Olfactory input to the parahippocampal region of the isolated guinea pig brain reveals weak entorhinal-to-perirhinal interactions.

The processing of olfactory inputs by the parahippocampal region has a central role in the organization of memory in mammals. The olfactory input is relayed to the hippocampus via interposed synapses located in the piriform and entorhinal cortices. Whether olfactory afferents directly or indirectly project to other areas of the parahippocampal region beside the entorhinal cortex (EC) is uncertain. We performed an electrophysiological and imaging study of the propagation pattern of the olfactory input carried by the fibres that form the lateral olfactory tract (LOT) into the parahippocampal region of the in vitro isolated guinea pig preparation. Laminar analysis was performed on field potential depth profiles recorded with 16-channel silicon probes at different sites of the insular-parahippocampal cortex. The LOT input induced a large amplitude polysynaptic response in the lateral EC. Following appropriate LOT stimulation, a late response generated by the interposed activation of the hippocampus was observed in the medial EC. LOT stimulation did not induce any local response in area 36 of the perirhinal cortex (PRC), while a small amplitude potential with a delay similar to the lateral EC response was inconsistently observed in PRC area 35. No PRC potentials were observed following the responses evoked by LOT stimulation in either the lateral or the medial EC. These findings were substantiated by current source density analysis of PRC laminar profiles. To further verify the absence of EC-to-PRC field interactions after LOT stimulation, high-resolution optical imaging of neuronal activity was performed after perfusion of the isolated brain with the voltage-sensitive dye RH-795. The optical recordings confirmed that olfactory-induced activity in the EC does not induce massive PRC activation. The present findings suggest that the olfactory input into the parahippocampal region is confined to the entorhinal cortex. The results also imply that, as demonstrated for the PRC-to-EC pathway, the propagation of neuronal activity from the EC to the PRC is hindered, possibly by a powerful inhibitory control generated within the EC.

Voltage-sensitive dye versus intrinsic signal optical imaging: comparison of optically determined functional maps from rat barrel cortex.

Using intrinsic and voltage-sensitive dye optical imaging methods, somatosensory-evoked neural activity and the consequent metabolic activity were visualized in the barrel cortex at high temporal and spatial resolution. We compared maps of neural and metabolic activity from the perspective of spatial distribution in the cortex. There was good agreement between the two functional maps, if the extent of metabolic activity before a prominent increase in cerebral blood volume (CBV) was assessed. This result indicates that oxygen consumption occurs before CBV changes, in approximately the same cortical area as that in which the preceding neural activity was evoked. This also suggests that the intrinsic signal reflects subthreshold synaptic activity, as well as spiking activity, which is similar to the dye-related signals.

Isolation and characterisation of a mutation in the PMR1 gene encoding a Golgi membrane ATPase, which causes hypersensitivity to over-expression of Clb3 in Saccharomyces cerevisiae.

We screened for mutant strains of Saccharomyces cerevisiae that are sensitive to overexpression of specific cyclins, and identified mutations in two genes that caused growth inhibition in response to mild overexpression of Clb3. One was the ANP1 gene, which encodes a glycosyltransferase previously identified by a similar strategy using Clb2 instead of Clb3. This paper describes the second strain of S. cerevisiae that is hypersensitive to Clb3 expression. The gene mutated in this strain was identified as PMR1, which encodes a Ca2+-ATPase located in the Golgi membrane. The protein product of pmr1-1 was truncated at residue 409 and thus lacked the C-terminal ATPase domain. The pmr1-1 strain was hypersensitive to over-expression of Clb3, but not Cln2, Clb5 or Clb2. The lethality due to Clb3 expression in pmr1-1 could be suppressed by adding Ca2+ ions to the medium. The pmr1-1 strain proved to be defective in glycosylation, and the defects in glycosylation were exacerbated by high levels of Clb3. On induction of Clb3 expression in the pmr1-1 strain, the cells arrested at anaphase with an elongated daughter bud. We discuss possible interpretations of this synthetic lethal phenotype.

