Mechanism of acute tryptophan depletion: is it only serotonin?

The method of acute tryptophan depletion (ATD), which reduces the availability of the essential amino acid tryptophan (TRP), the dietary serotonin (5-hydroxytryptamine (5-HT)) precursor, has been applied in many experimental studies. ATD application leads to decreased availability of TRP in the brain and its synthesis into 5-HT. It is therefore assumed that a decrease in 5-HT release and subsequent blunted neurotransmission is the underlying mechanism for the behavioural effects of ATD. However, direct evidence that ATD decreases extracellular 5-HT concentrations is lacking. Furthermore, several studies provide support for alternative underlying mechanisms of ATD. This may question the utility of the method as a selective serotonergic challenge tool. As ATD is extensively used for investigating the role of 5-HT in cognitive functions and psychiatric disorders, the potential of alternative mechanisms and possible confounding factors should be taken into account. It is suggested that caution is required when interpreting ATD effects in terms of a selective serotonergic effect.

Urocortin 3 transgenic mice exhibit a metabolically favourable phenotype resisting obesity and hyperglycaemia on a high-fat diet.

AIMS/HYPOTHESIS: Urocortins are the endogenous ligands for the corticotropin-releasing factor receptor type 2 (CRFR2), which is implicated in regulating energy balance and/or glucose metabolism. We determined the effects of chronic CRFR2 activation on metabolism in vivo, by generating and phenotyping transgenic mice overproducing the specific CRFR2 ligand urocortin 3. METHODS: Body composition, glucose metabolism, insulin sensitivity, energy efficiency and expression of key metabolic genes were assessed in adult male urocortin 3 transgenic mice (Ucn3(+)) under control conditions and following an obesogenic high-fat diet (HFD) challenge. RESULTS: Ucn3(+) mice had increased skeletal muscle mass with myocyte hypertrophy. Accelerated peripheral glucose disposal, increased respiratory exchange ratio and hypoglycaemia on fasting demonstrated increased carbohydrate metabolism. Insulin tolerance and indices of insulin-stimulated signalling were unchanged, indicating these effects were not mediated by increased insulin sensitivity. Expression of the transgene in Crfr2 (also known as Crhr2)-null mice negated key aspects of the Ucn3(+) phenotype. Ucn3(+) mice were protected from the HFD-induced hyperglycaemia and increased adiposity seen in control mice despite consuming more energy. Expression of uncoupling proteins 2 and 3 was higher in Ucn3(+) muscle, suggesting increased catabolic processes. IGF-1 abundance was upregulated in Ucn3(+) muscle, providing a potential paracrine mechanism in which urocortin 3 acts upon CRFR2 to link the altered metabolism and muscular hypertrophy observed. CONCLUSIONS/INTERPRETATION: Urocortin 3 acting on CRFR2 in skeletal muscle of Ucn3(+) mice results in a novel metabolically favourable phenotype, with lean body composition and protection against diet-induced obesity and hyperglycaemia. Urocortins and CRFR2 may be of interest as potential therapeutic targets for obesity.

Enhanced contractility of intraparenchymal arterioles after global cerebral ischaemia in rat - new insights into the development of delayed cerebral hypoperfusion.

AIM: Delayed cerebral hypoperfusion is a secondary complication found in the days after transient global cerebral ischaemia that worsens the ischaemic damage inflicted by the initial transient episode of global cerebral ischaemia. A recent study demonstrated increased cerebral vasoconstriction in the large arteries on the brain surface (pial arteries) after global cerebral ischaemia. However, smaller arterioles inside the brain (parenchymal arterioles) are equally important in the regulation of cerebral blood flow and yet their pathophysiology after global cerebral ischaemia is largely unknown. Therefore, we investigated whether increased contractility occurs in the intraparenchymal arterioles. METHODS: Global cerebral ischaemia was induced in male Wistar rats by bilateral common carotid occlusion for 15 min combined with hypovolaemia. Regional cerebral blood flow was determined by quantitative autoradiography. Intraparenchymal arterioles were isolated and pressurized, and concentration-response curves to endothelin-1 with and without the endothelin B receptor-selective antagonist BQ788 was generated. Endothelin B receptor expression was investigated by quantitative flow cytometry and immunohistochemistry. RESULTS: We observed increased endothelin-1-mediated contractility of parenchymal arterioles correlating with reduced cerebral blood flow of the cortex, hippocampus and caudate nucleus 48 h after global cerebral ischaemia. The increased endothelin-1-mediated contractility was abolished by BQ788, and the vascular smooth muscle cell-specific expression of endothelin B receptors was significantly increased after global cerebral ischaemia. CONCLUSION: Increased endothelin-1-mediated contractility and expression of endothelin B receptors in the intraparenchymal vasculature contributes to the development of delayed cerebral hypoperfusion after global cerebral ischaemia in combination with vascular changes of the pial vasculature.

