Rhythm Receptive Fields in Striatum of Mice Executing Complex Continuous Movement Sequences.

By the use of a novel experimental system, the step-wheel, we investigated the neural underpinnings of complex and continuous movements. We recorded neural activities from the dorsolateral striatum and found neurons sensitive to movement rhythm parameters. These neurons responded to specific combinations of interval, phase, and repetition of movement, effectively forming what we term "rhythm receptive fields." Some neurons even responsive to the combination of movement phases of multiple body parts. In parallel, cortical recordings in sensorimotor areas highlighted a paucity of neurons responsive to multiple parameter combinations, relative to those in the striatum. These findings have implications for comprehending motor coordination deficits seen in brain disorders including Parkinson's disease. Movement encoding by rhythm receptive fields should streamline the brain's capacity to encode temporal patterns, help to resolve the degrees of freedom problem. Such rhythm fields hint at the neural mechanisms governing effective motor control and processing of rhythmic information.

Emergence of rhythmic chunking in complex stepping of mice.

Motor chunking is important for motor execution, allowing atomization and efficiency of movement sequences. However, it remains unclear why and how chunks contribute to motor execution. To analyze the structure of naturally occurring chunks, we trained mice to run in a complex series of steps and identified the formation of chunks. We found that intervals (cycle) and the positional relationship between the left and right limbs (phase) of steps inside the chunks, unlike those outside the chunks, were consistent across occurrences. Further, licking by the mice was also more periodic and linked to the specific phases of limb movements within the chunk. Based on these findings, we propose the rhythm chunking hypothesis, whereby within chunks, the repetitive movements of many body parts are linked by the rhythm parameters: cycle and phase. The computational complexity of movement may thereby be reduced by adjusting movements as the combination of rhythms.

[Generation of Gait Rhythm in Mice].

The arrangement of a series of repetitive motions on the time axis should be planned while constructing continuous movement. To understand the mechanism for planning continuous movement and its neural basis, mice were trained to perform complicated continuous step running. The repeated foot movements and neural activities of the animals were recorded. The scaffolding peg arrangement was complex; however, the steps turned out to be more rhythmic, indicating that the mice adjusted to repeat their movements periodically rather than fitting their foot timing with the peg arrangement. In addition, neural activity recorded from the striatum was also found to be rhythmic, suggesting that the striatum may be involved in converting complex inputs into more rhythmic outputs.

Lesion of the hippocampus selectively enhances LEC's activity during recognition memory based on familiarity.

The sense of familiarity for events is crucial for successful recognition memory. However, the neural substrate and mechanisms supporting familiarity remain unclear. A major controversy in memory research is whether the parahippocampal areas, especially the lateral entorhinal (LEC) and the perirhinal (PER) cortices, support familiarity or whether the hippocampus (HIP) does. In addition, it is unclear if LEC, PER and HIP interact within this frame. Here, we especially investigate if LEC and PER's contribution to familiarity depends on hippocampal integrity. To do so, we compare LEC and PER neural activity between rats with intact hippocampus performing on a human to rat translational task relying on both recollection and familiarity and rats with hippocampal lesions that have been shown to then rely on familiarity to perform the same task. Using high resolution Immediate Early Gene imaging, we report that hippocampal lesions enhance activity in LEC during familiarity judgments but not PER's. These findings suggest that different mechanisms support familiarity in LEC and PER and led to the hypothesis that HIP might exert a tonic inhibition on LEC during recognition memory that is released when HIP is compromised, possibly constituting a compensatory mechanism in aging and amnesic patients.

CalDAG-GEFI mediates striatal cholinergic modulation of dendritic excitability, synaptic plasticity and psychomotor behaviors.

CalDAG-GEFI (CDGI) is a protein highly enriched in the striatum, particularly in the principal spiny projection neurons (SPNs). CDGI is strongly down-regulated in two hyperkinetic conditions related to striatal dysfunction: Huntington's disease and levodopa-induced dyskinesia in Parkinson's disease. We demonstrate that genetic deletion of CDGI in mice disrupts dendritic, but not somatic, M1 muscarinic receptors (M1Rs) signaling in indirect pathway SPNs. Loss of CDGI reduced temporal integration of excitatory postsynaptic potentials at dendritic glutamatergic synapses and impaired the induction of activity-dependent long-term potentiation. CDGI deletion selectively increased psychostimulant-induced repetitive behaviors, disrupted sequence learning, and eliminated M1R blockade of cocaine self-administration. These findings place CDGI as a major, but previously unrecognized, mediator of cholinergic signaling in the striatum. The effects of CDGI deletion on the self-administration of drugs of abuse and its marked alterations in hyperkinetic extrapyramidal disorders highlight CDGI's therapeutic potential.

