[Roundtable discussion: advances in neuroscience and therapeutic research on neurological diseases--the present status and future prospects].
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Biomedical Research↗
Biomedical subjects
Tadashi Isa
Publications and source records attributed to Tadashi Isa.
Electrophysiological abnormalities precede apparent histological demyelination in the central nervous system of mice overexpressing proteolipid protein.
Myelin proteolipid protein (plp), a major myelin protein in the CNS, has been proposed to function in myelin assembly. Transgenic mice overexpressing the plp gene by introduction of two extra wild-type (Wt) mouse plp genes (plp(tg/-)) exhibit normal myelination and ion channel clustering at the age of 2 months. However, at the age of 5 months, demyelination becomes observable, accompanied by a reduction in the number of K+ channel clusters at Ranvier's node and a progressive increase in motor deficit. To clarify how these age-dependent changes are related to nerve conduction in the CNS, we analyzed the conduction velocity (CV) and relative refractory period (RRP) of identified spinal ascending or descending tracts, such as the dorsal column pathway, the vestibulospinal and reticulospinal tracts, and the pyramidal tract, in plp(tg/-) mice 2, 5, and 8 months of age. We found that CVs decreased as age increased. Importantly, CVs were significantly reduced and prolonged RRPs were observed in 2-month-old (2M) plp(tg/-) mice that had no apparent demyelination. Immunohistological examination revealed that densities of Na+ and K+ channel clusters decreased as plp(tg/-) and Wt mice aged. However, a clear correlation was not observed between CVs and mean channel cluster densities or between mean channel cluster densities and progress of demyelination. Performance in the rotarod test was normal in 2M plp(tg/-) mice but deteriorated in mice older than age 5 months. These results suggest that electrophysiological analysis can detect the abnormalities of the plp(tg/-) mice earlier than histological or behavioral measures.
Animals↗
Properties of propriospinal neurons in the C3-C4 segments mediating disynaptic pyramidal excitation to forelimb motoneurons in the macaque monkey.
Candidate propriospinal neurons (PNs) that mediate disynaptic pyramidal excitation to forelimb motoneurons were studied in the C3-C4 segments in anesthetized macaque monkeys (n = 10). A total of 177 neurons were recorded (145 extracellularly, 48 intracellularly, and 16 both) in laminae VI-VII. Among these, 86 neurons (73 extracellularly, 14 intracellularly and 1 both) were antidromically activated from the forelimb motor nucleus or from the ventrolateral funiculus just lateral to the motor nucleus in the C6/C7 segments and thus are identified as PNs. Among the 73 extracellularly recorded PNs, 60 cells were fired by a train of four stimuli to the contralateral pyramid with segmental latencies of 0.8-2.2 ms, with most of them (n = 52) in a monosynaptic range (<1.4 ms including one synaptic delay and time to firing). The firing probability was only 21% from the third pyramidal volley but increased to 83% after intravenous injection of strychnine. In most of the intracellularly recorded PNs, stimulation of the contralateral pyramid evoked monosynaptic excitatory postsynaptic potentials (EPSPs, 12/14) and disynaptic inhibitory postsynaptic potentials (14/14), which were found to be glycinergic. In contrast, cells that did not project to the C6-Th1 segments where forelimb motoneurons are located were classified as segmental interneurons. These were fired from the third pyramidal volley with a probability of 71% before injection of strychnine. It is proposed that some of these interneurons mediate feed-forward inhibition to the PNs. These results suggest that the C3-C4 PNs receive feed-forward inhibition from the pyramid in addition to monosynaptic excitation and that this inhibition is stronger in the macaque monkey than in the cat. Another difference with the cat was that only 26 of the 86 PNs (30%, as compared with 84% in the cat) with projection to the forelimb motor nuclei send ascending collaterals terminating in the lateral reticular nucleus (LRN) on the ipsilateral side of the medulla. Thus we identified C3-C4 PNs that could mediate disynaptic pyramidal excitation to forelimb motoneurons in the macaque monkey. The present findings explain why it was difficult in previous studies of the macaque monkey to evoke disynaptic pyramidal excitation via C3-C4 PNs in forelimb motoneurons and why-as compared with the cat-the monosynaptic EPSPs evoked from the LRN via C3-C4 PNs were smaller in amplitude.
