and quantitative evaluation of neuronal interactions including excitatory-inhibitory categorization. This study accurately defined recording brain anatomical targets, such as brain regions and cortical layers, by inter-referring MRI and immunostaining recordings. The interaction networks enabled us to quantify topological influence of individual neurons, in terms of controlling ability to other neurons. Especially, the result indicated that highly influential inhibitory neurons show higher controlling ability of other neurons than excitatory neurons, and are relatively often distributed in deeper layers of the cortex. Furthermore, the neurons having high controlling ability are more effectively limited in number than central nodes of k-cores, and these neurons also participate in more clustered motifs. In summary, this study suggested that the high controlling ability of inhibitory neurons is a key mechanism to keep balance with a large number of other excitatory neurons beyond simple higher firing rate. Application of the selection method of limited important neurons would be also applicable for the ability to effectively and selectively stimulate E/I imbalanced disease states.

A spike analysis method for characterizing neurons based on phase locking and scaling to the interval between two behavioral events.

Standard analysis of neuronal functions assesses the temporal correlation between animal behaviors and neuronal activity by aligning spike trains with the timing of a specific behavioral event, e.g., visual cue. However, spike activity is often involved in information processing dependent on a relative phase between two consecutive events rather than a single event. Nevertheless, less attention has so far been paid to such temporal features of spike activity in relation to two behavioral events. Here, we propose "Phase-Scaling analysis" to simultaneously evaluate the phase locking and scaling to the interval between two events in task-related spike activity of individual neurons. This analysis method can discriminate conceptual "scaled"-type neurons from "nonscaled"-type neurons using an activity variation map that combines phase locking with scaling to the interval. Its robustness was validated by spike simulation using different spike properties. Furthermore, we applied it to analyzing actual spike data from task-related neurons in the primary visual cortex (V1), posterior parietal cortex (PPC), primary motor cortex (M1), and secondary motor cortex (M2) of behaving rats. After hierarchical clustering of all neurons using their activity variation maps, we divided them objectively into four clusters corresponding to nonscaled-type sensory and motor neurons and scaled-type neurons including sustained and ramping activities, etc. Cluster/subcluster compositions for V1 differed from those of PPC, M1, and M2. The V1 neurons showed the fastest functional activities among those areas. Our method was also applicable to determine temporal "forms" and the latency of spike activity changes. These findings demonstrate its utility for characterizing neurons.NEW & NOTEWORTHY Phase-Scaling analysis is a novel technique to unbiasedly characterize the temporal dependency of functional neuron activity on two behavioral events and objectively determine the latency and form of the activity change. This powerful analysis can uncover several classes of latently functioning neurons that have thus far been overlooked, which may participate differently in intermediate processes of a brain function. The Phase-Scaling analysis will yield profound insights into neural mechanisms for processing internal information.

Differential Changes in the Lateralized Activity of Identified Projection Neurons of Motor Cortex in Hemiparkinsonian Rats.

In the parkinsonian state, the motor cortex and basal ganglia (BG) undergo dynamic remodeling of movement representation. One such change is the loss of the normal contralateral lateralized activity pattern. The increase in the number of movement-related neurons responding to ipsilateral or bilateral limb movements may cause motor problems, including impaired balance, reduced bimanual coordination, and abnormal mirror movements. However, it remains unknown how individual types of motor cortical neurons organize this reconstruction. To explore the effect of dopamine depletion on lateralized activity in the parkinsonian state, we used a partial hemiparkinsonian model [6-hydroxydopamine (6-OHDA) lesion] in Long-Evans rats performing unilateral movements in a right-left pedal task, while recording from primary (M1) and secondary motor cortex (M2). The lesion decreased contralateral preferred activity in both M1 and M2. In addition, this change differed among identified intratelencephalic (IT) and pyramidal tract (PT) cortical projection neurons, depending on the cortical area. We detected a decrease in lateralized activity only in PT neurons in M1, whereas in M2, this change was observed in IT neurons, with no change in the PT population. Our results suggest a differential effect of dopamine depletion in the lateralized activity of the motor cortex, and suggest possible compensatory changes in the contralateral hemisphere.

