Sachiko Kakuta

Preferential neuron loss in the rat piriform cortex following pilocarpine-induced status epilepticus

Structures within the piriform cortex (PC) including the endopiriform nucleus (DEN) and pre-endopiriform nucleus (pEn) have been implicated to be involved in seizure genesis in models of temporal lobe epilepsy. We used stereological methods to examine the specificity and extent of neuron loss in the PC of pilocarpine-treated rats. Both 7 days and 2 months post-status epilepticus rats showed significant neuron loss in the pEn and DEN, layer III of the intermediate PC, and layers II and III of the caudal PC. Total losses in the PC were 40 and 46% in 7 days and 2 months post-status epilepticus rats, respectively (p<0.01). The numbers of parvalbumin (PV)- and cholecystokinin (CCK)-immunopositive neuron profiles significantly decreased, and somatostatin (SS)-immunopositive neuron profiles tended to decrease. A large decrease in the number of PV-immunopositive neuron profiles occurred in the pEn, adjoining parts of the DEN and deep layer III of the PC, portions of the DEN bordering the claustrum and agranular insular cortex, and layer III of the caudal PC. The regions with decreased numbers of PV-, CCK-, and SS-immunopositive neuron profiles overlapped with those where many Nissl-stained neurons were lost and many degenerating cell bodies were detected. These results suggest that the decreases in the numbers of PV/SS/CCK-immunopositive neurons are related to neuron loss rather than to a low rate of synthesis of their peptides or proteins.

Calbindin-D28k and calretinin immunoreactive neurons in the olfactory bulb of the musk shrew, Suncus murinus.

The distribution, morphological features, and postnatal development of calbindin-D28k (CB) and calretinin (CR) immunoreactive neurons in the main olfactory bulb (MOB) of the musk shrew, Suncus murinus, were studied by immunostaining to determine the degree of colocalization of CB and CR, and the relationship of CB and CR to neuron development in the MOB of animals of the order Insectivora. In adults, CB-positive neurons were identified as periglomerular and perinidal cells in the periglomerular region, as superficial short-axon cells in the external plexiform layer, and as four types of interneurons (Cajal, horizontal, Golgi, and bitufted cells) in the mitral cell, internal plexiform, and granule cell layers. CR-positive neurons were identified as projection neurons (tufted and mitral cells) and interneurons (periglomerular, perinidal, and granule cells). On postnatal days 1 and 3, CB-positive neurons revealed numerous processes finely arborized near the somata, and were morphologically unidentifiable. At the same time, CR-positive neurons were identified as young periglomerular and granule cells, and as migrating bipolar cells extending leading processes with growth cones in each layer of the MOB and the subependymal layer between the anterior lateral ventricle and the center of the MOB. On postnatal day 28, mature CB-positive and CR-positive interneurons were distributed in their corresponding layers, whereas migrating CR-positive bipolar cells were rarely detected. No cells colocalized CB and CR. The results suggest that perinidal cells in the shrew MOB may develop postnatally, together with glomerular and granule cells. We suggest that CB is associated with mechanisms of the outgrowth of neuronal processes, whereas CR is involved in mechanisms of cell migration and outgrowth of neuronal processes, in some types of neurons in the developing stage of the shrew MOB.

Parvalbumin Immunoreactive Neurons in the Main Olfactory Bulb of the House Musk Shrew, Suncus murinus

The distribution, morphological features, and postnatal development of parvalbumin (PV) immunoreactive neurons in the main olfactory bulb (MOB) of the house musk shrew, Suncus murinus, were studied to report for the first time on PV positive bulbar interneurons in the order Insectivora. In adult animals, PV neurons are distributed in the glomerular layer (GL), the external plexiform layer (EPL), the internal plexiform layer (IPL) and the granule cell layer (GCL) of the MOB. These neurons were identified as a subpopulation of periglomerular cells and perinidal cells [Alonso et al., 1995] in the GL and at the GL-EPL border, respectively, and as bipolar and multipolar neurons in the EPL and four types of the interneurons (horizontal cells, Cajal cells, Golgi cells, and bitufted cells) in the layers deeper than the mitral cell layer. During development of PV neurons, neurons exhibiting extremely faint PV immunoreactivity first appeared in the GCL at postnatal day 14 and increased markedly in number and intensity of their PV immunoreactivity from postnatal days 14 to 28. At postnatal day 21, PV neurons were identified as periglomerular cells in the GL, perinidal cells at the GL-EPL border, and morphologically unidentifiable neurons in the EPL, IPL and GCL. At postnatal day 28, PV neurons exhibited a nearly adult pattern with respect to distribution and structural features. The present results strongly suggest that a wide variety of PV positive neurons in the MOB of the house musk shrew may develop postnatally.