Early detection of cryptic memory and glucose uptake deficits in pre-pathological APP mice.

Earlier diagnosis and treatment of Alzheimer's disease would greatly benefit from the identification of biomarkers at the prodromal stage. Using a prominent animal model of aspects of the disease, we here show using clinically relevant methodologies that very young, pre-pathological PDAPP mice, which overexpress mutant human amyloid precursor protein in the brain, exhibit two cryptic deficits that are normally undetected using standard methods of assessment. Despite learning a spatial memory task normally and displaying normal brain glucose uptake, they display faster forgetting after a long delay following performance to a criterion, together with a strong impairment of brain glucose uptake at the time of attempted memory retrieval. Preliminary observations suggest that these deficits, likely caused by an impairment in systems consolidation, could be rescued by immunotherapy with an anti-ß-amyloid antibody. Our data suggest a biomarker strategy for the early detection of ß-amyloid-related abnormalities.

Basal ganglia calcification in BB/E rats with diabetes.

Human diabetes is associated with cognitive impairment and structural abnormalities in the brain such as cerebral atrophy. The aetiology of these abnormalities is not known. The BB/E rat is a well-established model of type 1 (insulin dependent) diabetes. A cohort of 34 BB/E rats with diabetes was divided into three sub-groups according to age (and duration of diabetes). Basal ganglia calcification (BGC) was present in the brains of more than 50% of diabetic animals, but not in any of 37 non-diabetic BB/E rats. BGC occurred more commonly in those animals which had the longest duration of diabetes (p=0.001), such that BGC was present in only 8% of animals with diabetes for 20 weeks, but in 100% of animals with diabetes for 60 weeks. There were no other significant light microscopic neuropathologic changes in diabetic animals. It will be important to investigate the mechanism of brain calcification, whether a similar process occurs in humans with diabetes, and its possible relationship to cognitive decline.

Differential regulation of corticosteroid receptors by monoamine neurotransmitters and antidepressant drugs in primary hippocampal culture.

Hyperactivity of the hypothalamic-pituitary-adrenal axis is a characteristic feature of depressive illness. The centrally located corticosteroid receptors, the glucocorticoid and mineralocorticoid receptors, are thought to be important modulators of this axis and changes in the levels of these receptors, particularly in the hippocampus, may underlie the hyperactivity observed. Various antidepressant drugs increase hippocampal mineralocorticoid and glucocorticoid receptor levels in vivo. These effects are thought to be mediated via alterations in monoaminergic neurotransmission. We examined whether serotonin (5HT) and noradrenaline (NA) have direct effects on glucocorticoid receptor and mineralocorticoid receptor expression in primary hippocampal neurones, and whether antidepressants also exert direct effects on target neurones. Exposure of hippocampal cells to 5HT for 4 days increased both glucocorticoid and mineralocorticoid receptor mRNA and protein expression. The induction of mineralocorticoid receptor mRNA was completely blocked by the 5HT(7) receptor antagonist SB 269970. In contrast glucocorticoid receptor induction was insensitive to the 5HT(7) receptor, whilst studies with the 5HT(1A) receptor agonist 8-hydroxy-2-(di-n-proplamino) tetralin hydrochloride and the 5HT(1A) receptor antagonist N-[2-[4-2-[O-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane carboxamide trihydrochloride (WAY 100635) suggest a partial role for 5HT(1A) receptors in hippocampal glucocorticoid receptor regulation. Treatment with NA for 4 days also increased glucocorticoid receptor expression but had no effect on mineralocorticoid receptor expression. This was blocked by propanolol suggesting action via beta-adrenergic receptors. Similarly to NA, fluoxetine and amitriptyline also selectively increased glucocorticoid receptor mRNA and protein levels over this time course. However, glucocorticoid receptor induction by fluoxetine or amitriptyline was not blocked by WAY 100635 or propanolol. These results show that 5HT, NA and antidepressants act directly but via distinct mechanisms on hippocampal neurones to regulate mineralocorticoid and glucocorticoid receptor expression. Thusly, manipulation of neurotransmitter or antidepressant levels in the brain may aid in reversing hypothalamic-pituitary-adrenal axis hyperactivity by restoring hippocampal corticosteroid receptor balance.

