Izumi Sugihara

Inwardly rectifying currents in hair cells and supporting cells in the goldfish sacculus.

1. Inwardly rectifying ionic currents were studied using patch‐clamp recording methods in oscillatory‐type and spike‐type hair cells and supporting cells dissociated from the goldfish sacculus. These cells had different types of inwardly rectifying currents. The biophysical properties of these currents were investigated. 2. A unique potassium current (Isc) was the sole ionic current recognized in supporting cells. Isc was active throughout the membrane potential range between +30 and ‐170 mV, but showed weak inward rectification and no inactivation. 3. In spike‐type hair cells, inwardly rectifying current (Ik1) was selectively permeable to K+ (K+:Na+ permeability ratio, 1:0.0021). Ik1 could underlie the high negative resting potential of these hair cells because it is partially active at this potential. The strong inward rectification of Ik1 contributed to the low negative plateau potential seen in spike‐type hair cells. 4. In oscillatory‐type hair cells, hyperpolarization‐activated potassium‐sodium current (Ih), which had properties similar to that in photoreceptor and other neurons, was present instead of inwardly rectifying K+ current. 5. In the cell‐attached and inside‐out modes with 125 microM external K+ ([K+]o), IK1 channel had a unitary conductance of 27 pS and showed inactivation with increasing hyperpolarization. Putative Ih and Iso single channels had unitary conductances of 7 and 61 pS, respectively, in the cell‐attached mode with 125 microM Ko+.

Close correlation between the birth date of purkinje cells and the longitudinal compartmentalization of the mouse adult cerebellum

The adult cerebellum is organized into longitudinal compartments that are revealed by specific axonal projections (olivocerebellar and corticonuclear projections). These compartments in the adult cerebellum are closely correlated with the striped expression of zebrin II (aldolase C), a late‐onset marker of Purkinje cells. Similarly, the embryonic cerebellum is organized into longitudinal compartments that are revealed by striped expression of other genes (early‐onset markers). The cerebellar compartments are thought to be the basic and functional subdivisions of the cerebellum. However, the relationship between the embryonic (early‐onset) and the adult (late‐onset) compartments has remained unknown, because the pattern of the embryonic compartments is distinct from that of the adult compartments. To examine this issue, we labeled Purkinje cells (PCs) born at embryonic day (E) 10.5, E11.5, and E12.5 by using an adenoviral vector and traced their fated positions in the adult cerebellum. By comparing the striped distribution of each cohort of birth date‐related PCs with the striped pattern of zebrin II immunoreactivity (zebrin II bands) in the entire adult cerebellum, we found that the striped distribution of PCs correlated strikingly with zebrin II bands. Generally, a single early‐onset compartment was transformed directly into a single late‐onset compartment. Therefore, our observation also indicated the close correlation between the compartments formed by birth date‐related PCs and olivocerebellar projections. Furthermore, we found that the cerebellum was composed of three units showing lateral‐to‐medial developmental gradients, as revealed by the birth dates of PCs. The results suggest that PC birth dates play an important role in organizing cerebellar compartmentalization. J. Comp. Neurol. 519:2594–2614, 2011.

Post-lesion transcommissural growth of olivary climbing fibres creates functional synaptic microzones

In the adult mammalian central nervous system, reinnervation and recovery from trauma is limited. During development, however, postlesion plasticity may generate alternate paths, providing models to investigate reinnervating axon–target interactions. After unilateral transection of the neonatal rat olivocerebellar path, axons from the ipsilateral inferior olive grow into the denervated hemicerebellum and develop climbing fibre (CF)‐like arbors on Purkinje cells (PCs). However, the synaptic function and extent of PC reinnervation remain unknown. In adult rats pedunculotomized on postnatal day 3 the morphological and electrophysiological properties of reinnervating olivocerebellar axons were studied, using axonal reconstruction and patch‐clamp PC recording of CF‐induced synaptic currents. Reinnervated PCs displayed normal CF currents, and the frequency of PC reinnervation decreased with increasing laterality. Reinnervating CF arbors were predominantly normal but 6% branched within the molecular layer forming smaller secondary arbors. CFs arose from transcommissural olivary axons, which branched extensively near their target PCs to produce on average 36 CFs, which is six times more than normal. Axons terminating in the hemisphere developed more CFs than those terminating in the vermis. However, the precise parasagittal microzone organization was preserved. Transcommissural axons also branched, although to a lesser extent, to the deep cerebellar nuclei and terminated in a distribution indicative of the olivo‐cortico‐nuclear circuit. These results show that reinnervating olivocerebellar axons are highly plastic in the cerebellum, compensating anatomically and functionally for early postnatal denervation, and that this reparation obeys precise topographic constraints although axonal plasticity is modified by target (PC or deep nuclear neurons) interactions.

