The aim of the present work was to study the involvement of the dopaminergic system of the telencephalic and diencephalic areas of the vertebrate brain in the organization of the sleep-waking cycle in cold-blooded and warm-blooded vertebrates. Immunohistochemical studies of tyrosine hydroxylase content, this being the key enzyme in dopamine synthesis, in the striatum, supraoptic and arcuate nuclei, and zona incerta of the hypothalamus of sturgeon and mammals (rats) of three age groups (14 and 30 days and adults), in conditions of tactile and sleep-deprivation stressors. In fish, transient stress was followed by the detection of tyrosine hydroxylase-immunoreactive cells in all parts of the brain. In prolonged stress, tyrosine hydroxylase-immunoreactive cells and fibers were not found in the forebrain, though they were well represented in the hypothalamic nuclei. In 14-day-old rat pups, 2-h sleep deprivation increased the tyrosine hydroxylase content of fibers in the caudate nucleus and cells in the zona incerta of the hypothalamus, while 30-day-old animals subjected to 6-h sleep deprivation showed increases in tyrosine hydroxylaseimmunoreactive material contents in cells in the paraventricular nucleus and decreases in the quantity in fibers. In adult rats, the arcuate nucleus and zona incerta showed decreases in the content of tyrosine hydroxylase-immunoreactive material on the background of sleep deprivation, with increases during postdeprivation sleep. These data are discussed in the light of the phylo- and ontogenetic development of the neurosecretory and neurotransmitter functions of the dopaminergic system in the evolutionarily ancient diencephalic and evolutionarily young telencephalic areas of the vertebrate brain as major systems triggering and maintaining the functional states of the body during the sleep-waking cycle.

Northern blot analysis was utilized to distinguish between catalytic and truncated TrkB mRNA on the basis of transcript size. Repeated (10 days), but not acute, immobilization stress significantly increased levels of catalytic TrkB mRNA, but did not influence expression of truncated TrkB transcripts in rat hippocampus. Exposure to another paradigm, a combination of different, unpredictable stressors, also increased levels of catalytic, but not truncated, TrkB mRNA. In situ hybridization analysis demonstrated that chronic stress up-regulated TrkB mRNA in CA1 and CA3 pyramidal and dentate gyrus granule cells layers of hippocampus. As previously reported, both acute and chronic immobilization stress decreased expression of BDNF mRNA, suggesting that up-regulation of catalytic TrkB mRNA may be a compensatory adaptation to repeated stress.

The underlying mechanisms by which physical or psychological stress causes neurodegeneration are still unknown. We have demonstrated that the high-output and long-lasting synthesizing source of nitric oxide (NO), inducible NO synthase (iNOS), is expressed in brain cortex after three weeks of repeated stress and that its overexpression accounts for the neurodegenerative changes found in this situation. Now we have found that a short duration of stress (immobilization for 6 h) also induces the expression of iNOS in brain cortex in adult male rats. In order to elucidate the possible mechanisms involved in iNOS expression, we have studied the role of the cytokine tumor necrosis factor-alpha (TNF-alpha) released in brain during stress. We have shown that there is an increase in soluble TNF-alpha levels after 1 h of stress in cortex and that this is preceded by an increase in TNF-alpha-convertase (TACE) activity in brain cortex as soon as 30 min after immobilization. Stress-induced increase in both TACE activity and TNF-alpha levels seems to be mediated by excitatory amino acids since they can be blocked by MK-801 (dizocilpine)(0.2 mg/kg i.p.), an antagonist of the N-methyl-D-aspartate subtype of glutamate receptor. In order to study the role of TACE and TNF-alpha in iNOS induction, a group of animals were i.p. injected with the preferred TACE inhibitor BB1101 (2 and 10 mg/kg). Indeed, BB1101 inhibited iNOS expression induced by six hours of stress. In addition, we studied the role of the transcription factor nuclear factor kappaB (NF-kappaB), which is required for iNOS expression. We have found that the administration of the TACE inhibitor BB1101 inhibited the stress-stimulated translocation of NF-kappaB to the nucleus. Taken together, these findings indicate that glutamate receptor activation induces TACE up-regulation and subsequent increase in TNF-alpha levels, and this account for stress-induced iNOS expression via NF-kappaB activation, supporting a possible neuroprotective role for specific TACE inhibitors in this situation.