Optical recording study of granule cell activities in the hippocampal dentate gyrus of kainate-treated rats.

In the epileptic hippocampus, newly sprouted mossy fibers are considered to form recurrent excitatory connections to granule cells in the dentate gyrus and thereby increase seizure susceptibility. To study the effects of mossy fiber sprouting on neural activity in individual lamellae of the dentate gyrus, we used high-speed optical recording to record signals from voltage-sensitive dye in hippocampal slices prepared from kainate-treated epileptic rats (KA rats). In 14 of 24 slices from KA rats, hilar stimulation evoked a large depolarization in almost the entire molecular layer in which granule cell apical dendrites are located. The signals were identified as postsynaptic responses because of their dependence on extracellular Ca(2+). The depolarization amplitude was largest in the inner molecular layer (the target area of sprouted mossy fibers) and declined with increasing distance from the granule cell layer. In the inner molecular layer, a good correlation was obtained between depolarization size and the density of mossy fiber terminals detected by Timm staining methods. Blockade of GABAergic inhibition by bicuculline enlarged the depolarization in granule cell dendrites. Our data indicate that mossy fiber sprouting results in a large and prolonged synaptic depolarization in an extensive dendritic area and that the enhanced GABAergic inhibition partly masks the synaptic depolarization. However, despite the large dendritic excitation induced by the sprouted mossy fibers, seizure-like activity of granule cells was never observed, even when GABAergic inhibition was blocked. Therefore, mossy fiber sprouting may not play a critical role in epileptogenesis.

Functional organization of chromaffin cells and cholinergic synaptic transmission in rat adrenal medulla.

Optical recordings of membrane depolarization and whole-cell patch-clamp recordings of membrane potentials and currents were obtained from chromaffin cells in slices of rat adrenal medulla. The stimulation of splanchnic nerve fibers caused a discontinuous spread of electrical activity across the slice. Cells in clusters with diameters of about 80 microns were excited simultaneously, suggesting that the adrenal medulla is organized into descrete cell complexes with common innervation. The electrical properties of chromaffin cells in situ were in agreement with previous reports on cultured cells. A fraction of the recorded cells displayed excitatory postsynaptic currents (EPSCs) of 0.2-1 nA upon the stimulation of presynaptic nerve fibers. The EPSC was blocked by hexamethonium, suggesting that nicotinic ACh receptors were involved. The decay phase of the EPSC was well fit by the sum of two exponentials with time constants of 6.3 and 57.3 ms. The relative amplitude of the fast component was 84.1%. These two exponentials may reflect activation of both fast and slow time-constant ACh receptor channels by presynaptic release of ACh. There were multiple peaks in the EPSC amplitude histograms in low-[Ca2+] saline, the first peak was at 37 pA. To resolve the quantal size, miniature EPSCs were recorded in a tetrodotoxin-containing high-[K+] solution. The miniature EPSC amplitude histograms were also multimodal with the first peak at 25 pA, which probably represents the quantal size of the synapse. The second and third peaks were at the integer multiples of the first one.

Entorhinal-hippocampal interactions revealed by real-time imaging.

The entorhinal cortex provides the major cortical input to the hippocampus, and both structures have been implicated in memory processes. The dynamics of neuronal circuits in the entorhinal-hippocampal system were studied in slices by optical imaging with high spatial and temporal resolution. Reverberation of neural activity was detected in the entorhinal cortex and was more prominent when the inhibition due to gamma-aminobutyric acid was slightly suppressed. Neural activity was transferred in a frequency-dependent way from the entorhinal cortex to the hippocampus. The entorhinal neuronal circuit could contribute to memory processes by holding information and selectively gating the entry of information into the hippocampus.

Occurrence of autoimmune antibodies to liver microsomal proteins associated with lethal hepatitis in LEC rats: effects of TJN-101 ((+)-(6S,7S,R-biar)- 5,6,7,8-tetrahydro-1,2,3,12-tetramethoxy-6,7-dimethyl-10,11- methylenedioxy-6-dibenzo[a,c]cyclooctenol) on the development of hepatitis and the autoantibodies.

