C-fos mRNA increases in the ground squirrel suprachiasmatic nucleus during arousal from hibernation.

During hibernation the body temperature of the golden-mantled ground squirrel, Spermophilus lateralis, may drop below 5 degrees C for a few hours to a week or more. Animals cycle between euthermia and deep hibernation many times over the course of the hibernation season. Expression of the transcription factor c-fos increased in the suprachiasmatic nucleus (SCN) of the hypothalamus, the mammalian circadian clock, during deep hibernation and peaked during the arousal from hibernation. The pattern of increase in c-fos messenger RNA seen in the SCN by in situ hybridization was similar to that seen by Northern blot analysis of total hypothalamic RNA. The induction of c-fos may reflect a wake-up signal, increasing transcription of genes required in the euthermic state.

Light-induced gene expression in the suprachiasmatic nucleus of young and aging rats.

We investigated age-related changes in a molecular mechanism associated with synchronization of circadian rhythms to the environment. In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus contains a circadian pacemaker that regulates a variety of physiological and behavioral rhythms. Recent studies have demonstrated photic induction of immediate early genes (IEGs) such as c-fos in the SCN in a circadian-phase dependent manner, suggesting that IEGs may be part of the pathway for entrainment of circadian rhythms. We find that there is a decreased response of the IEGs c-fos, and NGFI-A but not NGFI-B in the SCN of old animals after photic stimulation. Changes in gene transcription indicated by IEGs may provide insights into the molecular machinery of the biological clock and ultimately elucidate mechanisms underlying the age-dependent decay of circadian organization.

Melatonin influences Fos expression in the rat suprachiasmatic.

Administration of the pineal hormone melatonin to rats induces expression of Fos, the protein product of the c-fos proto-oncogene, in the suprachiasmatic nucleus (SCN), the putative biological clock of mammals. Expression of the Fos protein is dependent on circadian phase: injections in the late subjective night (circadian time (CT) 22) induce Fos expression in cells within the ventral SCN whereas injections during the subjective day are ineffective. Since melatonin injections in the late subjective day have previously been shown to phase advance circadian rhythms, these results indicate that phase-advances of the circadian system can occur without increased expression of Fos protein in the SCN, at least at levels detectable by immunohistochemistry. In support of in situ hybridization histochemical evidence obtained previously, immunocytochemical data from vehicle-injected control rats suggest that the Fos protein undergoes an endogenous fluctuation with peak levels in the SCN occurring during the subjective night. These observations indicate that melatonin can affect immediate early gene expression within the SCN.

Circadian and light-induced expression of immediate early gene mRNAs in the rat suprachiasmatic nucleus.

Recent work has suggested that immediate early genes are involved in the phase-shifting response of the circadian pacemaker to light. In mammals, the endogenous pacemaker is located in the suprachiasmatic nucleus (SCN). We report here a systematic examination of the expression of the immediate early genes fos, NGFI-A, and NGFI-B in the rat SCN, in basal and light-stimulated conditions across the circadian cycle. The photic induction of all three genes examined was found to be gated by the circadian system with maximal induction observed during the mid to late subjective night. The photic-induced expression of NGFI-B in the SCN was considerably less than that of NGFI-A, despite previous observations suggesting greater induction of NGFI-B by membrane depolarization. The levels of immediate early gene expression induced by light do not directly correspond to the magnitude of previously reported light-induced phase-shifts of the activity rhythm. The results also suggest that fos may undergo a circadian rhythm of expression in the rat SCN.

Neurochemicals in the dorsal pontine tegmentum.

Detailed maps of neurochemicals in the locus coeruleus and adjacent dorsal tegmental areas are discussed in this chapter. The locus coeruleus appears to be one of the most complex brain regions with six neurochemicals (acetylcholinesterase, tyrosine hydroxylase, galanin, neuropeptide Y, neurotensin, and vasoactive intestinal protein) contained within the cell bodies.

Detection of CCK mRNA in the motor nucleus of the rat trigeminal nerve with in situ hybridization histochemistry.

The regional distribution of dorsal pontine tegmental neurons expressing cholecystokinin (CCK) messenger RNA (mRNA) was examined using in situ hybridization histochemical techniques. A distribution of neurons containing CCK mRNA was observed in the motor nucleus of the trigeminal nerve, a region which has not been shown to contain CCK by immunocytochemical means. In addition, CCK mRNA was seen in the caudal portion of the dorsal raphe nucleus which correlates with prior immunocytochemical observations. The presence of CCK mRNA in the cholinergic motor trigeminal nucleus suggests that these cells express the CCK gene and have gone undetected in previous immunocytochemical studies. It is also suggested that CCK may have an influence on muscular contraction at sites of trigeminal nerve innervation (e.g. muscles of mastication).

Localization of substance P mRNA in cholinergic cells of the rat laterodorsal tegmental nucleus: in situ hybridization histochemistry and immunocytochemistry.