Animals↗
Properties of cholinergic responses in neurons in the intermediate grey layer of rat superior colliculus.
The intermediate grey layer (SGI) of superior colliculus (SC) receives cholinergic innervation from brainstem parabrachial region. To clarify the action of cholinergic inputs to local circuits in the SGI, we investigated the effect of cholinergic agonists and antagonists on a large number of randomly sampled neurons in Wistar rat SGI (n=246) using whole-cell patch clamp technique in slices of the rat SC. Responses of the recorded cells (n=98) to bath application of carbachol were classified into five patterns: (i) nicotinic inward only (n=14); (ii) nicotinic inward+muscarinic inward (n=26); (iii) nicotinic inward+muscarinic inward+muscarinic outward (n=39); (iv) nicotinic inward+muscarinic outward (n=13) and (v) muscarinic outward only (n=4). Among these, a majority of morphologically identified projection neurons exhibited either response pattern (ii) (9/28) or (iii) (15/28), which suggested that the primary action of cholinergic inputs on the SGI output is excitatory. Nicotinic receptor subtypes involved in the nicotinic current were examined by testing the effects of antagonists on the currents induced by bath application of 1,1-dimethyl-4-phenyl-piperazinium or transient pressure application of acetylcholine (ACh). Muscarinic receptor subtypes involved in the muscarinic inward and outward currents were investigated by examining the effects of antagonists on muscarine-induced currents. The results showed that nicotinic inward currents are mediated mainly by alpha4beta2 and partly by alpha7 nicotinic receptors and that muscarinic inward and outward currents are mediated by M3 (plus M1) and M2 muscarinic receptors, respectively.
Action Potentials↗
[New guidelines and standards in revised Animal Rights Act].
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Animal Experimentation↗
Nicotinic acetylcholine receptor subtypes involved in facilitation of GABAergic inhibition in mouse superficial superior colliculus.
The superficial superior colliculus (sSC) is a key station in the sensory processing related to visual salience. The sSC receives cholinergic projections from the parabigeminal nucleus, and previous studies have revealed the presence of several different nicotinic acetylcholine receptor (nAChR) subunits in the sSC. In this study, to clarify the role of the cholinergic inputs to the sSC, we examined current responses induced by ACh in GABAergic and non-GABAergic sSC neurons using in vitro slice preparations obtained from glutamate decarboxylase 67-green fluorescent protein (GFP) knock-in mice in which GFP is specifically expressed in GABAergic neurons. Brief air pressure application of acetylcholine (ACh) elicited nicotinic inward current responses in both GABAergic and non-GABAergic neurons. The inward current responses in the GABAergic neurons were highly sensitive to a selective antagonist for alpha3beta2- and alpha6beta2-containing receptors, alpha-conotoxin MII (alphaCtxMII). A subset of these neurons exhibited a faster alpha-bungarotoxin-sensitive inward current component, indicating the expression of alpha7-containing nAChRs. We also found that the activation of presynaptic nAChRs induced release of GABA, which elicited a burst of miniature inhibitory postsynaptic currents mediated by GABA(A) receptors in non-GABAergic neurons. This ACh-induced GABA release was mediated mainly by alphaCtxMII-sensitive nAChRs and resulted from the activation of voltage-dependent calcium channels. Morphological analysis revealed that recorded GFP-positive neurons are interneurons and GFP-negative neurons include projection neurons. These findings suggest that nAChRs are involved in the regulation of GABAergic inhibition and modulate visual processing in the sSC.
Acetylcholine↗
Electrophysiological and morphological properties of identified crossed tecto-reticular neurons in the rat superior colliculus.
