Dynamic and structural evidence of mesoscopic aggregation in phosphonium ionic liquids.

Mesoscopic aggregation in aprotic ionic liquids due to the microphase separation of polar and non-polar components is expected to correlate strongly with the physicochemical properties of ionic liquids and therefore their potential applications. The most commonly cited experimental evidence of such aggregation is the observation of a low-q pre-peak in the x-ray and neutron scattering profiles, attributed to the polarity alternation of polar and apolar phases. In this work, a homologous series of phosphonium ionic liquids with the bis(trifluoromethylsulfonyl)imide anion and systematically varying alkyl chain lengths on the phosphonium cation are investigated by small and wide-angle x-ray scattering, dynamic-mechanical spectroscopy, and broadband dielectric spectroscopy. A comparison of the real space correlation distance corresponding to the pre-peak and the presence or absence of the slow sub-α dielectric relaxation previously associated with the motion of mesoscale aggregates reveals a disruption of mesoscale aggregates with increasing symmetry of the quaternary phosphonium cation. These findings contribute to the broader understanding of the interplay of molecular structures, mesoscale aggregation, and physicochemical properties in aprotic ionic liquids.

FK506-protective effects against trimethyltin neurotoxicity in rats: hippocampal expression analyses reveal the involvement of periarterial osteopontin.

There is little information on the molecular mechanisms in FK506-mediated neuroprotection. In the present study, we investigated the protective effect of FK506, an immunosuppressant and neuroprotectant, on trimethyltin (TMT)-induced neurotoxicity in the rat hippocampus. Histologically, TMT-induced neuronal damage was partially prevented by FK506 in the hippocampal CA1 region, but not in CA3. FK506 treatment significantly reduced the number of apoptotic cells in CA1, but not in CA3, and also prevented induction of cognitive deficits by TMT. Microarray analysis of the rat hippocampus detected 14 genes with TMT-induced alteration of mRNA expression that was rescued by FK506 treatment. Subsequent quantitative RT-PCR analysis confirmed elevated mRNA levels for four inflammatory genes, glutathione S-transferase, lysozyme, matrix Gla protein, and osteopontin after TMT treatment. Upregulation of these genes was reversed by FK506 treatment at 5 days postgavage. Immunohistochemistry revealed that FK506 reduced osteopontin (OPN) induction by TMT in the periarterial area at 5 days postgavage. Our data suggest that inflammatory gene expression is involved in TMT-induced damage to the hippocampal CA1 region, resulting in apoptosis, and that this process is initiated by periarterial OPN activation, and can be alleviated by FK506.

Cytokines participate in neuronal death induced by trimethyltin in the rat hippocampus via type II glucocorticoid receptors.

We investigated the role of IL-1alpha and IL-1beta expressed in the reactive gliosis following hippocampal damage induced by trimethyltin (TMT). IL-1alpha immunoreactivity was expressed earlier in small glial cells on day 4 post-TMT, while IL-1beta expression was obvious in large swollen glial cells on day 14 post-TMT. Both IL-1alpha and IL-1beta immunoreactivities were double-labeled with astrocyte marker, vimentin, but not with a microglia marker, OX-42. The expression of both IL-1alpha/beta was enhanced by adrenalectomy (ADX) prior to TMT administration. Corticosterone (CORT) or dexamethasone (DEX) supplementation not only cancelled effects of ADX, but also partially reversed TMT-induced enhancement of IL-1alpha/beta expressions. These changes coincided with TMT-induced neuronal death in CA3 pyramidal cells of the hippocampus. It is suggested that IL-1alpha/beta expressed in reactive astrocytes participate in TMT neurotoxicity via type II glucocorticoid receptors.

Cytokines participate in neuronal death induced by trimethyltin in the rat hippocampus via type II glucocorticoid receptors.

