Shigeo Okoyama

In vivo transduction of murine cerebellar Purkinje cells by HIV-derived lentiviral vectors.

Cerebellar Purkinje cells are key elements in motor learning and motor coordination, and therefore, it is important to clarify the mechanisms by which Purkinje cells integrate information and control cerebellar function. Gene transfer into neurons, followed by the assessment of the effects on neural function, is an effective approach for examining gene function. However, this method has not been used fully in the study of the cerebellum because adenovirus vectors, the vectors most commonly used for in vivo gene transfer, have very low affinity for Purkinje cells. In this study, we used a human immunodeficiency virus (HIV)-derived lentiviral vector and examined the transduction profile of the vector in the cerebellum. A lentiviral vector carrying the GFP gene was injected into the cerebellar cortex. Seven days after the injection, Purkinje cells were efficiently transduced without significant influence on the cell viability and synaptic functions. GFP was also expressed, though less efficiently, in other cortical interneurons and Bergmann glias. In contrast to reported findings with other viral vectors, no transduced cells were observed outside of the cerebellar cortex. Thus, when HIV-derived lentiviral vectors were injected into the cerebellar cortex, transduction was limited to the cells in the cerebellar cortex, with the highest tropism for Purkinje cells. These results suggest that HIV-derived lentiviral vectors are useful for the study of gene function in Purkinje cells as well as for application as a gene therapy tool for the treatment of diseases that affect Purkinje cells.

Synaptic organization of the cat entopeduncular nucleus with special reference to the relationship between the afferents to entopedunculothalamic projection neurons: an electron microscope study by a combined degeneration and horseradish peroxidase tracing technique.

The synaptic organization of the feline entopeduncular nucleus was studied electron microscopically. After horseradish peroxidase injections into the ventral anterior and ventral lateral nuclear complex of the thalamus, normal axon terminals synapsing with entopedunculothalamic projection neurons were classified into four types on the basis of the size and shape of synaptic vesicles in them, and types of the postsynaptic membrane differentiation. Type I and type II axon terminals were characterized by symmetrical synaptic contacts, and large ovoid or small ovoid synaptic vesicles, respectively. Type II axon terminals were further classified into two subtypes as to their sizes: one was small (IIa), the other large (IIb). Type III and type IV axon terminals were characterized by asymmetrical synaptic contacts, and large ovoid or small ovoid synaptic vesicles, respectively. To determine the origin of each type of terminal, electrolytic lesions of the caudate nucleus or the subthalamic nuclear region were combined with horseradish peroxidase injections into the thalamus or the subthalamic nuclear region. After electrolytic lesions of the caudate nucleus, degeneration was seen in type I axon terminals contacting entopedunculothalamic projection neurons. Following electrolysis or horseradish peroxidase injection into the subthalamic nuclear region, type IIa and type IV axon terminals showed degenerations or horseradish peroxidase labelling. Such terminals also synapsed with entopedunculothalamic projection neurons. It was demonstrated that these projection neurons relay the striatal or subthalamic inputs directly to the thalamus. After horseradish peroxidase injection into the thalamus, many labelled type II axon terminals were observed to synapse with entopedunculothalamic projection neurons. Type III axon terminals were left unchanged throughout these experiments. In addition, the entopeduncular neuron was observed to receive convergent inputs from both the caudate nucleus and probably the subthalamic nucleus. Axoaxonal synapses were also found to be involved in the synaptic triad. These results indicate that type I axon terminals originate from the caudate nucleus, part of type IIa and type IV axon terminals originate from the subthalamic nucleus or caudal to the subthalamic nuclear region, and part of type IIa and type IIb terminals come from intrinsic axon collaterals.

Projections from the pericruciate cortex to the nucleus of Darkschewitsch and other structures at the mesodiencephalic junction in the cat.

After injecting a mixture of tritiated amino acids into various regions of the frontal cortices of cats, autoradiograms of the mesodiencephalic junctional region were processed. When the injections were placed in the lateral part of the motor area of the hand or arm regions, silver grains were manifested in the nucleus of Darkschewitsch (ND) in its whole rostrocaudal extent, and they were observed also in the ventrolateral part of the anterior pretectal nucleus (PA) and in the caudal portion of the posterior pretectal nucleus (PP). Following injections made into the medialmost part of the anterior sigmoid gyrus (area 6), medial to middle parts of the posterior sigmoid gyrus, the proreal gyrus or the medial surface of the frontal cortex (area 32), no silver grains were observed in ND, PA and PP.