Males housed in mixed sex groups quickly form dominance hierarchies; subordinates can be further subdivided into stress responsive subordinates (SRS) and non-responsive subordinates (NRS) based on corticosterone responses to a novel stressor. Tyrosine hydroxylase (TH) mRNA levels measured with in situ hybridization were elevated in locus coeruleus (LC) of NRS compared to singly or pair-housed controls; NRS also had higher TH levels than dominants. TH protein levels determined by immunoautoradiography were also higher in LC of NRS and SRS versus pair-housed controls.

Assessment of sympathoadrenal medullary system (SAM) activity is only possible to date via measurement of catecholamines in blood plasma or via electrophysiological methods. Both ways of measurement are restricted to endocrinological or psychophysiological laboratories, as both require either immediate freezing of blood samples or complex recording devices. Efforts have therefore been undertaken to find a method comparable to salivary cortisol measurements, in which noninvasive samples can be taken at any place and stored at room temperature for sufficient time before later analysis in the laboratory. Salivary alpha-amylase (sAA) is a candidate that may prove useful in this context. We show here that sAA activity is increased by acute psychosocial stress (Trier Social Stress Test) and that increases in sAA correlate with increases in norepinephrine. We further report that sAA exhibits a stable circadian pattern that mirrors that of salivary cortisol. In conclusion, the current data show that salivary alpha-amylase may serve as an easy-to-use index for SAM activity. However, some questions remain to be answered; for example, what impact does salivary flow rate exert on stress-induced sAA activity?

BACKGROUND: The locus coeruleus (LC), a target for CRH neurons, is critically involved in responses to stress. Various physiological stresses increase norepinephrine turnover, tyrosine hydroxylase (TH) enzymatic activity, protein and mRNA levels in LC cell bodies and terminals; however, the effect of stress on other enzymes involved in norepinephrine biosynthesis in the LC is unknown. METHODS: Rats were exposed to single (2 hour) or repeated (2 hour daily) immobilization stress (IMO). Recombinant rat dopamine b-hydroxylase (DBH) cDNA was expressed in E. coli and used to generate antisera for immunohistochemistry and immunoblots in LC. Northern blots were used to assess changes in mRNA levels for TH, DBH, and GTP cyclohydrolase I (GTPCH) in the LC in response to the stress. Conditions were found to isolate nuclei from LC and to use them for run-on assays of transcription. RESULTS: Repeated stress elevated the DBH immunoreactive protein levels in LC. Parallel increases in TH, DBH and GTPCH mRNA levels of about 300% to 400% over control levels were observed with single IMO, and remained at similar levels after repeated IMO. This effect was transcriptionally mediated, and even 30 min of a single IMO significantly increased the relative rate of transcription. CONCLUSIONS: This study is the first to reveal transcriptional activation of the genes encoding catecholamine biosynthetic enzymes in the LC by stress. In addition to TH, changes in DBH and GTPCH gene expression may also contribute to the development of stress-triggered affective disorders.

Tyrosine hydroxylase is considered to be the rate-limiting enzyme in the synthesis of catecholamines in both the central and peripheral nervous system. Increased or decreased neuronal activity, stress, lesions, drug effects, endocrinological manipulations and experimental models of hypertension are associated with alterations in tyrosine hydroxylase activity in the central nervous system. In many of these instances, the changes in the activity of tyrosine hydroxylase in the central nervous system that occur are localized to discrete catecholaminergic pathways and nuclei in the brain. The purpose of this review is to summarize and assess this information and to provide insight into the function of catecholamine systems in the brain and their interactions with other putative neurotransmitter systems.

Congenitally helpless rats have been selectively bred to display an immediate helpless response to stress in order to model hereditary brain differences that contribute to depression vulnerability. Differences in regional brain metabolism between congenitally helpless and non-helpless rats were investigated using quantitative cytochrome oxidase histochemistry. The results indicated that congenitally helpless rats had 64-71% elevated metabolism in the habenula and a 25% elevation in the related interpeduncular nucleus. In contrast, helpless rats had 28% reduced metabolism in the ventral tegmental area (VTA) and 14-16% reductions in the basal ganglia and basolateral and central amygdala. The opposite metabolic changes in the habenula and ventral tegmental area may be especially important for determining the congenitally helpless rat's global pattern of brain activity, which resembles the metabolic activity pattern produced by dopamine antagonism.