We investigated the role of IL-1alpha and IL-1beta expressed in the reactive gliosis following hippocampal damage induced by trimethyltin (TMT). IL-1alpha immunoreactivity was expressed earlier in small glial cells on day 4 post-TMT, while IL-1beta expression was obvious in large swollen glial cells on day 14 post-TMT. Both IL-1alpha and IL-1beta immunoreactivities were double-labeled with astrocyte marker, vimentin, but not with a microglia marker, OX-42. The expression of both IL-1alpha/beta was enhanced by adrenalectomy (ADX) prior to TMT administration. Corticosterone (CORT) or dexamethasone (DEX) supplementation not only cancelled effects of ADX, but also partially reversed TMT-induced enhancement of IL-1alpha/beta expressions. These changes coincided with TMT-induced neuronal death in CA3 pyramidal cells of the hippocampus. It is suggested that IL-1alpha/beta expressed in reactive astrocytes participate in TMT neurotoxicity via type II glucocorticoid receptors.

Temporal change of hippocampal enkephalin and dynorphin mRNA following trimethyltin intoxication in rats: effect of anticonvulsant.

Trimethyltin (TMT), an organic metal, has been known to induce behavioral abnormalities including seizures and aggression. We administered TMT to rats, then, behavioral changes as well as the changes of dynorphin and Met-enkephalin mRNA were observed with or without phenobarbital treatment in order to reveal the role of neuropeptides in seizure-generating mechanisms. Met-enkephalin mRNA was significantly increased at the 2nd to 6th day after TMT administration when seizure was frequently observed. Meanwhile, dynorphin mRNA was decreased significantly from the 2nd day to 16th day during aggression score remained high. Phenobarbital abolished not only seizures and aggression, but also the changes of neuropeptide expressions. These results suggest that the changes of dynorphin mRNA are more strongly associated with aggression than seizures, while Met-enkephalin changes correlate more with seizures.

Altered hippocampal expression of neuropeptide Y, somatostatin, and glutamate decarboxylase in Ihara's epileptic rats and spontaneously epileptic rats.

By in situ hybridization and immunocytochemistry, expression of neuropeptide Y (NPY), somatostatin and glutamate decarboxylase 65 (GAD65) was studied in the hippocampus of two different epileptic mutant rats, Ihara's epileptic rat (IER) and the spontaneously epileptic rat (SER). GAD65 mRNA expression was enhanced in interneurons of the hippocampus in young IER, that had not yet developed generalized seizures. In older IER and older SER that both showed spontaneous seizures, marked increases of NPY mRNA in hippocampal granule cells and interneurons were found, as well as elevated GAD65 mRNA levels in interneurons. NPY immunoreactivity was enhanced in hilar interneurons and the dentate gyrus of older IER. In addition, some older IER stained heavily for NPY in mossy fibers. These findings suggest that up-regulation of NPY and GAD65 synthesis may be important in epileptogenesis.

Increased expression of zif268 mRNA in rat retrosplenial cortex following administration of phencyclidine.

Phencyclidine (PCP) has been shown to cause neurotoxicity in rat retrosplenial cortex following a single administration, although the precise mechanism underlying PCP-induced neurotoxicity is unclear. Using in situ hybridization and immunohistochemistry, we studied the effects of PCP on expression of immediate early gene zif268 mRNA and zif268 protein in the rat brain. High constitutive levels of zif268 mRNA and zif268 immunoreactivity were observed in the brain of control rats. Administration of PCP (12.5, 25 or 50 mg/kg, i.p., 6 h) caused marked induction of zif268 mRNA in the rat retrosplenial cortex, in a dose-dependent manner. However, the basal levels of zif268 mRNA in the other regions of cerebral cortex were decreased by administration of PCP. Emulsion-autoradiographical study suggested that marked expression of zif268 mRNA was observed in the layers III and IV of retrosplenial cortex where the neurotoxicity of PCP was detected. Furthermore, zif268 immunoreactivity in the layer IV of retrosplenial cortex was not changed by administration of PCP (25 mg/kg, i.p., 5 h), but that in the other layers of retrosplenial cortex was reduced by PCP. These results suggest that immediate early gene zif268 may, in part, play a role in the neurotoxicity of NMDA receptor antagonists such as PCP.