Ipsilateral-Dominant Control of Limb Movements in Rodent Posterior Parietal Cortex.

It is well known that the posterior parietal cortex (PPC) and frontal motor cortices in primates preferentially control voluntary movements of contralateral limbs. The PPC of rats has been defined based on patterns of thalamic and cortical connectivity. The anatomical characteristics of this area suggest that it may be homologous to the PPC of primates. However, its functional roles in voluntary forelimb movements have not been well understood, particularly in the lateralization of motor limb representation; that is, the limb-specific activity representations for right and left forelimb movements. We examined functional spike activity of the PPC and two motor cortices, the primary motor cortex (M1) and the secondary motor cortex (M2), when head-fixed male rats performed right or left unilateral movements. Unlike primates, PPC neurons in rodents were found to preferentially represent ipsilateral forelimb movements, in contrast to the contralateral preference of M1 and M2 neurons. Consistent with these observations, optogenetic activation of PPC and motor cortices, respectively, evoked ipsilaterally and contralaterally biased forelimb movements. Finally, we examined the effects of optogenetic manipulation on task performance. PPC or M1 inhibition by optogenetic GABA release shifted the behavioral limb preference contralaterally or ipsilaterally, respectively. In addition, weak optogenetic PPC activation, which was insufficient to evoke motor responses by itself, shifted the preference ipsilaterally; although similar M1 activation showed no effects on task performance. These paradoxical observations suggest that the PPC plays evolutionarily different roles in forelimb control between primates and rodents.SIGNIFICANCE STATEMENT In rodents, the primary and secondary motor cortices (M1 and M2, respectively) are involved in voluntary movements with contralateral preference. However, it remains unclear whether and how the posterior parietal cortex (PPC) participates in controlling multiple limb movements. We recorded functional activity from these areas using a behavioral task to monitor movements of the right and left forelimbs separately. PPC neurons preferentially represented ipsilateral forelimb movements and optogenetic PPC activation evoked ipsilaterally biased forelimb movements. Optogenetic PPC inhibition via GABA release shifted the behavioral limb preference contralaterally during task performance, whereas weak optogenetic PPC activation, which was insufficient to evoke motor responses by itself, shifted the preference ipsilaterally. Our findings suggest rodent PPC contributes to ipsilaterally biased motor response and/or planning.

Area-specific Modulation of Functional Cortical Activity During Block-based and Trial-based Proactive Inhibition.

Animals can suppress their behavioral response in advance according to changes in environmental context (proactive inhibition: delaying the start of response), a process in which several cortical areas may participate. However, it remains unclear how this process is adaptively regulated according to contextual changes on different timescales. To address the issue, we used an improved stop-signal task paradigm to behaviorally and electrophysiologically characterize the temporal aspect of proactive inhibition in head-fixed rats. In the task, they must respond to a go cue as quickly as possible (go trial), but did not have to respond if a stop cue followed the go cue (stop trial). The task alternated between a block of only go trials (G-block) and a block of go-and-stop trials (GS-block). We observed block-based and trial-based proactive inhibition (emerging in GS-block and after stop trial, respectively) by behaviorally evaluating the delay in reaction time in correct go trials depending on contextual changes on different timescales. We electrophysiologically analyzed task-related neuronal activity in the primary and secondary motor, posterior parietal, and orbitofrontal cortices (M1, M2, PPC, and OFC, respectively). Under block-based proactive inhibition, spike activity of cue-preferring OFC neurons was attenuated continuously, while M1 and M2 activity was enhanced during motor preparation. Subsequently, M1 activity was attenuated during motor decision/execution. Under trial-based proactive inhibition, the OFC activity was continuously enhanced, and PPC and M1 activity was also enhanced shortly during motor decision/execution. These results suggest that different cortical mechanisms underlie the two types of proactive inhibition in rodents.

In Vivo Spiking Dynamics of Intra- and Extratelencephalic Projection Neurons in Rat Motor Cortex.