1. In situ hybridization histochemical techniques in combination with immunocytochemistry and acetylcholinesterase (AChE) histochemistry were used to study the colocalization of messenger RNA (mRNA) encoding the neuropeptide substance P (SP) in cholinergic cells of the laterodorsal tegmental nucleus (LDT) of the rat pontine brain stem. 2. Alternate serial sections were hybridized with a 48-base, 35S-labeled synthetic oligonucleotide probe encoding SP using in situ hybridization histochemistry and processed either histochemically for AChE or immunocytochemically for choline acetyltransferase (ChAT). 3. In addition, serial section analysis was used to demonstrate the correlation between SP and SP mRNA in the same cells of the LDT. 4. These studies reveal that the cholinergic neurons of the LDT synthesize SP.

A synthetic oligonucleotide probe encoding for atrial natriuretic peptide detects specific mRNA transcripts in rat heart but not brain using in situ hybridization histochemistry.

In situ hybridization histochemical techniques were used in an attempt to demonstrate atrial natriuretic peptide (ANP) messenger RNA (mRNA) in the rat brain. A synthetic oligonucleotide derived from previously reported ANF cDNA sequence was used as a probe. Northern blot analysis of total RNA isolated from rat heart demonstrated that the oligonucleotide recognized a single species of RNA (0.9 kb), a size consistent with previous reports. Rat heart sections revealed dense accumulations of ANF mRNA in the cardiac atria and lesser densities in the ventricles. Rat brain sections hybridized with the same oligonucleotide did not label ANF mRNA accumulations in any neuronal cell bodies. A possible explanation for this latter observation is either sparsely distributed expressing neurons or low expression and high turnover of ANF mRNA in brain.

Immunocytochemical localization of peptides and other neurochemicals in the rat laterodorsal tegmental nucleus and adjacent area.

The laterodorsal tegmental nucleus (ntdl) contains a cluster of cells located just medial to the locus coeruleus in the pontine brainstem. The ntdl has been shown to project both rostrally to the forebrain and diencephalon and caudally to the spinal cord. In an effort to characterize this region neurochemically, the present study was conducted to identify a variety of neurochemicals localized within perikarya and fibers of the ntdl and surrounding nuclei. Rats were perfused with formalin, and brain sections were processed for fluorescence immunocytochemistry and acetylcholinesterase (AChE). Of the neurochemicals screened, atrial natriuretic factor (ANF), choline acetyltransferase (ChAT), cholecystokinin (CCK), calcitonin gene-related peptide (CGRP), dynorphin B (Dyn B), galanin, somatostatin, substance P, neurotensin (NT), neuropeptide Y (NPY), vasopressin, vasoactive intestinal polypeptide (VIP), serotonin (5HT), glutamic acid decarboxylase (GAD), and tyrosine hydroxylase (TH) were studied. AChE and ChAT staining revealed that the ntdl contains mostly cholinergic neurons. In addition, brightly reactive substance P and galanin and paler staining CRF, ANF, CGRP, NT, VIP, and Dyn B cell bodies were found within the ntdl. Varicose fibers in this nucleus also contained these peptides in addition to CCK, GAD, TH, 5HT, and NPY. The dorsal tegmental nucleus, dorsal raphe nucleus, locus coeruleus, and the parabrachial region contained a dense and varied assortment of peptides with distinct positions and patterns. This multiplicity of neurochemicals within this area suggests a possible influence on a variety of functions modulated by the ntdl and other closely associated tegmental nuclei.

Rapid-eye movement sleep and muscarinic receptor binding in rats are augmented during withdrawal from chronic scopolamine treatment.

The purpose of this study was to determine whether a seven day, once-daily morning administration of scopolamine produces upregulation of muscarinic receptors and augments REMS during withdrawal. After obtaining two, six-hour baseline sleep recordings, beginning at 0900, independent groups of rats were administered either scopolamine or saline every morning for seven days. Six hour sleep recordings were obtained following the first and seventh day of injection and during the two subsequent withdrawal days. After obtaining the last sleep recording the rats were sacrificed and the following brain areas removed: cerebral cortex, hippocampii, caudate nuclei, brainstem, and cerebellum. 3H-QNB was used as the ligand to assess for changes in muscarinic receptor binding. Compared to baseline, scopolamine produced a significant decrease in REMS during the period of drug administration. During the withdrawal days, however, REMS increased during the morning period. Compared to the saline group, the scopolamine treated animals had increased muscarinic receptor binding in the caudate and hippocampus; no significant change in receptor density was observed in the cortex, brainstem or cerebellum.

Stress-induced immobility in rats with cholinergic supersensitivity.

Immobility during forced swimming or after mild footshock (1 mA for 2 sec) was observed in five groups of rats. The Flinders Sensitive Line (FSL) of rats, known to be more sensitive to cholinergic agonists, exhibited the greatest degree of immobility in the forced swim test. Rats chronically treated with, and subsequently withdrawn from, either scopolamine (2 mg/kg, once daily) or amitriptyline (10 mg/kg, once daily) were also significantly more immobile than either a control group treated chronically with isotonic saline or the Flinders Resistant Line (FRL) of rats in the forced swim test. Similar trends were observed for locomotor depression in the open field following exposure to footshock. Receptor binding studies indicated significantly greater concentrations of muscarinic acetylcholine receptors in the hippocampus of the scopolamine, and amitriptyline, withdrawn rats. These findings indicate that rats with increased cholinergic sensitivity are more sensitive to the immobility-inducing effects of mild stressors. Thus, they may prove to be useful models for studying the relationship between affective disorders and the cholinergic system.