Symptom classification of schizophrenia changes with the duration of illness.

Our starting hypothesis was that schizophrenic symptomatology changes over time. This hypothesis explains conflicting reports on schizophrenic symptom structures as a consequence of different durations of illness in the samples studied to date. Therefore a sample of 258 schizophrenic in-patients (with ICD-10 diagnoses F20) was categorized according to illness duration. A factor analysis was performed on the 8 items of the Manchester Scale for three subgroups (duration < 10 years, 10> or =20 years and > or = 20 years). For those patients whose illness duration was less than 10 years, 'formal thought disorder' was not related to any other mental state, whereas for those whose duration was 10 years or longer, it was correlated with 'negative symptoms'. In the < 10 years group, 'anxiety and depression syndrome' and 'positive symptoms' formed one complex, but these symptoms were separated into two distinct syndromes in the > or = 20 years group. Thus we were able to demonstrate that the classification of symptoms changes with increasing duration of illness.

Elevated neuropeptide Y and corticotropin-releasing factor in the brain of a novel epileptic mutant rat: Noda epileptic rat.

Noda epileptic rat (NER) is a new epileptic rat strain, which was developed by inbreeding rats with spontaneous tonic-clonic seizures in a stock of Crj:Wistar. In the present study, possible changes of two neuropeptides, neuropeptide Y (NPY) and corticotropin-releasing factor (CRF), in the brains of NER were investigated. Increased contents of immunoreactive (IR) NPY were found in the striatum and amygdala of 8-week NERs with partial seizure, while these changes extended to the limbic region including hippocampus in 16-week NERs with fully developed generalized tonic-clonic seizure. IR-CRF were elevated only in the entorhinal and pyriform cortex of both 8-week and 16-week NERs. Generalized tonic-clonic seizure in NERs induced a transient increase of NPY mRNA in the granular layer of dentate gyrus. These results suggest that NPY metabolism in the limbic brain contributes to the seizure susceptibility in this model of epilepsy.

Criteria defining refractory schizophrenia.

To date there has been no agreement with regard to the criteria that define refractory schizophrenia. In this study, we intended to clarify the criteria which psychiatrists use to judge schizophrenic patients as being refractory in Japan. Based on 258 schizophrenic in-patients (ICD-10) and their likelihood of discharge, level of psychosocial functioning, mental state and use of medication, the common features of patients who are viewed as non-dischargeable because of their severe mental state, compared to those of dischargeable patients, were extracted and used as the criteria. The criteria proposed necessitate (i) diagnosis of schizophrenia by standard operational criteria, (ii) continuous hospitalization for at least the past 2 years, (iii) a level of psychosocial functioning of < or = 40 as measured by the Global Assessment Scale, and (iv) an intensity score of 'marked' or 'severe' on at least three of the six Manchester Scale items (flattened affect, psychomotor retardation, delusions, hallucinations, incoherence of speech and poverty of speech).

Trimethyltin-induced expression of neuropeptide Y Y2 receptors in rat dentate gyrus.

Trimethyltin (TMT) causes prominent neuronal damage and enhanced expression of neuropeptide Y in the hippocampus. We investigated expression of neuropeptide Y Y2 receptors after TMT intoxication. Markedly elevated (by 470%) concentrations of Y2 receptor mRNA were found in the suprapyramidal blade of the dentate granule cell layer after 5 days. Increases in the infrapyramidal blade were less prominent (by 198%). After 16 days, mRNA levels in both blades of the granule cell layer showed no significant difference from those in controls. Quantification of Y2 receptor-specific binding revealed no significant change at both 5 and 16 days after TMT intoxication. It is suggested, together with a previous report describing a similar increase of neuropeptide Y expression, that a transient expression of Y2 receptors in the dentate gyrus in the initial phase of TMT intoxication may be involved in mediating TMT-induced hippocampal damage.