Morphology of axon collaterals of single climbing fibers in the deep cerebellar nuclei of the rat

Projection of inferior olive (IO) neurons to the deep cerebellar nuclei (CN) was investigated in the rat by reconstructing single axons that were labeled with biotinylated dextran amine injected into the IO. All reconstructed terminal arborizations in the CN (n = 18) arose as collaterals from climbing fibers (CFs). One to six nuclear collaterals were given off from each of six CFs that were reconstructed along the nearly entire pathway backward from cortical terminal arborizations to the IO. Nuclear collaterals were much thinner (0.2-0.3 micron in diameter) than stem axons projecting to Purkinje cells (0.7-1.4 microns). The number of swellings per a single nuclear collateral ranged from 24 to 118 (n = 18). Terminal arborizations of nuclear collateral originating from a single CF spread for some hundreds of micrometers and occupied a localized portion within the CN.

Potassium currents underlying the oscillatory response in hair cells of the goldfish sacculus.

1. Ionic currents underlying the oscillatory response of membrane potential were studied in oscillatory‐type hair cells isolated from the goldfish sacculus with the whole‐cell recording method using a patch pipette. 2. Bath application of 4‐aminopyridine (4‐AP; 10 mM) reversibly produced moderate depolarization of the resting potential along with complete suppression of the oscillatory response. Sustained injection of a small depolarizing current also suppressed the oscillatory response. 3. A 4‐AP‐sensitive atypical A‐type K+ current which had a high threshold voltage for inactivation (IA(H)) was found to be a major outward current underlying the oscillatory response. 4. IA(H) was activated with a time constant of 0.4‐10 ms and was inactivated slowly with a time constant of 0.6‐2 s. IA(H) activation and inactivation occurred mostly at membrane potentials more positive than ‐70 mV. 5. There was a clear correlation between activation speed of IA(H) and the frequency of pulse‐evoked oscillation. A ‘hump’‐type response was produced in about one‐quarter of the oscillatory‐type hair cells.

Isochrony in the olivocerebellar system underlies complex spike synchrony

In a recent issue of The Journal of PhysiologyBaker & Edgley (2006) published results which they interpreted as showing a non-uniform olivocerebellar conduction time in the rat. Baker and Edgley used the climbing fibre reflex to measure conduction time to different points along a folial wall. This reflex is initiated by electrical stimulation of the cerebellar white matter, which triggers antidromic spikes in olivocerebellar axons that invade the inferior olive (IO). Current generated by these spikes spreads to neighbouring IO neurones via the gap junctions that electrically couple IO neurones (Llinas et al. 1974). This spreading excitation can trigger orthodromic spikes that return to the cerebellar cortex and trigger Purkinje cell complex spikes (CSs). Baker and Edgley found that climbing fibre reflex latency varied with recording electrode depth, and inferred from this that conduction time between the IO and cerebellar cortex varies systematically with cortical location. This study conflicts with results by us and others that indicate a near uniform olivocerebellar conduction time in rats and turtles (Sugihara et al. 1993; Lang & Rosenbluth, 2003; Ariel, 2005). Below we discuss several troubling issues with this new study, but first we would like to make the point that spontaneous CS synchrony patterns almost necessitate a uniform olivocerebellar conduction time.

Spatial rearrangement of Purkinje cell subsets forms the transverse and longitudinal compartmentalization in the mouse embryonic cerebellum: Vibulyaseck et al.