Previously we classified randomly sampled neurons in the intermediate layer (SI) of the rat superior colliculus (SC) into six subclasses according to their firing responses to depolarizing current pulses and five subclasses based on their morphological properties in slice preparations. In the present study, we investigated properties of a major output cell group of the rat SC (PND 17-24), crossed tecto-reticular neurons (cTRNs), which project to the contralateral medial pontine reticular formation. The cTRNs were identified by retrograde labeling with a fluorescent tracer (n=112). We compared their properties with those of presumed interneurons (n=127). We found that a majority of cTRNs were regular spiking neurons with moderate firing frequency (73%) and were multipolar-shaped (66%). The cTRNs had larger membrane capacitance, larger soma size and lower input impedance than presumed interneurons. Electrical stimulation of the superficial gray layer induced oligosynaptic EPSPs in the cTRNs. When bicuculline was added to the extracellular solution, the EPSPs were markedly enhanced and bursting spike responses were induced. The bursting responses were suppressed by applying D-2-amino-5-phosphonovalerate. These results suggest that the cTRNs exhibit NMDA receptor-dependent bursting responses to visual inputs. These observations give insights into the neuronal mechanism of generating burst activity in cTRNs, which triggers orienting behaviors.
Animals↗
Organization of interlaminar interactions in the rat superior colliculus.
Our previous studies have shown that when slices of the rat superior colliculus (SC) are exposed to a solution containing 10 microM bicuculline and a low concentration of Mg2+ (0.1 mM), most neurons in the intermediate gray layer (stratum griseum intermediale; SGI), wide-field vertical (WFV) cells in the optic layer (stratum opticum; SO), and a minor population of neurons in the superficial gray layer (stratum griseum superficiale; SGS) exhibit spontaneous depolarization and burst firing, which are synchronous among adjacent neurons. These spontaneous and synchronous depolarizations were thought to share common mechanisms with presaccadic burst activity in SGI neurons. In the present study, we explored the site responsible for generation of synchronous depolarization of SGI neurons by performing dual whole cell recordings under different slice conditions. A pair of SGI neurons recorded in a small rectangular piece of the SGI punched out from the SC slice showed synchronous depolarization but far less frequently than those recorded in a small rectangular piece including SGS and SO. This suggests that the superficial layers are needed for triggering synchronous depolarization in the SGI. Furthermore, we recorded spontaneous depolarizations in pairs of neurons belonging to the different layers. Analysis of their synchronicity revealed that WFV cells in the SO exhibit synchronous depolarizations with both SGS and SGI neurons, and the onset of spontaneous depolarization in WFV cells precedes those of neurons in other layers. Further, when SGS and SGI neurons exhibit synchronous depolarizations, SGI neurons usually precede the SGS neurons. These observations give further evidence to the existence of interlaminar interaction between superficial and deeper layers of the SC. In addition, it is suggested that WFV cells can trigger burst activity in other layers of the SC and that there is an excitatory signal transmission from the deeper layers to the superficial layers.
Animals↗
Effects of local nicotinic activation of the superior colliculus on saccades in monkeys.
To examine the role of competitive and cooperative neural interactions within the intermediate layer of superior colliculus (SC), we elevated the basal SC neuronal activity by locally injecting a cholinergic agonist nicotine and analyzed its effects on saccade performance. After microinjection, spontaneous saccades were directed toward the movement field of neurons at the injection site (affected area). For visually guided saccades, reaction times were decreased when targets were presented close to the affected area. However, when visual targets were presented remote from the affected area, reaction times were not increased regardless of the rostrocaudal level of the injection sites. The endpoints of visually guided saccades were biased toward the affected area when targets were presented close to the affected area. After this endpoint effect diminished, the trajectories of visually guided saccades remained modestly curved toward the affected area. Compared with the effects on endpoints, the effects on reaction times were more localized to the targets close to the affected area. These results are consistent with a model that saccades are triggered by the activities of neurons within a restricted region, and the endpoints and trajectories of the saccades are determined by the widespread population activity in the SC. However, because increased reaction times were not observed for saccades toward targets remote from the affected area, inhibitory interactions in the SC may not be strong enough to shape the spatial distribution of the low-frequency preparatory activities in the SC.
Animals↗
Laminar specific distribution of lateral excitatory connections in the rat superior colliculus.