Dopamine D1 and muscarinic acetylcholine receptors in dorsal striatum are required for high speed running.

Dopamine (DA) signaling in the basal ganglia plays important roles in motor control. Motor deficiencies were previously reported in dopamine receptor D1 (D1R) and D2 (D2R) knockout mice. While these results indicate the involvement of DA receptors in motor execution, the null knockout (KO) mouse lacks the specificity necessary to determine when and where in the brain D1R and D2R function in motor execution. To address these questions, we restricted the loss of function temporally and spatially by using D1R conditional knockdown (cKD) mice and mice injected with antagonists against DA receptors directly into the dorsal striatum. In addition, we address the DA and acetylcholine (ACh) balance hypothesis by using antagonists against ACh receptors. We tested the motor ability of the mice with a newly devised task named the accelerating step-wheel. In this task, the maximum running speed was measured in a situation where the wheel rotation speed was gradually accelerated in one trial. We found significant decreases in the maximum running speed of D1R cKD mice and the mice injected with the antagonist against D1R or muscarinic ACh receptor. These results indicated that D1R and muscarinic ACh receptor in the dorsal striatum play pivotal roles in the execution of walking/running.

Single-cell memory trace imaging with immediate-early genes.

For the past decades, an increasing number of studies has taken advantage of molecular imaging methods involving the detection of immediate-early genes' (IEGs) expression for investigating neural substrates underlying plasticity processes and memory function. The detection of IEGs RNA by Fluorescent In-Situ Hybridization (FISH) yields single-cell as well as high temporal resolution and has recently enabled the mapping of medial temporal lobe subareas/subnetworks activity induced by single or multiple behavioural events in the same animal. After briefly reviewing the function and the ties of the typical IEGs (Fos, Zif268, Arc, Homer1a) used for mapping plasticity, we focus on discussing technical considerations vital for the successful detection of IEGs with FISH with emphasis on the design of RNA probes, the optimization of experimental conditions and the necessity for controls. Finally, we discuss recent developments in brain clearing methods that in combination with FISH detection of IEGs' expression allow for 3D imaging with single cell resolution as well as whole brain analyses. This, in parallel with the recent development of fMRI cognitive tasks in awake rats and the use of high resolution fMRI in humans, holds great promises for bridging further memory in humans and animals.

Recognition memory: Cellular evidence of a massive contribution of the LEC to familiarity and a lack of involvement of the hippocampal subfields CA1 and CA3.

A highly debated issue in memory research is whether familiarity is supported by the parahippocampal region, especially the lateral (LEC) and the perirhinal (PER) cortices, or whether it is supported by the same brain structure as recollection: the hippocampus. One reason for this is that conflicting results have emerged regarding the contribution of the hippocampus to familiarity. This might stem from the lack of dissociation between hippocampal subfields CA1 and CA3 as these areas are involved to a different extent in processes which are pertinent to familiarity. Another reason is that empirical evidence for a contribution of the LEC is still missing. Furthermore, it is unclear whether the superficial and the deep layers of the LEC would equally contribute to this process as these layers are differentially recruited during memory retrieval which partly relies on familiarity. To identify the specific contribution of the LEC, CA1, and CA3, we imaged with cellular resolution activity in the brain of rats performing a version of a standard human memory task adapted to rats that yields judgments based on familiarity. Using this translational approach, we report that in striking contrast to CA1 and CA3, the LEC is recruited for familiarity-judgments and that its contribution is comparable to that of the PER. These results show for the first time that the LEC, specifically its deep layers, contributes to familiarity and constitute the first cellular evidence that the hippocampus does not, thus establishing that familiarity does not share the same neural substrate as recollection.

Learning new sequential stepping patterns requires striatal plasticity during the earliest phase of acquisition.

