Rie Kimura

Protein kinase CK2 modulates synaptic plasticity by modification of synaptic NMDA receptors in the hippocampus

Synaptic plasticity is the foundation of learning and memory. The protein kinase CK2 phosphorylates many proteins related to synaptic plasticity, but whether it is directly involved in it has not been clarified. Here, we examined the role of CK2 in synaptic plasticity in hippocampal slices using the CK2 selective inhibitors 5,6‐dichloro‐1‐β‐d‐ribofuranosylbenzimidazole (DRB) and 4,5,6,7‐tetrabromobenzotriazole (TBB). These significantly inhibited N‐methyl‐d‐aspartate (NMDA) receptor‐dependent long‐term potentiation (LTP). DRB also inhibited NMDA receptor‐mediated synaptic transmission, while leaving NMDA receptor‐independent LTP unaffected. NMDA receptors thus appear to be the primary targets of CK2. Although both long‐term depression (LTD) and LTP are induced by the influx of Ca2+ through NMDA receptors, surprisingly, LTD was not affected by CK2 inhibitors. We postulated that the LTP‐selective modulation by CK2 is due to selective modulation of NMDA receptors, and tested two hypotheses concerning the modulation of NMDA receptors: (i) CK2 selectively modulates NR2A subunits possibly related to LTP, but not NR2B subunits possibly related to LTD; and (ii) CK2 selectively affects synaptic but not extrasynaptic NMDA receptors whose activation is sufficient to induce LTD. DRB decreased NMDA receptor‐mediated synaptic transmission in the presence of selective NR2A subunit antagonist. The former hypothesis thus appears unlikely to be correct. However, DRB decreased synaptic NMDA receptor responses in cultured hippocampal neurons without affecting extrasynaptic NMDA receptor current. These findings support the latter hypothesis, that CK2 selectively affects LTP by selective modification of synaptic NMDA receptors in a receptor‐location‐specific manner.

Reinforcing operandum: rapid and reliable learning of skilled forelimb movements by head-fixed rodents.

Stereotaxic head fixation plays a necessary role in current physiological techniques, such as in vivo whole cell recording and two-photon laser-scanning microscopy, that are designed to elucidate the cortical involvement in animal behaviors. In rodents, however, head fixation often inhibits learning and performance of behavioral tasks. In particular, it has been considered inappropriate for head-fixed rodents to be operantly conditioned to perform skilled movements with their forelimb (e.g., lever-press task), despite the potential applicability of the task. Here we have solved this problem conceptually by integrating a lever (operandum) and a rewarding spout (reinforcer) into one ″spout-lever″ device for efficient operant learning. With this device, head-fixed rats reliably learned to perform a pull manipulation of the spout-lever with their right forelimb in response to an auditory cue signal (external-trigger trial, namely, Go trial) within several days. We also demonstrated stable whole cell recordings from motor cortex neurons while the rats were performing forelimb movements in external-trigger trials. We observed a behavior-related increase in the number of action potentials in membrane potential. In the next session, the rats, which had already learned the external-trigger trial, effortlessly performed similar spout-lever manipulation with no cue presentation (internal-trigger trial) additionally. Likewise, some of the rats learned to keep holding the spout-lever in response to another cue signal (No-go trial) in the following session, so that they mastered the Go/No-go discrimination task in one extra day. Our results verified the usefulness of spout-lever manipulation for behavioral experiments employing cutting-edge physiological techniques.

Integrative spike dynamics of rat CA1 neurons: a multineuronal imaging study

The brain operates through a coordinated interplay of numerous neurons, yet little is known about the collective behaviour of individual neurons embedded in a huge network. We used large‐scale optical recordings to address synaptic integration in hundreds of neurons. In hippocampal slice cultures bolus‐loaded with Ca2+ fluorophores, we stimulated the Schaffer collaterals and monitored the aggregate presynaptic activity from the stratum radiatum and individual postsynaptic spikes from the CA1 stratum pyramidale. Single neurons responded to varying synaptic inputs with unreliable spikes, but at the population level, the networks stably output a linear sum of synaptic inputs. Nonetheless, the network activity, even though given constant stimuli, varied from trial to trial. This variation emerged through time‐varying recruitment of different neuron subsets, which were shaped by correlated background noise. We also mapped the input‐frequency preference in spiking activity and found that the majority of CA1 neurons fired in response to a limited range of presynaptic firing rates (20–40 Hz), acting like a band‐pass filter, although a few neurons had high pass‐like or low pass‐like characteristics. This frequency selectivity depended on phasic inhibitory transmission. Thus, our imaging approach enables the linking of single‐cell behaviours to their communal dynamics, and we discovered that, even in a relatively simple CA1 circuit, neurons could be engaged in concordant information processing.