Neuronal organization of the rat inferior colliculus participating in four major auditory pathways.

The central nucleus of the inferior colliculus (CNIC) contains different types of neurons and is a source of ascending projection to the medial geniculate body (MGB), commissural projection to the contralateral IC, direct descending projection to the cochlea nucleus (CN) and indirect projection to the CN via the superior olivary complex (SOC). Using a retrograde tracing technique, we examined what kind of neurons and what percentage of neurons of each type recognized in the CNIC participated in the above-mentioned four projection pathways. We also examined whether the individual CNIC neurons send the collateral to the MGB, the contralateral IC, the CN and the SOC. In the present study, we demonstrated that the neurons participating in the four projections could be morphologically classified into two types of neurons with soma size variation. The percentages of neurons of each type differed among the four projection pathways. Using a double-labeling technique, we found very few double-labeled neurons, indicating the collateral projections to the ipsilateral MGB and the contralateral IC. There were no double-labeled neurons in the collateral projections between the other combinations of targets. Therefore, we conclude that the ascending projection, the commissural projection and the descending projection to these targets arise from separate populations of neurons.

Electron microscopic study of synaptogenesis and myelination of the olfactory centers in developing rats

Development of the central olfactory system was studied in the rat with an electron microscope at three main structures: the olfactory bulb, the lateral olfactory tract, and the primary olfactory cortex (the piriform cortex). As a parameter of development, the synaptic density was examined quantitatively in the bulbar glomerulus and layer Ia (termination of bulbofugal fibers) of the piriform cortex, which are the key stations of the olfactory pathway. The synaptic densities in the glomerulus and those in layer Ia were 5.7% and 4.6% on embryonic day 19, 15.8% and 12.5% on postnatal day (P) 0, and 57.3% and 37.2% on P10, as compared with the adult (100%). As another parameter of development, the density of myelinated axons in the lateral olfactory tract was examined quantitatively. The densities of myelinated axons in the tract were 0% on P5, 15.1% on P10, and 73.5% on P21 of the adult density. Maturation in the tract was still progressing, even at P21, in terms of bundle formation and the thickness of myelin sheaths. The results show that synaptogenesis in the bulbar glomerulus is followed by synaptogenesis in layer Ia of the piriform cortex, and that myelination in the lateral olfactory tract occurs over a prolonged period, even in the stages after P21.

Postnatal development of the projection from the medial superior olive to the inferior colliculus in the rat.

Normal projection development from the medial superior olive (MSO) to the inferior colliculus (IC) was examined by injecting Fluoro-Gold (FG), a retrograde tracer, into the IC unilaterally at postnatal days 0 (P0), P3, P7 and maturity. The rats were killed 1 day after FG injection. At all ages, labeled neurons in the MSO appeared on the ipsilateral side only, as in adult controls. The total number of MSO neurons counted in Nissl-stained sections was constant throughout the postnatal periods. The labeled frequency index of MSO neurons was increased stepwise (from 35% to 90%) with increasing postnatal stages (from P0 to adulthood), suggesting differential growth of early- and late-developing axons.

Crossed projection neurons are generated prior to uncrossed projection neurons in the lateral superior olive of the rat

The present study examined in the lateral superior olive (LSO) of the rat whether LSO neurons projecting to the ipsilateral inferior colliculus (IC) might be generated later than those projecting to the contralateral IC. Rat fetuses were exposed in utero to 5-bromodeoxyuridine (BrdU), a thymidine analogue, to label neurons proliferating at different embryonic stages from day E11 through to E20. Upon reaching adulthood, the rats were given unilateral injections of fluoro-gold (FG), a retrograde fluorescent tracer, into the IC. Subsequently, the tissue sections of the brains obtained from the rats were immunostained for BrdU to simultaneously detect neurons that were BrdU-positive and/or FG-positive. BrdU-positive LSO neurons were found in the rats which had been exposed to BrdU during E12-E16. In E12 and E13 BrdU-exposure cases, the vast majority of doubled-labeled (BrdU-positive and FG-positive) neurons were seen on the contralateral side to the FG injection. In E14, E15 and E16 BrdU-exposure cases, in contrast, all double-labeled neurons were found on the ipsilateral side to the FG injection. The distribution of these double-labeled neurons within the nucleus was diffuse in all the BrdU-exposure cases. Thus, the results indicate that LSO neurons are generated during E12-E16, that the crossed projection neurons are generated 1-4 days earlier than the uncrossed projection neurons, and that no topographical relationships exist between the early- and the late-generated populations of the LSO neurons.