Antiemetic effect of a tachykinin NK1 receptor antagonist GR205171 on cisplatin-induced early and delayed emesis in the pigeon.

Cisplatin (4 mg/kg, i.v.) induced both early emesis, which appears within the first 8-h period, and delayed emesis, which appears between 8 and 48 h after its administration to pigeons. GR205171 ([(2S-cis)-N-((2-methoxy-5(5-(trifluoromethyl)-1H-tetrazol-1-yl)-phenyl) methyl)-2-phenyl-3-piperidinamine dihydrochloride]) administered intramuscularly (1-10 mg/kg) reduced significantly the number of emetic response to cisplatin: this reduction was 60-81% (P < 0.05) for early emesis and 48-64% (P < 0.05) for the delayed response. Intracerebroventricularly administered GR205171 (30 microg/kg) also reduced the number of emetic responses: 53% (P < 0.05) in early emesis and 88% (P < 0.05) in the delayed response. However, the latency time to the first emesis was not affected by GR205171. Direct injection of cisplatin (10 microg/kg) into the fourth ventricle produced emesis, which was reduced by GR205171 administered via the peripheral or central route. Substance P-immunoreactive fibres were distributed throughout the dorsal vagal complex. These results suggest that the antiemetic effect of GR205171 on both emetic responses to cisplatin acts on a central site, and that the onset of the emetic response may be mediated partly via GR205171-insensitive mechanisms.

Ultrastructure of ascending cholinergic terminals in the anteroventral thalamic nucleus of the rat: A comparison with the mammillothalamic terminals

In this study, to identify the ultrastructure and distribution of ascending cholinergic afferent terminals in the anteroventral thalamic nucleus, we used an anti-vesicular acetylcholine transporter antibody as marker of cholinergic afferents, and characterized the immunoreactive terminals at the ultrastructural level. We then compared the distribution pattern of the cholinergic terminals and that of the mammillothalamic terminals identified by anterograde transport of a tracer injected into the mammillary body. The cholinergic terminals were small, and formed both symmetrical and asymmetrical synaptic contacts throughout the dendritic arborizations, particularly in the distal region. This distribution pattern differed from that of mammillothalamic terminals, that were of LR (large terminal containing round synaptic vesicles) type and were preferentially distributed in the proximal region of dendrites. We also found relatively numerous cholinergic terminals making contact directly with immunonegative excitatory terminals, both LR and SR (small terminal containing round vesicles) terminals, without clear postsynaptic specialization. A few cholinergic terminals even seemed to form a synaptic complex with the LR or SR terminals. These findings suggest that the ascending cholinergic afferents in the anteroventral thalamic nucleus can effectively modulate excitatory inputs from both the mammillothalamic and corticothalamic terminals, in close vicinity to a synaptic site.

Differential immunolocalization of m2 and m3 muscarinic receptors in the anteroventral and anterodorsal thalamic nuclei of the rat.

In this study, to identify the precise localization of m2 and m3 muscarinic receptors in the anteroventral and anterodorsal thalamic nuclei of the rat, we used receptor-subtype-specific antibodies and characterized their immunolocalization patterns by light and electron microscopy. Many m2-positive neurons were distributed throughout these nuclei. Ultrastructural analysis showed that more than 30% of m2-positive dendritic profiles in these nuclei are proximal dendritic shafts. Moreover, a few m2-positive fiber terminals were found only in the anterodorsal thalamic nucleus. These m2-positive terminals were large (1.10+/-0.30 microm in diameter) and formed asymmetrical synapses with dendritic profiles. The m3-positive neurons were also distributed in both nuclei, and the m3-positive neuropil exhibited a significant staining gradient, with the most intense staining in the ventrolateral part of the anteroventral thalamic nucleus. This region receives the densest cholinergic input originating from the dorsal tegmental region. At the ultrastructural level, the majority of m3-positive dendritic profiles were more distal regions of the dendrites compared to the m2 receptors in the anteroventral thalamic nucleus. However, no significant difference in the intradendritic distribution pattern between m2 and m3 receptors was found in the anterodorsal thalamic nucleus, which receives no cholinergic input. These findings show the differential localization of m2 and m3 receptors in the anteroventral and anterodorsal thalamic nuclei, and suggest that the m3 receptors are spatially more closely associated with ascending cholinergic afferent fibers in the anteroventral thalamic nucleus.