Animals including humans execute motor behavior to reach their goals. For this purpose, they must choose correct strategies according to environmental conditions and shape many parameters of their movements, including their serial order and timing. To investigate the neurobiology underlying such skills, we used a multi-sensor equipped, motor-driven running wheel with adjustable sequences of foothold pegs on which mice ran to obtain water reward. When the peg patterns changed from a familiar pattern to a new pattern, the mice had to learn and implement new locomotor strategies in order to receive reward. We found that the accuracy of stepping and the achievement of water reward improved with the new learning after changes in the peg-pattern, and c-Fos expression levels assayed after the first post-switch session were high in both dorsolateral striatum and motor cortex, relative to post-switch plateau levels. Combined in situ hybridization and immunohistochemistry of striatal sections demonstrated that both enkephalin-positive (indirect pathway) neurons and substance P-positive (direct pathway) neurons were recruited specifically after the pattern switches, as were interneurons expressing neuronal nitric oxide synthase. When we blocked N-methyl-D-aspartate (NMDA) receptors in the dorsolateral striatum by injecting the NMDA receptor antagonist, D-2-amino-5-phosphonopentanoic acid (AP5), we found delays in early post-switch improvement in performance. These findings suggest that the dorsolateral striatum is activated on detecting shifts in environment to adapt motor behavior to the new context via NMDA-dependent plasticity, and that this plasticity may underlie forming and breaking skills and habits as well as to behavioral difficulties in clinical disorders.

Establishment of high reciprocal connectivity between clonal cortical neurons is regulated by the Dnmt3b DNA methyltransferase and clustered protocadherins.

BACKGROUND: The specificity of synaptic connections is fundamental for proper neural circuit function. Specific neuronal connections that underlie information processing in the sensory cortex are initially established without sensory experiences to a considerable extent, and then the connections are individually refined through sensory experiences. Excitatory neurons arising from the same single progenitor cell are preferentially connected in the postnatal cortex, suggesting that cell lineage contributes to the initial wiring of neurons. However, the postnatal developmental process of lineage-dependent connection specificity is not known, nor how clonal neurons, which are derived from the same neural stem cell, are stamped with the identity of their common neural stem cell and guided to form synaptic connections. RESULTS: We show that cortical excitatory neurons that arise from the same neural stem cell and reside within the same layer preferentially establish reciprocal synaptic connections in the mouse barrel cortex. We observed a transient increase in synaptic connections between clonal but not nonclonal neuron pairs during postnatal development, followed by selective stabilization of the reciprocal connections between clonal neuron pairs. Furthermore, we demonstrate that selective stabilization of the reciprocal connections between clonal neuron pairs is impaired by the deficiency of DNA methyltransferase 3b (Dnmt3b), which determines DNA-methylation patterns of genes in stem cells during early corticogenesis. Dnmt3b regulates the postnatal expression of clustered protocadherin (cPcdh) isoforms, a family of adhesion molecules. We found that cPcdh deficiency in clonal neuron pairs impairs the whole process of the formation and stabilization of connections to establish lineage-specific connection reciprocity. CONCLUSIONS: Our results demonstrate that local, reciprocal neural connections are selectively formed and retained between clonal neurons in layer 4 of the barrel cortex during postnatal development, and that Dnmt3b and cPcdhs are required for the establishment of lineage-specific reciprocal connections. These findings indicate that lineage-specific connection reciprocity is predetermined by Dnmt3b during embryonic development, and that the cPcdhs contribute to postnatal cortical neuron identification to guide lineage-dependent synaptic connections in the neocortex.

Similarity in Neuronal Firing Regimes across Mammalian Species.