Expression of GABA(A) receptor subunits in the hippocampus of the rat after kainic acid-induced seizures.

The GABA(A) receptor is a ligand gated chloride channel consisting of five membrane spanning proteins for which 13 different genes have been identified in the mammalian brain. The present review summarizes recent work from our laboratory on the characterization of the immunocytochemical distribution of these GABA(A) receptor subunits in the rat brain and changes in immunoreactivity and mRNA expression after kainic acid-induced status epilepticus. A heterogeneous distribution of immunoreactive GABA(A) receptor subunits was observed. The most abundant ones were: alpha1, alpha2, alpha4, alpha5, beta2, beta3, gamma2, and delta. Alpha1, beta2, and gamma2 were about equally distributed in all subfields of the hippocampus; alpha4- and delta-subunits were preferentially found in the dentate molecular layer and in CA1; alpha2 was localized to the dentate molecular layer and CA3; alpha5 was found in the dendritic areas of CA1 to CA3; and beta1 was preferentially seen in CA2. Alpha1, beta2, gamma2 and delta were highly concentrated in interneurons. Kainic acid-induced seizures caused acute and chronic changes in the expression of mRNAs and immunoreactive proteins. Acute changes included decreases in alpha2, alpha5, beta1, beta3, gamma2 and delta mRNA levels (by about 25-50%), accompanied by increases (by about 50%) in alpha1, alpha4, and beta2 messages in granule cells (after 6-12 h). Chronic changes, characterized by losses in mRNA and immunoreactive proteins in CA1 and CA3, are undoubtedly due to seizure-related cell damage. However, compensatory expression of alpha2 and beta3 subunits, especially in CA3b/c, was observed. Furthermore, increases in mRNAs and immunoreactive proteins were seen for alpha1, alpha2 alpha4, beta1, beta2, beta3 and gamma2 in granule cells and in the molecular layer of the dentate gyrus at 7-30 days after kainic acid injection. The changes in the expression of GABA(A) receptor subunits, observed in practically all hippocampal subfields, may reflect altered GABA-ergic transmission during development of the epileptic syndrome. Increased expression of GABA(A) receptor subunits in the dendritic field of granule cells and CA3 suggest that GABA-ergic inhibition may be augmented at these levels. However, the lasting preservation of alpha1-, beta2-, and gamma2-subunits in interneurons could provide a basis for augmented inhibition of GABA-ergic interneurons, leading to net disinhibition.

Trimethyltin intoxication induces marked changes in neuropeptide expression in the rat hippocampus.

In situ hybridization and immunocytochemistry were applied to investigate changes in the expression of somatostatin, neuropeptide Y, neurokinin B, cholecystokinin, dynorphin, and Met-enkephalin in the rat hippocampus after administration of a single peroral dose of trimethyltin hydroxide (9 mg/kg). Two time intervals were investigated: 5 days after trimethyltin treatment, when CA3 damage becomes manifest and is associated with increased aggression, seizure susceptibility, and memory deficit, and 16 days after trimethyltin, when neuronal damage is almost maximal and seizure susceptibility is declining. Robust but transient increases of neuropeptide Y, neurokinin B, and Met-enkephalin mRNA levels were revealed in the granule cell layer of the dentate gyrus and increased neuropeptide Y and neurokinin B immunoreactivities were found in mossy fibers. In reverse, dynorphin mRNA and immunoreactivity were decreased transiently in the dentate gyrus and mossy fibers, respectively. Strong over-expression of NPY mRNA was also observed in hilar interneurons and in CA1 and CA3 pyramidal cells as well as in the cortex at 5 days postdosing. Cholecystokinin- or neurokinin B-containing basket cells were preserved, while somatostatin-bearing interneurons were damaged by trimethyltin exposure. These neurochemical changes induced by trimethyltin intoxication strikingly parallel to those observed in animal models of temporal lobe epilepsy and may reflect activation of endogenous protective mechanisms. It is also suggested that hilar interneurons respond differently to trimethyltin exposure, for which neuropeptides are valuable markers.