Long Evans Cinnamon (LEC) rats, that spontaneously develop hepatitis, were found to possess autoantibodies to liver microsomal proteins (anti-LM) before the development of hepatitis. Anti-LM antibody was assumed to appear in association with the lethal hepatitis in the LEC rats. Thus, the purpose of this study was to investigate the effects of an anti-hepatitis drug on the development of hepatitis and the occurrence of the antibody in LEC rats. Mortality, blood biochemical parameters and the titer of serum anti-LM antibody were measured. In control LEC rats, 4 of 8 rats died before 20 weeks of age. In rats treated with TJN-101 ((+)-(6S,7S,R-biar)-5,6,7,8-tetrahydro-1,2,3,12-tetramethoxy -6,7-dimethyl-10,11 - methylenedioxy-6-dibenzo[a,c]cyclooctenol), 4 of 7 rats died of hepatitis, but the time of death was delayed by 7-10 weeks compared to the control rats. The titer of the anti-LM antibody increased 3-7 weeks before death in the non-survivors in control and TJN-101-treated rats, supporting the idea that anti-LM antibody occurs in association with acute lethal hepatitis.

Effects of cyclosporin-A and D-penicillamine on the development of hepatitis and the production of antibody to protein disulfide isomerase in LEC rats.

Long Evans Cinnamon (LEC) rats, which spontaneously develop hepatitis, produce an autoantibody to protein disulfide isomerase (PDI) before the development of clinical signs of hepatitis. Anti-PDI antibody may be associated with immunological hepatitis. Thus, the purpose of this study was to investigate the effects of some drugs on the development of hepatitis and the occurrence of the antibody in LEC rats. Cyclosporin-A, an immunosuppressant, and D-penicillamine, which promotes copper excretion, were orally administered to LEC rats for 23 weeks. Mortality, blood biochemical parameters and the titer of serum anti-PDI antibody were measured. In control LEC rats, four of eight rats died before 20-weeks-old. Only one of seven rats in the cyclosporin-A-treated group died at the age of 20 weeks. When rats were treated with D-penicillamine, the development of clinical signs of hepatitis was inhibited, and all rats survived. Cyclosporin-A-treated rats showed increases in blood biochemical parameters similar to those in control rats. The titer of anti-PDI antibody in control rats was higher the non-survivors than survivors. These findings suggest the association of the anti-PDI antibody with lethality, but not with the apparent development and progression of hepatitis as measured by blood biochemical parameters in LEC rats.

Identification of protein disulfide isomerase and calreticulin as autoimmune antigens in LEC strain of rats.

Long Evans Cinnamon (LEC) rats, showing spontaneous hereditary hepatitis and hepatic carcinoma, were found to possess autoimmune antibodies to liver microsomal proteins, particularly to proteins with the molecular weight of 56kD and 55kD. The antibodies occurred in association with acute lethal hepatitis in the LEC rats in our previous study. Two-dimensional immunoblot analysis of the antigenic proteins revealed that the 56kDa and 55kDa proteins showed 4.2 and 4.0 pI values and were estimated to be protein disulfide isomerase (PDI) and calreticulin, respectively, from NH2-terminal amino acid sequence analysis. These proteins were further identified by immunoblot analyses using purified proteins and specific antibodies. PDI was a major autoimmune antigenic protein.

Immunological studies on herpetic keratitis: effect of IL-2 on HSV-specific CTL.

The effect of interleukin 2 (IL-2) on cytotoxic T lymphocytes (CTL) was examined in mice with corneal infection of herpes simplex virus (HSV). The corneas of the mice were inoculated with HSV after scratching the corneal epithelium. Ten days after the inoculation, CTL were induced in vitro from spleen cells of the mice. IL-2 was added for the initiation of CTL induction. CTL induced without IL-2 served as controls. After the culture, viable cells were counted and cytotoxicity of CTL was measured by 3H-proline releasing assay. IL-2 promoted proliferation of HSV specific CTL without increasing the cytotoxicity of HSV specific CTL.