Effect of vagal autotransplantation and bifemelane hydrochloride on cholinergic markers and event-related potentials in rats with lesions of the nucleus basalis magnocellularis

In rats lesioned by injecting the ibotenic acid (8 micrograms/site) into the unilateral nucleus basalis magnocellularis (NBM), the effect of treatment with bifemelane hydrochloride (BIF) or autotransplantation of the vagal nodosal ganglion was studied electrophysiologically by serial measurement of the event-related potential (ERP, P300) for 4 weeks. In addition, the effects on cholinergic markers were assessed by determining the specific binding of [3H]QNB (quinuclidinyl benzilate) to the muscarinic acetylcholine receptor (mAChR) as well as the activity of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE). The P300 latency was delayed and its amplitude remained low for 4 weeks in NBM-lesioned rats. In contrast, a return to normal occurred after 2-3 weeks in rats given daily intraperitoneal injections of BIF (15 mg/kg) and in autotransplanted rats. In lesioned rats, the cortical ChAT and AChE activities on the affected side did not recover, but the postsynaptic receptor response was transiently activated soon after lesioning. BIF increased specific mAChR binding (an early increase of affinity and a subsequent increase of receptor density) as well as presynaptic ChAT activity. Transplantation achieved the early activation of mAChR binding (increased receptor density) and continuously increased ChAT activity. Thus, the postsynaptic compensatory receptor mechanism of denervation supersensitivity acted as an early response to the depression of presynaptic cholinergic activity, but it could not improve the P300 response until the subsequent increase of cortical ChAT activity. Improvement of P300 combined with cortical cholinergic recovery after nodosal ganglion grafting or administration of BIF suggests that the neocortical ACh level may play an important role in regulating ERP.

Neurogenetical segregation of the vestibulospinal neurons in the rat

The time of origin of the vestibulospinal projection neurons was determined by a double-labeling method using 5-bromodeoxyuridine (BrdU), the thymidine analogue, and Fluoro-Gold (FG), a retrograde fluorescent tracer. Rat fetuses were exposed to BrdU in utero to label the vestibular neurons on one of the embryonic (E) days between E12 and E15. Upon reaching adulthood, the rats were given unilateral injections of FG into the cervical cord to identify the spinal projection neurons. Brainstem sections were immunohistochemically processed for BrdU and then examined for neurons that were both BrdU-positive and FG-positive in the vestibular nuclei. In the lateral vestibular nucleus (LVe), most of the vestibulospinal neurons were generated on E12. In the inferior vestibular nucleus (IVe), the vestibulospinal neurons were produced almost equally on both E12 and E13. In the medial vestibular nucleus (MVe), the vestibulospinal neurons were generated consistently on days between E12 and E14 with a mild peak on E13. The present results thus demonstrate that genesis of the vestibulospinal neurons occurs sequentially in the following order: firstly in the LVe, secondly in the IVe, and finally in the MVe. The different sequential generation of vestibulospinal neurons among the LVe, MVe and IVe may reflect the fact that the vestibulospinal projections are differentially organized depending on the nature of each subnucleus.

Cortico-Darkschewitsch-olivary projection in the cat: an electron microscope study with the aid of horseradish peroxidase tracing technique.

The nucleus of Darkschewitsch (ND) of the cat was observed electron microscopically after surgical ablation of the motor cortex and horseradish peroxidase (HRP) injection into the inferior olive of the same animals. Degenerated axon terminals containing pleomorphic synaptic vesicles were observed to synapse chiefly with medium-sized or small dendritic processes, some of which were labeled with HRP retrogradely. Therefore, a cortico-olivary projection which was relayed at the ND was revealed at an ultrastructural level.