Tatsuya Umeda

Kinase-Dead Knock-In Mouse Reveals an Essential Role of Kinase Activity of Ca2+/Calmodulin-Dependent Protein Kinase IIα in Dendritic Spine Enlargement, Long-Term Potentiation, and Learning

Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) is an essential mediator of activity-dependent synaptic plasticity that possesses multiple protein functions. So far, the autophosphorylation site-mutant mice targeted at T286 and at T305/306 have demonstrated the importance of the autonomous activity and Ca2+/calmodulin-binding capacity of CaMKIIα, respectively, in the induction of long-term potentiation (LTP) and hippocampus-dependent learning. However, kinase activity of CaMKIIα, the most essential enzymatic function, has not been genetically dissected yet. Here, we generated a novel CaMKIIα knock-in mouse that completely lacks its kinase activity by introducing K42R mutation and examined the effects on hippocampal synaptic plasticity and behavioral learning. In homozygous CaMKIIα (K42R) mice, kinase activity was reduced to the same level as in CaMKIIα-null mice, whereas CaMKII protein expression was well preserved. Tetanic stimulation failed to induce not only LTP but also sustained dendritic spine enlargement, a structural basis for LTP, at the Schaffer collateral–CA1 synapse, whereas activity-dependent postsynaptic translocation of CaMKIIα was preserved. In addition, CaMKIIα (K42R) mice showed a severe impairment in inhibitory avoidance learning, a form of memory that is dependent on the hippocampus. These results demonstrate that kinase activity of CaMKIIα is a common critical gate controlling structural, functional, and behavioral expression of synaptic memory.

Simultaneous observation of stably associated presynaptic varicosities and postsynaptic spines: morphological alterations of CA3–CA1 synapses in hippocampal slice cultures

Dendritic spines are highly motile structures, but the extent and mode of coordination in motility between spines and presynaptic varicosities with synaptic contacts is not clear. To analyze movements of dendritic spines and axonal varicosities simultaneously, we labeled CA1 pyramidal cells with green fluorescent protein and CA3 pyramidal cells with rhodamine-dextran in hippocampal slice cultures. Varicosities and spines were visualized using two-photon microscopy to detect close association of two components. Time-lapse imaging revealed that they performed rapid morphological changes without losing their contacts. The extent of overall structural changes between varicosities and spines was correlated, while the direction of short-term volume changes was regulated independently. Furthermore, alterations of dendritic morphology induced by strong electrical stimulation had little effects on their association. These results indicate the presence of local regulatory mechanisms to coordinate presynaptic and postsynaptic motility.

Large-scale reorganization of corticofugal fibers after neonatal hemidecortication for functional restoration of forelimb movements.

As an experimental model to study the mechanism of large‐scale network plasticity of the juvenile brain, functional compensation after neonatal brain damage was studied in rats that received unilateral decortication at postnatal day 5. These animals exhibited a marked ability in reaching and grasping movements in the contralesional side of the forelimb when tested at 10–14 weeks of age. Additional lesion of the sensorimotor cortex in the remaining contralesional hemisphere at this stage resulted in severe impairment of both forelimbs. It was suggested that the sensorimotor cortex on the contralesional side was controlling the movements of both forelimbs. Following the injection of an anterograde tracer into the remaining sensorimotor cortex, the corticofugal axons from the remaining sensorimotor cortex were found to issue aberrant projections to the contralateral red nucleus, contralateral superior colliculus, contralateral pontine nuclei, ipsilateral dorsal column nucleus and ipsilateral gray matter of the cervical spinal cord, all of which appeared to be necessary for the control of contralesional forelimb movements. These results suggest that the forelimb movements on the contralesional side were compensated by large‐scale reorganization of the corticofugal axons from the remaining sensorimotor cortex.

Formation of descending pathways mediating cortical command to forelimb motoneurons in neonatally hemidecorticated rats.