Similarity in Neuronal Firing Regimes across Mammalian Species

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.

Large‐scale analysis reveals populational contributions of cortical spike rate and synchrony to behavioural functions

There have been few systematic population‐wide analyses of relationships between spike synchrony within a period of several milliseconds and behavioural functions. In this study, we obtained a large amount of spike data from > 23,000 neuron pairs by multiple single‐unit recording from deep layer neurons in motor cortical areas in rats performing a forelimb movement task. The temporal changes of spike synchrony in the whole neuron pairs were statistically independent of behavioural changes during the task performance, although some neuron pairs exhibited correlated changes in spike synchrony. Mutual information analyses revealed that spike synchrony made a smaller contribution than spike rate to behavioural functions. The strength of spike synchrony between two neurons was statistically independent of the spike rate‐based preferences of the pair for behavioural functions.

Hippocampal Polysynaptic Computation

Neural circuitry is a self-organizing arithmetic device that converts input to output and thereby remodels its computational algorithm to produce more desired output; however, experimental evidence regarding the mechanism by which information is modified and stored while propagating across polysynaptic networks is sparse. We used functional multineuron calcium imaging to monitor the spike outputs from thousands of CA1 neurons in response to the stimulation of two independent sites of the dentate gyrus in rat hippocampal networks ex vivo. Only pyramidal cells were analyzed based on post hoc immunostaining. Some CA1 pyramidal cells were observed to fire action potentials only when both sites were simultaneously stimulated (AND-like neurons), whereas other neurons fired in response to either site of stimulation but not to concurrent stimulation (XOR-like neurons). Both types of neurons were interlaced in the same network and altered their logical operation depending on the timing of paired stimulation. Repetitive paired stimulation for brief periods induced a persistent reorganization of AND and XOR operators, suggesting a flexibility in parallel distributed processing. We simulated these network functions in silico and found that synaptic modification of the CA3 recurrent excitation is pivotal to the shaping of logic plasticity. This work provides new insights into how microscopic synaptic properties are associated with the mesoscopic dynamics of complex microcircuits.

Ensemble spiking activities in rat motor cortex during externally- and internally-initiated movements

Previous study (Isomura et al., Nat. Neurosci., 12(12): 1586–1593, 2009) reported that the neurons of motor cortex of rats, which may be responsible for motor preparation, initiation, and execution, often discharge synchronously within several milliseconds. Here, we examined whether such coordinated ensemble activities in a local neuronal circuit are dynamically affected by external environment or internal brain state. We improved our behavioral task system to train head-restrained rats to learn an operant lever manipulation only in three days. After the behavioral task training, the animals efficiently learned to perform an externally-/internally-initiated forelimb movement task, in which they had to pull a lever using their forelimb when a cue tone was presented (externally-initiated trials) or pull it spontaneously without the cue tone (internally-initiated trials) to acquire a drop of saccharin water as a reward (typically total 1000–1500 success trials for 3 hours). Then, we analyzed the difference in functional neuronal activity between the externally-initiated and internally-initiated forelimb movements, by our multi-neuron recording and/or juxtacellular recording from the forelimb area of motor cortex in these task-performing animals. Research fund: CREST, KAKENHI(22120520, 21680036).

Remodeling of information computation by hippocampal polysynaptic circuits

To study cellular functions in situ in realtime, we developed a fibercoupled confocal microscope, (FCM) and observed fluorescently labeled cells in the organs of animal. The microscope system consisted of microlens-attached Nipkow disk (Yokogawa), and an imaging fiber (IF, FiberTech). The IF had 10,000 of single mode flexible fiber of 3.75 m in diameter, which was scanned with multiple laser beams to form a confocal image. The tip of IF was 0.75 mm thick, for easier approach to the target inside the organ without a large physical damage. When the incidental face of IF was placed below the surface of organs, many spots (50 cells) were clearly visible in the field of 300 m2, showing fluctuation in fluorescence intensity (breathing and some cellular activities). Such an in situ imaging by FCM is very promising for detailed studies on the relationship between cell structure and functions.