Transversely oriented lobules and longitudinally arrayed stripes of Purkinje cell subsets subdivide the cerebellar cortex into multiple compartments that are involved in diverse functions. In the mammalian cerebellum, anterior, and posterior lobules, which are involved in somatosensorimotor function, show an alternation of aldolase C (zebrin II) ‐positive and ‐negative stripes, whereas the central lobules (lobules VIb–VII and crus I), which are implicated in nonmotor functions, show a laterally expanded arrangement solely of aldolase C‐positive stripes. To understand the developmental process of this compartmental pattern, we identified groups of Purkinje cell subsets in the entire mouse cerebellum at embryonic day (E) 14.5 by staining Purkinje cell subset markers. We then tracked four major domains of Protocadherin 10 (Pcdh10)‐positive Purkinje cell subsets (medial, dorsal, central, and mid‐lateral subsets), which were clearly demarcated during E14.5–17.5. These domains of Purkinje cell subsets shifted predominantly in the longitudinal direction to be positioned in the anterior and posterior lobules. However, a particular portion of the medial and mid‐lateral domains, and the whole of the central domain shift in the lateral direction to be positioned in the central lobules. The results indicate that while the longitudinal shift of domains of Purkinje cell subsets forms the longitudinally striped compartments in the anterior and posterior cerebellum, the lateral shift of particular domains of Purkinje cell subsets underlies the laterally expanded arrangement of stripes in central lobules. Thus, the rearrangement of Purkinje cell subsets in the embryonic cerebellum is critically related to the compartmental organization in the mammalian cerebellum.

Systematic analysis of neuronal wiring of the rodent deep cerebellar nuclei reveals differences reflecting adaptations at the neuronal circuit and internuclear levels

A common view of the architecture of different brain regions is that, despite their heterogeneity, they have optimized their wiring schemes to make maximal use of space. Based on experimental findings, computational models have delineated how about two‐thirds of the neuropil is filled out with dendrites and axons optimizing cable costs and conduction time while keeping the connectivity at the highest level. However, whether this assumption can be generalized to all brain regions has not yet been tested. Here we quantified and charted the components of the neuropil in the four deep cerebellar nuclei (DCN) of the rats brain. We segmented and traced the neuropil stained with one of two antibodies, one antibody against dendritic microtubule‐associated proteins (MAP2a,b) and the second against the Purkinje cell axons (PCP2). We compared fiber length density, average fiber diameter, and volume fraction within different components of the DCN in a random, systematic fashion. We observed differences in dendritic and axonal fiber length density, average fiber diameters, and volume fraction within the four different nuclei that make up the DCN. We observe a relative increase in the length density of dendrites and Purkinje cell axons in two of the DCN, namely, the posterior interposed nucleus and the lateral nucleus. Furthermore, the DCN have a surprisingly low volume fraction of their dendritic length density, which we propose is related to their special circuitry. In summary, our results show previously unappreciated functional adaptations among these nuclei. J. Comp. Neurol. 522:2481–2497, 2014.

Quickly flickering inwardly rectifying K channels in goldfish hair cell membrane

The kinetics of the quickly flickering inwardly rectifying K channel were studied with the cell-attached patch clamp method in goldfish hair cells. The activity of the channel was very unique in repeating continuously open and closed events at an ultrafast rate. Moreover, open-close events of the channel showed a marked dependence on membrane potential; a shift toward hyperpolarized levels brought about an elongation of closed events resulting in a decrease in the open state probability, although no marked change was produced in open event duration itself. The unitary conductance of the channel was 109 pS as 123 mM KCl solution was used.

Off-oscillation in hair cell membrane is correlated with the decremental response at the hair cell-afferent fibre synapse in the goldfish sacculus.

In this study, we examined the decremental response in the hair cell-afferent fibre synapse of goldfish. This decremental response is a transient reduction in sound-evoked EPSPs associated with a step reduction in sound intensity. To determine its presynaptic correlates, a patch clamp study was performed on oscillatory-type hair cells isolated from the sacculus of goldfish. Injection of depolarizing current produced a damped oscillation in the membrane potential of such cells. When the current intensity was reduced in steps, instead of simply being turned off, very marked dips were produced as off-oscillation in the hair cell membrane potential. These dips correlated with the decremental response in EPSPs.