The purpose of the present study was to investigate the sites in the hypothalamus where the suprachiasmatic nucleus (SCN) may influence corticosteroid secretion. In spite of the well established, SCN-mediated, daily rhythms in adrenocorticotrophic hormone (ACTH) and corticosteroid secretion, previous studies determining the projections of the suprachiasmatic nucleus failed to illustrate direct connections with corticotrophin-releasing hormone neurons (CRH). In order to identify where in the central nervous system the SCN may influence corticosteroid secretion, areas were selected that contained SCN efferents contacting neurons involved in the stress response. To achieve this in the present study, SCN efferents were visualized by Pha-L tract-tracing, together with the neurons involved in the stress response by immunocytochemical staining for c-fos protein. The sites where these efferents contacted c-fos-positive neurons were established by light microscopic double staining and electron microscopic immunocytochemical studies. It appeared that apart from the medial parvocellular area of the paraventricular nucleus (PVN) of the hypothalamus, many more regions showed fos-positive neurons. Sites where SCN efferents contacted such neurons are limited only to areas immediately adjacent to these putative CRH neurons but are not concentrated on these neurons themselves. These areas consist of the periventricular and rostral PVN together with the dorsomedial hypothalamus: all three regions are known to project into the PVN. Therefore, it is concluded that the SCN transmits its information related to corticosteroid secretion via interneurons in and around the PVN to the CRH-containing neurons, rather than by a direct interaction with these neurons themselves.

GABAergic projections of the suprachiasmatic nucleus (SCN) were demonstrated in a double-labelling ultrastructural study which visualised the efferents of the SCN by PHA-L tracing, diaminobenzidine (DAB) immunocytochemistry, and GABA with immunogold postembedding staining. The results show a strong contralateral projection of the SCN that is partly GABA-containing. In addition, ipsilateral SCN projections to the dorsomedial hypothalamus and periventricular part of the paraventricular nucleus and sub-paraventricular nucleus were shown to contain GABA. The present results indicate that the SCN may utilize this inhibitory neurotransmitter to regulate and organize its own circadian rhythm as well as using GABA to transmit its diurnal information to other regions of the brain.

Rodent studies have shown that furan is a hepatocarcinogen. Previous studies conducted with high doses showed tumors at nearly 100% incidence at all doses. In this paper, a ninety-day gavage experiment conducted with lower doses (0.0, 0.03, 0.12, 0.5, 2.0, and 8.0 mg/kg bw) to identify a no-observed adverse effect level for hepatotoxicity and to characterize non-neoplastic effects including gross changes and histopathology, clinical biochemistry, hematology, and immunotoxicology is reported. As indicated by changes in serum biomarkers, increased liver weights and gross and histological lesions, the liver is the major target organ affected by furan. There were no changes in body weights, food consumption, or histology in other organs. Some of the serum electrolyte markers, including phosphorus, were altered. There was a significant increase in serum thyroxine and triidothyronine in males. This increase was not accompanied by histological thyroid changes. Immunophenotypic analysis showed that thymic lymphocyte maturation was altered in male rats. Although altered clinical biochemistry and hematological parameters were observed at a dose of > 0.5 mg/kg bw, mild histological lesions in the liver were observed at > 0.12 mg/kg bw. Based on this finding, a furan dose of 0.03 mg/kg bw was proposed as the no-observed adverse effect level for hepatic toxicity.

The seemingly contradictory observations in previous publications that gamma-aminobutyric acid (GABA) is detected in all cell bodies of the suprachiasmatic nucleus (SCN) and that terminals originating from the SCN are only 20-30% GABA positive prompted us to investigate whether this might be explained by a preference of colocalization in terminals of certain peptidergic neurons in the SCN or by a day/night rhythm in GABA synthesis. At three different circadian times, animals were perfusion fixed, and their SCNs were stained for vasopressin (VP), somatostatin (SOM), or vasoactive intestinal polypeptide (VIP). Subsequently, the number of GABA peptide-positive terminals was determined using GABA postembedding staining in ultrathin sections. It appeared that the highest percentage of colocalization with GABA was detected in VIP terminals (38%) and the lowest in VP terminals (15%). No differences in colocalization percentages could be observed in any parameter at any circadian time. In the dorsomedial hypothalamus, one of the target areas of the VP and VIP fibers from the SCN, a colocalization of GABA within VP and VIP terminals was found similar to that in the SCN. In the region of the somatostatin-containing neurons in the SCN, a number of axoaxonal contacts could be observed that sometimes exhibited synaptic specializations. In nearly all cases, the axoaxonic terminals contained GABA and/or SOM. The conclusion is that the high level of intrinsic GABAergic connections in the SCN represents a putatively powerful mechanism to synchronize or shut down the activity of the SCN. We discuss the possibility that, depending on the firing frequency of the neurons, the colocalization of GABA with all peptides under investigation allows for the selection of which transmitter is released, the peptidergic one or the amino acid.

Furan is a heterocyclic organic compound formed during heat treatment for processing and preservation of various types of food. Rodent studies have previously shown that furan is a hepatocarcinogen. Those studies were conducted over a high dose range, which induced tumors at nearly 100% incidence at all doses. This ninety-day gavage study in mice was conducted to extend the dose to a lower range (0.0, 0.03, 0.12, 0.5, 2.0, and 8.0 mg/ kg body weight [bw] per day) to identify a no-observed adverse effect level for hepatotoxicity and to characterize non-neoplastic effects, including those affecting clinical biochemistry, hematology, tissue morphology, and histopathology. The liver was the primary target organ with dosedependent toxicity. Liver weights were increased at the 8.0 mg/kg bw dose in females only. Levels of the serum enzyme alanine transaminase, representative of liver damage, were increased three-fold at the highest dose. Histological changes in the liver were observed at 2.0 and 8.0 mg/kg bw in both sexes. Although clinical parameters were also altered for the kidney, these differences were not accompanied by histological changes. Based on these clinical biochemical and histological changes, a no-observed adverse effect level of 0.12 mg/kg bw per day of furan in mice is suggested.