Premovement activities in neurons in the intermediate gray layer [stratum griseum intermediale (SGI)] of the mammalian superior colliculus (SC) are essential for initiation of orienting behaviors such as saccades. Our previous study demonstrated that burst activities are induced by synchronous activation of SGI neurons communicating within a local excitatory network, which depends on NMDA-receptor-dependent synaptic transmission and release from GABAA inhibition. Furthermore, dual whole cell recordings from adjacent neurons in SGI revealed that application of 10 microM bicuculline (Bic) and reduction of extracellular Mg2+ concentration (to 0.1 mM) induce spontaneous depolarization that is synchronous between neuron pairs, suggesting the recruitment of a large number of neurons communicating through intense excitatory connections. In the present study, we investigated the properties of synchronous depolarization and the fundamental structure of the lateral excitatory network that recruits a neuronal population in SC to synchronous activation, by analyzing the synchronicity of spontaneous depolarization induced in the presence of Bic plus low Mg2+. We found that 1) spontaneous depolarization exhibits bidirectional horizontal propagation among the SGI neuron pairs; 2) induction of spontaneous depolarization is not caused by activation of intrinsic voltage-dependent conductances; 3) neurons exposed to low Mg2+ alone exhibit spontaneous depolarization, although in this case the depolarization is less synchronous; and 4) neurons exposed to Bic alone exhibit synchronous depolarization, but less frequently than those exposed to both Bic and low Mg2+. Analysis of the synchronicity of spontaneous depolarization indicates that the distribution of lateral excitatory connections is markedly different among layers of SC; the SGI neurons form extensive lateral excitatory connections, whereas they are sparse or limited within subsets of neurons in the stratum griseum superficiale (SGS). Wide-field vertical neurons in the stratum opticum have features intermediate between neurons in the SGS and SGI. Such differences in the structure of lateral excitatory connections may reflect the different way signal processing is achieved in each layer of SC.
Animals↗
Dexterous finger movements in primate without monosynaptic corticomotoneuronal excitation.
It is generally accepted that the precision grip and independent finger movements (IFMs) in monkey and man are controlled by the direct (monosynaptic) corticomotoneuronal (CM) pathway. This view is based on previous observations that pyramidotomy causes near permanent deficits of IFMs. However, in addition to the direct CM pathway, pyramidotomy interrupts several corticofugal connections to the brain stem and upper cervical segments. Indirect (oligosynaptic) CM pathways, which are phylogenetically older, have been considered to be of little or no importance in prehension. In three adult macaque monkeys, complete transection of the direct CM pathway was made in C4/C5, which is rostral to the forelimb segments (C6-Th1). Electrophysiological recordings revealed lack of the direct lateral corticospinal tract (LCST) volley, monosynaptic extracellular field potentials in the motor nuclei, and monosynaptic CM excitation. However, a disynaptic volley, disynaptic field potentials and disynaptic CM excitation mediated via C3-C4 propriospinal neurons remained after the lesion. Thus the lesion interrupted the monosynaptic CM pathway and oligosynaptic LCST pathways mediated by interneurons in the forelimb segments. Precision grip and IFMs were observed already after 1-28 days postoperatively. Weakness in force and deficits in preshaping remained for an observation period of 3 mo. Indirect CM pathways may be important for neuro-rehabilitation.
Animals↗
Conduction properties of identified neural pathways in the central nervous system of mice in vivo.
Various lines of transgenic or knockout mice are now available that have abnormalities in neuron, glial cells or neuron-glial interaction. However, the techniques for quantitative analysis of their pathophysiological functions are still limited. We established an experimental model system to measure the properties of nerve conduction of identified neural pathways in the CNS using anesthetized and immobilized mice. Dorsal column (DC), vestibulospinal/reticulospinal tracts (VRST) and pyramidal tract (PT) were stimulated by inserting stimulating electrodes into the dorsal column nuclei, medial longitudinal fasciculus, and the medullary pyramid, respectively. Volleys were recorded at various segments in the cervical spinal cord with surface electrodes, and their conduction velocities (CVs) and relative refractory periods (RRPs) were measured. The CVs of the DC, VRST and PT were 26.25 +/- 4.96 m/s (n = 7), 51.55 +/- 4.65 m/s (n = 7), 8.89 +/- 1.81 m/s (n = 7), respectively. Data from paired stimulation indicated that the median values of RRPs of the DC, VRST and PT were 10, 2 and 4 ms, respectively, which suggested marked difference among individual tracts. This is the first attempt to measure the conduction properties of the central tracts in mice in vivo. This experimental procedure will give us a physiological measure of CNS functions in normal and genetically manipulated mice and contribute to clarifying the molecular mechanisms and pathophysiology of neurodegenerative diseases such as multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS).