Cerebrovascular responses to pathophysiological insult in diabetic rats.

Diabetes mellitus is associated with altered cerebrovascular responsiveness and this could contribute to the pathology of stroke in diabetic patients. In these studies, we used a model of haemorrhagic stroke (intrastriatal injection of 50 microl blood) to examine subacute perilesional perfusion and blood-brain barrier (BBB) integrity in spontaneously diabetic rats. Volumes of striatal oligaemia (blood flow < 35 ml 100 g(-1) min(-1)) were significantly increased (>300%) in diabetic rats with intrastriatal blood, compared to either non-diabetic rats with blood or control diabetic rats with striatal injection of silicon oil. However, the increase in BBB permeability was both qualitatively and quantitatively similar in diabetic and control rats. Poorer outcomes following haemorrhagic stroke in diabetic patients may thus result from dysfunctional cerebrovascular control, and particularly decreased dilatatory reserve.

Acute tryptophan depletion in rats alters the relationship between cerebral blood flow and glucose metabolism independent of central serotonin.

Acute tryptophan depletion (ATD) decreases the 5-HT precursor tryptophan (TRP) in blood and is used both clinically and preclinically to investigate the involvement of 5-HT in the development of depressive symptomatology. Depression is associated with both central 5-HT dysfunction and abnormalities in the normal relationship between regional cerebral blood flow (CBF) and glucose metabolism (CMRG). In this study, ATD was applied in Wistar rats to investigate the cerebrovascular effects of acute changes in peripheral TRP. Rats were orally fed with a protein-carbohydrate mixture, either containing or lacking TRP. Four hours later, CBF or CMRG was measured by quantitative autoradiographic imaging in 43 brain regions of interest (ROI). In plasma, ATD resulted in a 40% reduction in the ratio of TRP to the sum of other large neutral amino acids, but had no measurable effect upon TRP or 5-HT levels in hippocampus or prefrontal cortex. Nevertheless, ATD significantly reduced local CBF in 11 of the 43 brain ROIs, while local CMRG remained unchanged. Global analysis of all 43 ROIs revealed a close correlation between CBF and CMRG within both treatment groups. However, the overall ratio (=slope) after ATD (m=1.07) was significantly decreased compared to the control group (m=1.27), indicating a state of relative cerebral oligaemia. Since ATD induced a significant lowering of peripheral TRP, without affecting central TRP or 5-HT concentrations, the decrease in CBF and global change in the flow-metabolism relationship cannot be directly attributed to decreases in brain TRP availability. This could be explained if the raphe were selectively vulnerable to ATD, but the exact mechanism remains unknown. Nevertheless, these data suggest that cerebrovascular disturbances should be considered as a potential contributory factor in studies of serotonergic dysfunction, including depression, with important implications for imaging studies that use CBF alone as a measure of neuronal function.

The role of endothelin in the cerebrovascular response following intracerebral haemorrhage: experimental studies using the endothelin antagonist SB209670.

Primary intracerebral haemorrhage (ICH) is associated with considerable morbidity and mortality. Local endothelin release following ICH may contribute to the pathophysiology of perilesional ischaemia. In diabetics, endothelin release can be enhanced by hyperglycaemia and cerebrovascular dilation may be inhibited by vascular endothelial dysfunction. To examine the effects of endothelin-mediated vasoconstriction after spontaneous ICH in the normal and diabetic brain, regional cerebral blood flow (rCBF) was examined in insulin dependent BB-rats and non-diabetic BB control rats. These experiments were performed 24 h following experimental ICH in both groups of animals that were either given the endothelin antagonist SB209670 or saline. Perilesional oligaemia was similar in control and SB209670 treated diabetic rats, but SB209670 reduced perilesional oligaemia in normal rats. In brain contralateral to the experimental ICH, rCBF was increased by SB209670 in diabetic rats, but not in non-diabetic rats. These studies show that there are differences in the cerebrovascular effects of endothelin in perilesional and contralateral brain in non-diabetic and diabetic rats following ICH.