Expression of fos protein in rat brain following administration of a nicotinic acetylcholine receptor agonist epibatidine.

Epibatidine (exo-2-(6-chloro-3-pyridyl)-7-azabicyclo-[2.2.1]heptane), an extract of frog skin, is a novel and highly potent agonist for the nicotinic acetylcholine (ACh) receptor. The present study was undertaken to examine the expression of Fos protein in several rat brain regions following an acute administration of epibatidine. Furthermore, we also studied the role of the dopamine D1 and D2 receptors and the N-methyl-d-aspartate (NMDA) receptor, and nicotinic ACh receptor in the expression of Fos protein by epibatidine. A single administration of epibatidine (5, 10, 50 microgram/kg) caused a marked induction of Fos-immunoreactivity in the prefrontal cortex, medial striatum, nucleus accumbens, amygdala and superior colliculus of rat brain. In these regions, pretreatment with SCH 23390 (1.0 mg/kg), a dopamine D1 receptor antagonist, MK-801 (1.0 mg/kg), a NMDA receptor antagonist, and mecamylamine (5. 0 mg/kg), a nicotinic Ach receptor antagonist, inhibited the induction of Fos protein by epibatidine (10 microgram/kg). Pretreatment with sulpiride, a dopamine D2 receptor antagonist, blocked the induction of Fos protein in the prefrontal cortex and the core region of accumbens nucleus, but not in the medial striatum and the shell division of nucleus accumbens of rat brain. These results suggest that epibatidine induced the expression of Fos protein in several regions of rat brain, and that dopamine D1 receptor, NMDA receptor, and nicotinic ACh receptor may play a role in the expression of Fos protein by epibatidine in rat brain. Furthermore, dopamine D2 receptor may, in part, play a role in epibatidine induced expression of Fos protein in the prefrontal cortex and the core region of nucleus accumbens, but not in the medial striatum and the shell division of nucleus accumbens of rat brain.

Difference in chromatin packaging between active and inactive X chromosomes by fractionation and allele-specific detection.

Using a novel method consisting of chromatin fractionation and allele-specific detection, chromatin packaging is compared between active X (Xa) and inactive X (Xi) chromosomes for five tumor cell clones that were derived from inter-subspecific F1 female mice. Separation of heterochromatic (H) and euchromatic (E) fractions is monitored by hybridization with subtelomeric satellite DNA and ribosomal RNA gene and by PCR amplification of p53 gene/pseudogene with one primer set. The H fraction was enriched with satellite and p53 pseudogene probably existing in heterochromatic regions while the E fraction showed inverse, suggesting fair separation. Analysis with seven marker and three gene loci revealed concentration of alleles on Xi in the H fraction and those on Xa in the E fraction, though the concentration levels varied. This implies that the packaging level of Xi is higher than that of active or inactive euchromatin on Xa. Intriguingly, one cell line showed biallelic expression and chromatin relaxation of the Pgk-1 locus, suggesting that the relaxation occur regionally on X chromosome.

GABA(A) receptor subunits in the rat hippocampus I: immunocytochemical distribution of 13 subunits.