Animals↗
PC-based high-speed video-oculography for measuring rapid eye movements in mice.
We newly developed an infrared video-oculographic system for on-line tracking of the eye position in awake and head-fixed mice, with high temporal resolution (240 Hz). The system consists of a commercially available high-speed CCD camera and an image processing software written in LabVIEW run on IBM-PC with a plug-in video grabber board. This software calculates the center and area of the pupil by fitting circular function to the pupil boundary, and allows robust and stable tracking of the eye position in small animals like mice. On-line calculation is performed to obtain reasonable circular fitting of the pupil boundary even if a part of the pupil is covered with shadows or occluded by eyelids or corneal reflections. The pupil position in the 2-D video plane is converted to the rotation angle of the eyeball by estimating its rotation center based on the anatomical eyeball model. By this recording system, it is possible to perform quantitative analysis of rapid eye movements such as saccades in mice. This will provide a powerful tool for analyzing molecular basis of oculomotor and cognitive functions by using various lines of mutant mice.
Animals↗
Enhancement of excitatory postsynaptic potentials by preceding application of acetylcholine in mesencephalic dopamine neurons.
Previously, we reported that Ca(2+) influx through nicotinic acetylcholine (ACh) receptors (nAChRs) activates a fulfenamic acid (FFA)-sensitive inward current, presumably a Ca(2+)-activated nonselective cation current (I(CAN)), in mesencephalic dopamine (DA) neurons. This current exhibited a negative slope conductance in the voltage range between -80 and -40mV and its activation led to a dramatic change in the responses to a transient application of glutamate, from single spikes to burst discharges. In this study, to examine the effect of activation of the FFA-sensitive current on EPSPs, we applied ACh (1mM) by transient air pressure shortly before electrical stimulation to evoke EPSPs in DA neurons. Application of ACh enhanced the amplitude of EPSPs when it preceded the electrical stimulation by less than 2 s, but not when the interval was longer than 3 s. In addition, this enhancement was critically dependent on intracellular Ca(2+) and the membrane potentials of the postsynaptic cell. Furthermore, the enhancing effect of ACh on EPSPs was sensitive to FFA and phenytoin. These results suggest that Ca(2+) influx caused by cholinergic inputs enhances EPSPs via activation of the FFA- and phenytoin-sensitive current.
6-Cyano-7-nitroquinoxaline-2,3-dione↗
Switching between cortical and subcortical sensorimotor pathways.
It is well known that the reaction times of visually guided saccades exhibit a bimodal distribution. Those with extremely short reaction times are termed 'express saccades'. In their case, visual input appears to be transformed into motor output via a 'short-loop', brainstem-mediated pathway. In contrast, those with longer reaction times are called 'regular saccades'. The latter are presumably executed via a cortically mediated, 'long-loop' sensorimotor pathway. The 'gate' that switches signal flow between the short and long loop is thought to be located in between the superficial and deeper layers of the superior colliculus (SC). Nonlinear signal amplification mechanisms, which operate in local circuits of the deeper SC layers may underlie this gating function, with switching of the gate regulated in a context-dependent manner by inputs from the cerebral cortex and basal ganglia.
Animals↗
Pedunculo-pontine control of visually guided saccades.