Neonatally hemidecorticated rats show fairly normal reaching and grasping behaviors of the forelimb contralateral to the lesion at the adult stage. Previous experiments using an anterograde tracer showed that the corticospinal fibers originating from the sensorimotor cortex of the intact side projected aberrant collaterals to the spinal gray matter on the ipsilateral side. The present study used electrophysiological methods to investigate whether the aberrant projections of the corticospinal tract mediated the pyramidal excitation to the ipsilateral forelimb motoneurons and, if so, which pathways mediate the effect in the hemidecorticated rats. Electrical stimulation to the intact medullary pyramid elicited bilateral negative field potentials in the dorsal horn of the spinal cord. In intracellular recordings of forelimb motoneurons, oligosynaptic pyramidal excitation was detected on both sides of the spinal cord in the hemidecorticated rats, whereas pyramidal excitation of motoneurons on the side ipsilateral to the stimulation was much smaller in normal rats. By lesioning the dorsal funiculus at the upper cervical level, we clarified that the excitation was transmitted to the ipsilateral motoneurons by at least two pathways: one via the corticospinal tract and spinal interneurons and the other via the cortico-reticulo-spinal pathways. These results suggested that in the neonatally hemidecorticated rats, the forelimb movements on the side contralateral to the lesion were modulated by motor commands through the indirect ipsilateral descending pathways from the sensorimotor cortex of the intact side either via the spinal interneurons or reticulospinal neurons.

Electroporation-mediated gene transfer system applied to cultured CNS neurons

Electroporation is effective in transferring foreign genes into immature neurons in intact brain tissue. We utilized this approach to transfect genes into developing rodent hippocampi. Transfected hippocampi were subsequently dissociated and allowed to differentiate in culture. By optimizing developmental stage of the hippocampus, promoters to drive the marker cDNA, and culture conditions, neurons kept strong expression of multiple marker genes for more than two weeks after electroporation. We could also induce transient expression in mature neurons by combining electroporation of plasmids containing loxP-flanked stopper sequences and infection of Cre-producing recombinant adenoviruses. The system described here is useful in analyzing biological roles of multiple genes in specific stages of neuronal development.

Differential contributions of rostral and caudal frontal forelimb areas to compensatory process after neonatal hemidecortication in rats

Following brain damage, especially in juvenile animals, large‐scale reorganization is known to occur in the remaining brain structures to compensate for functional deficits. In rats with neonatal hemidecortication, corticospinal fibers originating from the undamaged side of the sensorimotor cortex issue collateral sprouts to the ipsilateral spinal gray matter that mediate cortical excitation to ipsilateral forelimb motoneurons and compensate for the deficit in forelimb movements. The present study was designed to investigate the origins of the ipsilateral corticospinal projection in neonatally hemidecorticated rats. Corticospinal neurons (CSNs) were labeled in adults by injecting retrograde neural tracers, cholera toxin subunit B with different fluorescent probes, into either side of the cervical spinal gray matter. In the undamaged cortex, double‐labeled neurons were rarely found. CSNs with contralateral projections (contra‐CSNs) and those with ipsilateral projections (ipsi‐CSNs) were distributed both in the rostral forelimb motor area (RFA) and the caudal forelimb motor area (CFA). However, there was a difference in the distributions of the ipsi‐CSNs between the two forelimb areas. Whereas the distribution of the ipsi‐CSNs largely overlapped with that of the contra‐CSNs in the RFA, the ipsi‐CSNs tended to be segregated from the contra‐CSNs in the CFA. The results suggested that the RFA and the CFA contribute to the compensatory process in different ways.

Visualizing synapse formation and remodeling: recent advances in real-time imaging of CNS synapses.

The formation and maintenance of synaptic connections are critical in the development and plasticity of the central nervous system (CNS). Until recently, there have been few studies that followed the molecular sequences of the CNS synapse formation and maintenance. This situation changed dramatically after the introduction of green fluorescent protein (GFP)-based fluorescent probes and the development of lipophilic tracers of endocytotic membranes. These techniques enabled us to visualize presynaptic and postsynaptic structures in living neurons and illustrated active transport and remodeling of synaptic components. Furthermore, recent attempts to identify correlation between presynaptic and postsynaptic morphogenesis suggested very rapid time course of synapse formation at the individual axo-dendritic contact sites. These recent works clearly demonstrated the power of real-time imaging studies. Development of a wide variety of fluorescent probes and advances in the imaging techniques in future will further extend our knowledge on the molecular events that take place in the process of the development and maturation of synaptic junctions.