UNLABELLED: The architectonic subdivisions of the brain are believed to be functional modules, each processing parts of global functions. Previously, we showed that neurons in different regions operate in different firing regimes in monkeys. It is possible that firing regimes reflect differences in underlying information processing, and consequently the firing regimes in homologous regions across animal species might be similar. We analyzed neuronal spike trains recorded from behaving mice, rats, cats, and monkeys. The firing regularity differed systematically, with differences across regions in one species being greater than the differences in similar areas across species. Neuronal firing was consistently most regular in motor areas, nearly random in visual and prefrontal/medial prefrontal cortical areas, and bursting in the hippocampus in all animals examined. This suggests that firing regularity (or irregularity) plays a key role in neural computation in each functional subdivision, depending on the types of information being carried. SIGNIFICANCE STATEMENT: By analyzing neuronal spike trains recorded from mice, rats, cats, and monkeys, we found that different brain regions have intrinsically different firing regimes that are more similar in homologous areas across species than across areas in one species. Because different regions in the brain are specialized for different functions, the present finding suggests that the different activity regimes of neurons are important for supporting different functions, so that appropriate neuronal codes can be used for different modalities.

Imaging a memory trace over half a life-time in the medial temporal lobe reveals a time-limited role of CA3 neurons in retrieval.

Whether retrieval still depends on the hippocampus as memories age or relies then on cortical areas remains a major controversy. Despite evidence for a functional segregation between CA1, CA3 and parahippocampal areas, their specific role within this frame is unclear. Especially, the contribution of CA3 is questionable as very remote memories might be too degraded to be used for pattern completion. To identify the specific role of these areas, we imaged brain activity in mice during retrieval of recent, early remote and very remote fear memories by detecting the immediate-early gene Arc. Investigating correlates of the memory trace over an extended period allowed us to report that, in contrast to CA1, CA3 is no longer recruited in very remote retrieval. Conversely, we showed that parahippocampal areas are then maximally engaged. These results suggest a shift from a greater contribution of the trisynaptic loop to the temporoammonic pathway for retrieval.

The transfer and transformation of collective network information in gene-matched networks.

Networks, such as the human society network, social and professional networks, and biological system networks, contain vast amounts of information. Information signals in networks are distributed over nodes and transmitted through intricately wired links, making the transfer and transformation of such information difficult to follow. Here we introduce a novel method for describing network information and its transfer using a model network, the Gene-matched network (GMN), in which nodes (neurons) possess attributes (genes). In the GMN, nodes are connected according to their expression of common genes. Because neurons have multiple genes, the GMN is cluster-rich. We show that, in the GMN, information transfer and transformation were controlled systematically, according to the activity level of the network. Furthermore, information transfer and transformation could be traced numerically with a vector using genes expressed in the activated neurons, the active-gene array, which was used to assess the relative activity among overlapping neuronal groups. Interestingly, this coding style closely resembles the cell-assembly neural coding theory. The method introduced here could be applied to many real-world networks, since many systems, including human society and various biological systems, can be represented as a network of this type.

Expression pattern of immediate early genes in the cerebellum of D1R KO, D2R KO, and wild type mice under vestibular-controlled activity.

We previously reported the different motor abilities of D1R knockout (KO), D2R KO and wild-type (WT) mice. To understand the interaction between the cerebellum and the striatal direct and indirect pathways, we examined the expression patterns of immediate early genes (IEG) in the cerebellum of these three genotypes of mice. In the WT naive mice, there was little IEG expression. However, we observed a robust expression of c-fos mRNA in the vermis and hemisphere after running rota-rod tasks. In the vermis, c-fos was expressed throughout the lobules except lobule 7, and also in crus 1 of the ansiform lobule (Crus1), copula of the pyramis (Cop) and most significantly in the flocculus in the hemisphere. jun-B was much less expressed but more preferentially expressed in Purkinje cells. In addition, we observed significant levels of c-fos and jun-B expressions after handling mice, and after the stationary rota-rod task in naive mice. Surprisingly, we observed significant expression of c-fos and jun-B even 30 min after single weighing. Nonetheless, certain additional c-fos and jun-B expressions were observed in three genotypes of the mice that experienced several sessions of motor tasks 24 h after stationary rota-rod task and on days 1 and 5 after rota-rod tasks, but no significant differences in expressions after the running rota-rod tasks were observed among the three genotypes. In addition, there may be some differences 24 h after the stationary rota-rod task between the naive mice and the mice that experienced several sessions of motor tasks.

Impaired clustered protocadherin-α leads to aggregated retinogeniculate terminals and impaired visual acuity in mice.