The cholinergic pedunculopontine tegmental nucleus (PPTN) is one of the major ascending arousal systems in the brainstem, and it is linked to motor, limbic and sensory centers. Despite an abundance of anatomical and physiological data, however, the functional role of PPTN neurons in behavioral control is still unresolved. In this chapter, we hypothesize that the PPTN is implicated in the integrative control of movement, particularly the reinforcement of tasks performed during conscious behavior. We present a new model of the PPTN's involvement in the control of arousal, attention and reinforcement aspects of motor behavior, with a focus on the control of saccadic eye movements.
Animals↗
Nigral GABAergic inhibition upon mesencephalic dopaminergic cell groups in rats.
Synaptic inhibition from the substantia nigra pars reticulata (SNr) to the mesencephalic dopaminergic neurons, which was mediated by gamma (gamma)-amino-butyric acid (GABA), was investigated in a midbrain slice preparation of Wistar rats. Whole-cell patch-clamp recordings were used to record synaptic potentials/currents from the dopaminergic neurons (n = 93) located in the retrorubral field (n = 22), the substantia nigra pars compacta (n = 47) and the ventral tegmental area (n = 24). In the presence of ionotropic glutamate receptor antagonists electrical stimulation of the SNr induced inhibitory postsynaptic potentials (IPSPs) and/or currents (IPSCs) in 83 neurons. The IPSPs/IPSCs were comprised early and late components. The early IPSPs/IPSCs were mediated by chloride currents through GABA(A) receptors. The late IPSPs/IPSCs were mediated by potassium currents through GABA(B) receptors. Both GABA(A)- and GABA(B)-IPSPs were amplified by repetitive stimuli with frequencies between 25 and 200 Hz. This frequency range covers the firing frequencies of SNr neurons in vivo. It was observed that an application of a GABA(B) receptor antagonist increased the amplitude of the GABA(A)-IPSPs. The amplification was followed by a rebound depolarization that induced transient firing of dopaminergic neurons. These properties of the IPSPs were common in all of the three dopaminergic nuclei. These results suggest that postsynaptic GABA(A)- and GABA(B)-inhibition contribute to transient and persistent alternations of the excitability of dopaminergic neurons, respectively. These postsynaptic mechanisms may be, in turn, regulated by presynaptic GABA(B)-inhibition. Nigral GABAergic input may provide the temporospatial regulation of the background excitability of mesencephalic dopaminergic systems.
2-Amino-5-phosphonovalerate↗
Presynaptic muscarinic acetylcholine receptors suppress GABAergic synaptic transmission in the intermediate grey layer of mouse superior colliculus.
The intermediate grey layer (the stratum griseum intermediale; SGI) of the superior colliculus (SC) receives cholinergic inputs from the parabrachial region of the brainstem. It has been shown that cholinergic inputs activate nicotinic acetylcholine (nACh) receptors on projection neurons in the SGI. Therefore, it has been suggested that they facilitate the initiation of orienting behaviours. In this study, we investigated the effect of muscarinic acetylcholine (mACh) receptor activation on GABAergic synaptic transmission to SGI neurons using the whole-cell patch-clamp recording technique in slice preparations from mice. The GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) evoked in SGI neurons by focal electrical stimulation were suppressed by bath application of 10 microm muscarine chloride. During muscarine application, both the paired-pulse facilitation index and the coefficient of variation of IPSCs increased; however, the current responses induced by a transient pressure application of 1 mm GABA were not affected by muscarine. Muscarine reduced frequencies of miniature IPSCs (mIPSCs) while the amplitudes of mIPSCs remained unchanged. These results suggested that mAChR-mediated inhibition of IPSCs was of presynaptic origin. The suppressant effect of muscarine was antagonized by an M1 receptor antagonist, pirenzepine dihydrochloride (1 microM), and a relatively specific M3 receptor antagonist, 4-DAMP methiodide (50 nM). By contrast, an M2 receptor antagonist, methoctramine tetrahydrochloride (10 microM), was ineffective. These results suggest that the cholinergic inputs suppress GABAergic synaptic transmission to the SGI neurons at the presynaptic site via activation of M1 and, possibly, M3 receptors. This may be an additional mechanism by which cholinergic inputs can facilitate tectofugal command generation.
Animals↗