Quantitative analysis of axon collaterals of single superficial pyramidal cells in layer IIb of the piriform cortex of the guinea pig

To understand the functional organization of the piriform cortex (PC), the axon collaterals of three pyramidal cells in layer IIb of the anterior PC and one pyramidal cell in layer IIb of the posterior PC were labeled and quantitatively analyzed by intracellular biocytin injection in the guinea pig. Single pyramidal cells in the anterior and posterior PCs have widely distributed axon collaterals, which exhibit little tendency for patchy concentrations inside as well as outside the PC. The total lengths of the axon collaterals of the three fully analyzed pyramidal cells ranged from 68 to 156 mm, more than 50% of which were distributed in the PC. The total number of boutons of the three cells ranged from 6000 to 14,000, 5000-7000 of which were distributed in the PC. It was estimated that individual pyramidal cells in layer IIb form synaptic contacts with 2200 to 3000 other pyramidal cells in the PC, indicating that single pyramidal cells in layer IIb receive input from a large number of other pyramidal cells. This high connectivity of the network of pyramidal cells in the PC can be regarded as the neural network operating parallel distributed processing, which may play an important role in experience-induced enhancement in odorant discrimination in the PC.

2010 The studies of calbindin-, parvalbumin- and calretinin-positive neurons in the olfactory bulb of the insectvore, suncus murinus

We have elucidated some patterns of signal propagation in slices of guinea pig pirifotm cortex (PC) stained with a voltage-sensitive dye (RH482) using optical imaging. In sagittal slices, neural activity evoked by elect&al stimulation of layer Ia propagates mainly to the caudal direction along layer II/IlI. This propagation is considered to be mediated by afferent fibers in layer Ia and association fibers in layer Ib. To estimate degrees of participation of these two groups of fibers in signal propagation, layer Ia 0.4-0.7 mm caudal to the site of stimulation was cut vertical@ to the cortical stir&e and patterns of propagation to Ia stimulation before and after the cut were compared. In about 60% of 59 samples, although layer lb was left. intact, excitability in the region caudal to the cut was reduced to less than 5% of that before the cut, which means that, in these samples, propagation to the region caudal to the cut suffered a severe damage or completely disappeared. In 29% of the samples, excitability was reduced to 5-25%. In only 7% of the samples, excitability of more than 50% of the control was maintained in the region caudal to the cut Thus, so tar, it is likely that the participation of afferent fibers to signal propagation in PC is much greater than that of association fibers. When layer lb was stimulated in intact slices, however, fairly strong propagation akmg layer II/Ill coukl be elicited. It would therefore be necessary to investigate effects of layer Ib cut on propagation evoked by stimulation of layer Ia and Ib, to evaluate properly the role of association fibers in signal propagation.

Effect of Ethanol on the Development and Maturation of Synapses in the Rat Hippocampus: A Quantitative Electron-Microscopic Study1

Rats of both sexes were exposed to ethanol during the entire period of the fetal life as well as the whole period of postnatal ages, and their brains were investigated at 2, 7, 14, 21, and 70 days postnatally. Control animals were examined at each age. Densities of synapses in the strata radiatum and lacunosum-moleculare of CA1 in the hippocampus were analyzed by quantitative ultrastructural techniques. Densities of all synapses in the strata radiatum and lacunosum-moleculare in ethanol-treated group were significantly lower than those of control group on 2, 14, 21 and 70 days. However, there were no significant differences in the ratio of axo-spinous to axo-shaftic synapses between control and ethanol-treated group. In addition, in regard to the rate of reduction in densities of all synapses, no change was detectable between both strata that receive different groups of afferent fibers. These data suggest that chronic administration of ethanol during the fetal and postnatal ages reduces the synaptic density in this area, and that this effect is neither specific to types of synapses nor to sorts of afferent fibers.