Clustered protocadherins (cPcdhs) comprising cPcdh-α, -ß, and -γ, encode a large family of cadherin-like cell-adhesion molecules specific to neurons. Impairment of cPcdh-α results in abnormal neuronal projection patterns in specific brain areas. To elucidate the role of cPcdh-α in retinogeniculate projections, we investigated the morphological patterns of retinogeniculate terminals in the lateral geniculate (LG) nucleus of mice with impaired cPcdh-α. We found huge aggregated retinogeniculate terminals in the dorsal LG nucleus, whereas no such aggregated terminals derived from the retina were observed in the olivary pretectal nucleus and the ventral LG nucleus. These aggregated terminals appeared between P10 and P14, just before eye opening and at the beginning of the refinement stage of the retinogeniculate projections. Reduced visual acuity was observed in adult mice with impaired cPcdh-α, whereas the orientation selectivity and direction selectivity of neurons in the primary visual cortex were apparently normal. These findings suggest that cPcdh-α is required for adequate spacing of retinogeniculate projections, which may be essential for normal development of visual acuity.

Distinct motor impairments of dopamine D1 and D2 receptor knockout mice revealed by three types of motor behavior.

Both D1R and D2R knock out (KO) mice of the major dopamine receptors show significant motor impairments. However, there are some discrepant reports, which may be due to the differences in genetic background and experimental procedures. In addition, only few studies directly compared the motor performance of D1R and D2R KO mice. In this paper, we examined the behavioral difference among N10 congenic D1R and D2R KO, and wild type (WT) mice. First, we examined spontaneous motor activity in the home cage environment for consecutive 5 days. Second, we examined motor performance using the rota-rod task, a standard motor task in rodents. Third, we examined motor ability with the Step-Wheel task in which mice were trained to run in a motor-driven turning wheel adjusting their steps on foothold pegs to drink water. The results showed clear differences among the mice of three genotypes in three different types of behavior. In monitoring spontaneous motor activities, D1R and D2R KO mice showed higher and lower 24 h activities, respectively, than WT mice. In the rota-rod tasks, at a low speed, D1R KO mice showed poor performance but later improved, whereas D2R KO mice showed a good performance at early days without further improvement. When first subjected to a high speed task, the D2R KO mice showed poorer rota-rod performance at a low speed than the D1R KO mice. In the Step-Wheel task, across daily sessions, D2R KO mice increased the duration that mice run sufficiently close to the spout to drink water, and decreased time to touch the floor due to missing the peg steps and number of times the wheel was stopped, which performance was much better than that of D1R KO mice. These incongruent results between the two tasks for D1R and D2R KO mice may be due to the differences in the motivation for the rota-rod and Step-Wheel tasks, aversion- and reward-driven, respectively. The Step-Wheel system may become a useful tool for assessing the motor ability of WT and mutant mice.

Spatial and stimulus-type tuning in the LEC, MEC, POR, PrC, CA1, and CA3 during spontaneous item recognition memory.

According to the "two streams" hypothesis, the lateral entorhinal (LEC) and the perirhinal (PrC) cortices process information related to items (a "what" stream), the postrhinal (POR) and the medial entorhinal cortices (MEC) process spatial information (a "where" stream), and both types of information are integrated in the hippocampus (HIP). However, within the framework of memory function, only the HIP is reliably shown to preferentially process spatial information, and the PrC items' features. In contrast, the role of the LEC and MEC in memory is virtually unexplored, and conflicting results emerge for the POR. Moreover, the specific contribution of the hippocampal subfields CA1 and CA3 to spatial and non-spatial memory is not thoroughly understood. To investigate which of these areas is specifically tuned to spatial demands or stimulus identity (odor or object), we assessed the pattern of activation of these areas during recognition memory by detecting the immediate-early gene Arc, commonly used as a marker of neuronal activation. We report that all MTL areas were recruited during the spatial and the non-spatial tasks. However, the LEC, MEC, POR, and CA1 were activated to a comparable level in spatial and non-spatial tasks, while the PrC was tuned to stimulus-type, not spatial demands, and CA3 to spatial demands but not stimulus-type. Results are discussed within the frame of a recent model suggesting that the MTL could be segregated in terms of memory processes, such as recollection and familiarity, rather than information content.

Proximodistal segregation of nonspatial information in CA3: preferential recruitment of a proximal CA3-distal CA1 network in nonspatial recognition memory.