The GABA(A) receptor is a ligand-operated chloride channel. It has a pentameric structure. In mammalian brain different subunits are recruited from four gene subfamilies. Using immunocytochemistry, we investigated the distribution of the 13 GABA(A) receptor subunits in the hippocampus of the rat. GABA(A) receptor subunits were heterogeneously distributed within different hippocampal subfields. High concentrations of alpha1-, alpha2-, alpha4-, beta3-, gamma2- and delta-immunoreactivities were observed within the molecular layer of the dentate gyrus, representing the dendritic area of the granule cells. In the hippocampus proper, the predominant GABA(A) receptor subunits were alpha1, alpha2, alpha5, beta3 and gamma2 that were located throughout the strata radiatum and oriens of CA1 to CA3. Immunocytochemical staining was there less prominent for alpha4-, beta1-, beta2- gamma3- and delta- subunits. In the hippocampus proper, the beta1 subunit was preferentially located in CA2. The alpha4- and delta-subunits were somewhat more abundant in CA1 than in CA3. Numerous local circuit neurons in the hippocampus proper and the hilus of the dentate gyrus contained alpha1-, beta2-, gamma2- and/or delta-subunits. Alpha3 and gamma1 were present only in minute amounts and no alpha6-IR was detected in the hippocampal formation. The distribution of the GABA(A) receptor subunits indicates the existence of heterogenously constituted GABA(A) receptor complexes within various hippocampal subfields, which may exert different physiological or pharmacological properties upon stimulation by GABA or its agonists.

GABA(A) receptor subunits in the rat hippocampus II: altered distribution in kainic acid-induced temporal lobe epilepsy.

Intraperitoneal injection of kainic acid in the rat represents a widely used animal model of human temporal lobe epilepsy. Injection of kainic acid induces acute limbic seizures which are accompanied by seizure-induced brain damage and late spontaneous recurrent seizures. There is considerable evidence for an altered transmission of GABA in human temporal lobe epilepsy and in the kainic acid model. We therefore investigated by immunocytochemistry the distribution of 13 GABA receptor subunits in the hippocampus of rats 12 h, 24 h, and two, seven and 30 days after injection of kainic acid. Within the molecular layer of the dentate gyrus, decreases in alpha2- and delta- and slight increases in alpha1, beta2- and beta3-immunoreactivities were observed at early intervals (12 to 24 h) after kainic acid injection. These changes were succeeded by marked increases in alpha1-, alpha2-, alpha4-, alpha5-, beta1-, beta3-, gamma2- and delta-immunoreactivities in the same area after seven to 30 days. Within the hippocampus proper, changes in expression of GABA(A) receptor subunits were demarcated by considerable neurodegeneration of CA1 and CA3 pyramidal neurons. All subunits present within dendritic areas of CA1 and CA3 were affected. These were alpha1, alpha2, alpha5, beta1-beta3, gamma2 and alpha4 (present only in CA1). Decreases in these subunits were followed by increased expression of alpha2-, alpha5-, beta3-, gamma2- and delta-subunits in the hippocampus proper notably in CA3 at later intervals (up to 30 days). Alpha1-, beta2-, gamma2- and delta-subunits were found in presumed GABA containing interneurons throughout the hippocampus. Their immunoreactivity was augmented after two to seven days. Some alpha4-, gamma3- and delta-immunoreactivity was also found in astrocytes 48 h after kainic acid injection. Our data indicate an impairment of GABA-mediated neurotransmission due to a lasting loss of GABA(A) receptor containing cells after kainic acid-induced seizures. The seizure-induced loss in GABA(A) receptors within the hippocampus may in part be compensated by increased expression of GABA(A) receptor subunits within the molecular layer of the dentate gyrus and in pyramidal cells.

GABA(A) receptor subunits in the rat hippocampus III: altered messenger RNA expression in kainic acid-induced epilepsy.