Previous studies have demonstrated that chronic stress increases and antidepressant treatments decrease levels of tyrosine hydroxylase (TH) in locus coeruleus (LC). In the present study, the influence of chronic antidepressant treatment on the induction of TH immunoreactivity in response to cold stress is examined. It was found that chronic imipramine pretreatment (18 days) attenuated the induction of TH in response to cold stress, resulting in levels of TH immunoreactivity not different from control. In contrast, imipramine pretreatment for 1 or 7 days was not sufficient to normalize the stress-induced elevation of TH immunoreactivity. These findings raise the possibility that the therapeutic action of antidepressants may be derived, in part, from the ability of these treatments to normalize levels of TH and thereby the function of the NE neurotransmitter system under conditions of stress.

RATIONALE: Stress-related glucocorticoid and glutamate release have been implicated in hippocampal atrophy evident in patients with post-traumatic stress disorder (PTSD). Glutamatergic mechanisms activate nitric oxide synthase (NOS), while gamma-amino-butyric acid (GABA) may inhibit both glutamatergic and nitrergic transmission. Animal studies support a role for NOS in stress. OBJECTIVES: We have studied the role of NOS and glucocorticoids, as well as inhibitory and excitatory transmitters, in a putative animal model of PTSD that emphasizes repeated trauma. METHODS: Hippocampal NOS activity, N-methyl-D-aspartate (NMDA) receptor binding characteristics and GABA levels were studied in Sprague-Dawley rats 21 days after exposure to a stress-restress paradigm, using radiometric analysis, radioligand studies and high-performance liquid chromatography (HPLC) analysis with electrochemical detection, respectively. The NOS isoform involved, and the role of stress-mediated corticosterone release in NOS activation, was verified with the administration of selective iNOS and nNOS inhibitors, aminoguanidine (50 mg/kg/day i.p.) and 7-nitroindazole (12.5 mg/kg/day i.p.), and the steroid synthesis inhibitor, ketoconazole (24 mg/kg/day i.p.), administered for 21 days prior to and during the stress procedure. RESULTS: Stress evoked a sustained increase in NOS activity, but reduced NMDA receptor density and total GABA levels. Aminoguanidine or ketoconazole, but not 7-nitroindazole or saline, blocked stress-induced NOS activation. CONCLUSIONS: Stress-restress-mediated glucocorticoid release activates iNOS, followed by a reactive downregulation of hippocampal NMDA receptors and dysregulation of inhibitory GABA pathways. The role of NO in neuronal toxicity, and its regulation by glutamate and GABA has important implications in stress-related hippocampal degeneration.

In order to quantify psychological stress and to distinguish eustress and distress, we have been investigating the establishment of a method that can quantify salivary amylase activity (SMA). Salivary glands not only act as amplifiers of a low level of norepinephrine, but also respond more quickly and sensitively to psychological stress than cortisol levels. Moreover, the time-course changes of the salivary amylase activity have a possibility to distinguish eustress and distress. Thus, salivary amylase activity can be utilized as an excellent index for psychological stress. However, in dry chemistry system, a method for quantification of the enzymatic activity still needs to be established that can provide with sufficient substrate in a testing tape as well as can control enzymatic reaction time. Moreover, it is necessary to develop a method that has the advantages of using saliva, such as ease of collection, rapidity of response, and able to use at any time. In order to establish an easy method to monitor the salivary amylase activity, a salivary transcription device was fabricated to control the enzymatic reaction time. A fabricated salivary amylase activity monitor consisted of three devices, the salivary transcription device, a testing-strip and an optical analyzer. By adding maltose as a competitive inhibitor to a substrate Ga1-G2-CNP, a broad-range activity testing-strip was fabricated that could measure the salivary amylase activity with a range of 0-200 kU/l within 150 s. The calibration curve of the monitor for the salivary amylase activity showed R2=0.941, indicating that it was possible to use this monitor for the analysis of the salivary amylase activity without the need to determine the salivary volume quantitatively. In order to evaluate the assay variability of the monitor, salivary amylase activity was measured using Kraepelin psychodiagnostic test as a psychological stressor. A significant difference of salivary amylase activity was recognized between the pre-stress and mid-stress periods. This study demonstrated that broad-range salivary amylase activity monitor was developed that could be used with only 5 microl of saliva.