A prevailing view in memory research is that CA3 principally supports spatial processes. However, few studies have investigated the contribution of CA3 to nonspatial memory function. Interestingly, the proximal part of CA3 (close to the dentate gyrus) predominantly projects to distal CA1 (away from the dentate gyrus), which preferentially processes nonspatial information. Moreover, the cytoarchitecture and connectivity patterns in the proximal and distal parts of CA3 strongly differ, suggesting a functional segregation in this area. Here, we tested whether CA3 is recruited during nonspatial recognition memory, and whether nonspatial information is differentially represented along the proximodistal axis of CA3. Furthermore, we investigated whether the pattern of activation within CA3 would mirror that of CA1. We used a high-resolution imaging technique specifically designed to analyze brain activity in distant areas that is based on the detection of the expression of the immediate-early gene Arc, used as a marker of neuronal activation. We showed that proximal CA3 is strongly recruited during a nonspatial delayed nonmatching-to-sample recognition memory task in rats, while distal CA3 is not. In addition, distal CA1 was more activated than proximal CA1 in the same task. These findings suggest a functional segregation of CA3 that mirrors that of CA1, and potentially indicate the existence of a proximal CA3-distal CA1 hippocampal subnetwork that would preferentially process nonspatial information during recognition memory.

Single-neuron diversity generated by Protocadherin-ß cluster in mouse central and peripheral nervous systems.

The generation of complex neural circuits depends on the correct wiring of neurons with diverse individual characteristics. To understand the complexity of the nervous system, the molecular mechanisms for specifying the identity and diversity of individual neurons must be elucidated. The clustered protocadherins (Pcdh) in mammals consist of approximately 50 Pcdh genes (Pcdh-α, Pcdh-ß, and Pcdh-γ) that encode cadherin-family cell surface adhesion proteins. Individual neurons express a random combination of Pcdh-α and Pcdh-γ, whereas the expression patterns for the Pcdh-ß genes, 22 one-exon genes in mouse, are not fully understood. Here we show that the Pcdh-ß genes are expressed in a 3'-polyadenylated form in mouse brain. In situ hybridization using a pan-Pcdh-ß probe against a conserved Pcdh-ß sequence showed widespread labeling in the brain, with prominent signals in the olfactory bulb, hippocampus, and cerebellum. In situ hybridization with specific probes for individual Pcdh-ß genes showed their expression to be scattered in Purkinje cells from P10 to P150. The scattered expression patterns were confirmed by performing a newly developed single-cell 3'-RACE analysis of Purkinje cells, which clearly demonstrated that the Pcdh-ß genes are expressed monoallelically and combinatorially in individual Purkinje cells. Scattered expression patterns of individual Pcdh-ß genes were also observed in pyramidal neurons in the hippocampus and cerebral cortex, neurons in the trigeminal and dorsal root ganglion, GABAergic interneurons, and cholinergic neurons. Our results extend previous observations of diversity at the single-neuron level generated by Pcdh expression and suggest that the Pcdh-ß cluster genes contribute to specifying the identity and diversity of individual neurons.

A novel instrumented multipeg running wheel system, Step-Wheel, for monitoring and controlling complex sequential stepping in mice.

Motor control is critical in daily life as well as in artistic and athletic performance and thus is the subject of intense interest in neuroscience. Mouse models of movement disorders have proven valuable for many aspects of investigation, but adequate methods for analyzing complex motor control in mouse models have not been fully established. Here, we report the development of a novel running-wheel system that can be used to evoke simple and complex stepping patterns in mice. The stepping patterns are controlled by spatially organized pegs, which serve as footholds that can be arranged in adjustable, ladder-like configurations. The mice run as they drink water from a spout, providing reward, while the wheel turns at a constant speed. The stepping patterns of the mice can thus be controlled not only spatially, but also temporally. A voltage sensor to detect paw touches is attached to each peg, allowing precise registration of footfalls. We show that this device can be used to analyze patterns of complex motor coordination in mice. We further demonstrate that it is possible to measure patterns of neural activity with chronically implanted tetrodes as the mice engage in vigorous running bouts. We suggest that this instrumented multipeg running wheel (which we name the Step-Wheel System) can serve as an important tool in analyzing motor control and motor learning in mice.