Kainic acid-induced seizures in rats represent an established animal model for human temporal lobe epilepsy. The neuropathological sequelae include acute status epilepticus followed by neurodegeneration in the CA1 and CA3 sector of the Ammon's horn and of interneurons in the hilus of the dentate gyrus. After about three weeks spontaneous recurrent seizures become manifest. We investigated changes in messenger RNA expression of 13 GABA(A) receptor subunits in the hippocampus of rats in the initial phase (6 h, 12 h and 24 h) after acute kainic acid-induced status epilepticus and seizure-related neuronal cell damage during and after acquisition of spontaneous recurrent seizures (seven and 30 days after kainic acid injection). In the granule cell layer, initial (after 6 to 12 h) decreases in (alpha2, alpha3, alpha5, beta1, beta3, gamma2 and delta messenger RNAs (by about 25 to 50%) were accompanied by increases (by about 50%) in alpha1, alpha4, and beta2 messages. At later intervals (after seven to 30 days), expression of alpha2, alpha4, beta3 and gamma2 messenger RNAs recovered to control values, with alpha5 and delta messenger RNA still being reduced (by 15 and 40% below control levels, respectively). Concentrations of the transcripts encoding for alpha1, alpha3, beta1, beta2, became markedly enhanced (between 20 and 50% of controls). Within the pyramidal cell layers CA1 and CA3, decreases in alpha2, alpha4, alpha5, beta(1-3) and gamma2 messenger RNAs were detected after seven to 30 days, reflecting pronounced neurodegeneration in these areas. The alpha1 transcript was decreased in CA3 after 24 h and increased to control levels indicating compensatory up-regulation of this message after seven days. Messenger RNAs encoding for alpha3-, gamma1-, and gamma3-subunits were detected at rather low levels, alpha6 was not present in the hippocampus. Our data suggest a fast but transient change in the expression of messenger RNAs encoding for different subunits of the GABA(A) receptor in the granule cell layer of the dentate gyrus. This is followed by a lasting augmentation of messenger RNAs encoding different GABA(A) receptor subunits in the same cell layer indicating long-lasting GABAergic inhibition. Changes within the pyramidal cell layer are mostly determined by concomitant neurodegenerative processes.

Limbic seizures induce neuropeptide and chromogranin mRNA expression in rat adrenal medulla.

Rats treated with kainic acid develop limbic seizures and have elevated levels of circulating catecholamines resulting from an extensive stimulation of the adrenal gland. We investigated the levels of several constituents of chromaffin granules in rat adrenal medulla after injection of kainic acid. This treatment increased mRNA steady-state levels of enkephalin, neuropeptide Y and chromogranin B 2-6-fold. Elevated levels of these constituents were found as early as 2 h after treatment and lasted up to 24 h. Chromogranin A and secretogranin II mRNA levels, on the other hand, remained unchanged. Adrenal catecholamine concentrations were reduced by 80%. Pre-treatment of rats with thiopental prior to kainic acid prevented seizures, the decline in catecholamines and the elevation of enkephalin and neuropeptide Y mRNAs but not that of chromogranin B. On the other hand, the peripherally acting ganglionic blocker chlorisondamine did not protect from the kainic acid-induced up-regulation of chromogranin B mRNA, suggesting that chromogranin B mRNA may be regulated by a direct effect of kainic acid on chromaffin cells. The pattern of changes in mRNA expression differed from that seen after insulin hypoglycemia or reserpine treatment. Thus, stimulation of the splanchnic innervation in vivo by various means leads to an individual and independent regulation of granule constituents by quite different mechanisms.

A novel clonality assay for the mouse: application to hepatocellular carcinomas induced with diethylnitrosamine.

A polymerase chain reaction-based clonality assay was developed for mouse tumors and cellular proliferations of the mouse. This assay was based on a polymorphism of the phosphoglucokinase-1 (Pgk-1) gene on the X chromosome between two different mouse subspecies and the different methylation patterns of active and inactive X chromosomes. All 15 tumor cell lines examined showed one of the two allelic bands on gel electrophoresis, which is consistent with the theory that tumor cell lines are monoclonally derived. This suggests that the Pgk-1 system is useful for clonality studies that will give insight into cancer development. With this method, nine hepatocellular carcinomas were examined, and eight showed monoallelic patterns. The remaining tumor exhibited a biallelic pattern, which is suggestive of polyclonal origin; however, other possibilities are discussed.