In motor cortex, 2 types of deep layer pyramidal cells send their axons to other areas: intratelencephalic (IT)-type neurons specifically project bilaterally to the cerebral cortex and striatum, whereas neurons of the extratelencephalic (ET)-type, termed conventionally pyramidal tract-type, project ipsilaterally to the thalamus and other areas. Although they have totally different synaptic and membrane potential properties in vitro, little is known about the differences between them in ongoing spiking dynamics in vivo. We identified IT-type and ET-type neurons, as well as fast-spiking-type interneurons, using novel multineuronal analysis based on optogenetically evoked spike collision along their axons in behaving/resting rats expressing channelrhodopsin-2 (Multi-Linc method). We found "postspike suppression" (~100 ms) as a characteristic of ET-type neurons in spike auto-correlograms, and it remained constant independent of behavioral conditions in functionally different ET-type neurons. Postspike suppression followed even solitary spikes, and spike bursts significantly extended its duration. We also observed relatively strong spike synchrony in pairs containing IT-type neurons. Thus, spiking dynamics in IT-type and ET-type neurons may be optimized differently for precise and coordinated motor control.

Distinct Laterality in Forelimb-Movement Representations of Rat Primary and Secondary Motor Cortical Neurons with Intratelencephalic and Pyramidal Tract Projections.

Two distinct motor areas, the primary and secondary motor cortices (M1 and M2), play crucial roles in voluntary movement in rodents. The aim of this study was to characterize the laterality in motor cortical representations of right and left forelimb movements. To achieve this goal, we developed a novel behavioral task, the Right-Left Pedal task, in which a head-restrained male rat manipulates a right or left pedal with the corresponding forelimb. This task enabled us to monitor independent movements of both forelimbs with high spatiotemporal resolution. We observed phasic movement-related neuronal activity (Go-type) and tonic hold-related activity (Hold-type) in isolated unilateral movements. In both M1 and M2, Go-type neurons exhibited bias toward contralateral preference, whereas Hold-type neurons exhibited no bias. The contralateral bias was weaker in M2 than M1. Moreover, we differentiated between intratelencephalic (IT) and pyramidal tract (PT) neurons using optogenetically evoked spike collision in rats expressing channelrhodopsin-2. Even in identified PT and IT neurons, Hold-type neurons exhibited no lateral bias. Go-type PT neurons exhibited bias toward contralateral preference, whereas IT neurons exhibited no bias. Our findings suggest a different laterality of movement representations of M1 and M2, in each of which IT neurons are involved in cooperation of bilateral movements, whereas PT neurons control contralateral movements.SIGNIFICANCE STATEMENT In rodents, the primary and secondary motor cortices (M1 and M2) are involved in voluntary movements via distinct projection neurons: intratelencephalic (IT) neurons and pyramidal tract (PT) neurons. However, it remains unclear whether the two motor cortices (M1 vs M2) and the two classes of projection neurons (IT vs PT) have different laterality of movement representations. We optogenetically identified these neurons and analyzed their functional activity using a novel behavioral task to monitor movements of the right and left forelimbs separately. We found that contralateral bias was reduced in M2 relative to M1, and in IT relative to PT neurons. Our findings suggest that the motor information processing that controls forelimb movement is coordinated by a distinct cell population.

Three-state molecular shuttles operated using acid/base stimuli with distinct outputs.

This paper describes the acid/base-mediated three-state translational isomerization of two [2]rotaxanes, each containing N-alkylaniline and N,N-dialkylamine centers as binding sites for threaded dibenzo[24]crown-8 units. Under neutral conditions, the dialkylamine unit predominantly recognized the crown ether component through cooperative binding of a proton; when both amino units were protonated under acidic conditions, both translational isomers were generated; the addition of a strong base caused aniline-crown ether interactions to dominate. The three states of the [2]rotaxane featuring the 3,5-diphenylaniline terminus in its dumbbell-shaped component were accompanied by distinct absorptive outputs that were detectable using UV spectroscopy.