Population Coding of Forelimb Joint Kinematics by Peripheral Afferents in Monkeys

Various peripheral receptors provide information concerning position and movement to the central nervous system to achieve complex and dexterous movements of forelimbs in primates. The response properties of single afferent receptors to movements at a single joint have been examined in detail, but the population coding of peripheral afferents remains poorly defined. In this study, we obtained multichannel recordings from dorsal root ganglion (DRG) neurons in cervical segments of monkeys. We applied the sparse linear regression (SLiR) algorithm to the recordings, which selects useful input signals to reconstruct movement kinematics. Multichannel recordings of peripheral afferents were performed by inserting multi-electrode arrays into the DRGs of lower cervical segments in two anesthetized monkeys. A total of 112 and 92 units were responsive to the passive joint movements or the skin stimulation with a painting brush in Monkey 1 and Monkey 2, respectively. Using the SLiR algorithm, we reconstructed the temporal changes of joint angle, angular velocity, and acceleration at the elbow, wrist, and finger joints from temporal firing patterns of the DRG neurons. By automatically selecting a subset of recorded units, the SLiR achieved superior generalization performance compared with a regularized linear regression algorithm. The SLiR selected not only putative muscle units that were responsive to only the passive movements, but also a number of putative cutaneous units responsive to the skin stimulation. These results suggested that an ensemble of peripheral primary afferents that contains both putative muscle and cutaneous units encode forelimb joint kinematics of non-human primates.

Reorganization of sensory pathways after neonatal hemidecortication in rats

We investigated ascending somatosensory pathways in neonatally hemidecorticated rats. Injection of an anterograde tracer, biotinylated dextran amine (BDA), into the contralesional dorsal root ganglions revealed ipsilateral projections to the dorsal column nuclei (DCN) in hemidecorticated rats as well as in normal rats. Injection of BDA into the DCN on the same side revealed that while most axons projected to the contralateral thalamus, some axons were detected in the ipsilateral thalamus in hemidecorticated rats while such projections were rarely detected in normal rats. The results suggest that aberrant ipsilateral projections of DCN neurons contralateral to the lesion developed after the hemidecortication.

Decoding of the spike timing of primary afferents during voluntary arm movements in monkeys

Understanding the mechanisms of encoding forelimb kinematics in the activity of peripheral afferents is essential for developing a somatosensory neuroprosthesis. To investigate whether the spike timing of dorsal root ganglion (DRG) neurons could be estimated from the forelimb kinematics of behaving monkeys, we implanted two multi-electrode arrays chronically in the DRGs at the level of the cervical segments in two monkeys. Neuronal activity during voluntary reach-to-grasp movements were recorded simultaneously with the trajectories of hand/arm movements, which were tracked in three-dimensional space using a motion capture system. Sixteen and 13 neurons, including muscle spindles, skin receptors, and tendon organ afferents, were recorded in the two monkeys, respectively. We were able to reconstruct forelimb joint kinematics from the temporal firing pattern of a subset of DRG neurons using sparse linear regression (SLiR) analysis, suggesting that DRG neuronal ensembles encoded information about joint kinematics. Furthermore, we estimated the spike timing of the DRG neuronal ensembles from joint kinematics using an integrate-and-fire model (IF) incorporating the SLiR algorithm. The temporal change of firing frequency of a subpopulation of neurons was reconstructed precisely from forelimb kinematics using the SLiR. The estimated firing pattern of the DRG neuronal ensembles encoded forelimb joint angles and velocities as precisely as the originally recorded neuronal activity. These results suggest that a simple model can be used to generate an accurate estimate of the spike timing of DRG neuronal ensembles from forelimb joint kinematics, and is useful for designing a proprioceptive decoder in a brain machine interface.