Linda Ferrington

Phosphodiesterase inhibitors enhance object memory independent of cerebral blood flow and glucose utilization in rats

Phosphodiesterase (PDE) inhibitors prevent the breakdown of the second messengers, cyclic AMP (cAMP) and cyclic GMP (cGMP), and are currently studied as possible targets for cognitive enhancement. Earlier studies indicated beneficial effects of PDE inhibitors in object recognition. In this study we tested the effects of three PDE inhibitors on spatial memory as assessed in a place and object recognition task. Furthermore, as both cAMP and cGMP are known vasodilators, the effects of PDE inhibition on cognitive functions could be explained by enhancement of cerebrovascular function. We examined this possibility by measuring the effects of PDE5 and PDE4 inhibitor treatment on local cerebral blood flow and glucose utilization in rats using [14C]-iodoantipyrine and [14C]-2-deoxyglucose quantitative autoradiography, respectively. In the spatial location task, PDE5 inhibition (cGMP) with vardenafil enhanced only early phase consolidation, PDE4 inhibition (cAMP) with rolipram enhanced only late phase consolidation, and PDE2 inhibition (cAMP and cGMP) with Bay 60–7550 enhanced both consolidation processes. Furthermore, PDE5 inhibition had no cerebrovascular effects in hippocampal or rhinal areas. PDE4 inhibition increased rhinal, but not hippocampal blood flow, whereas it decreased glucose utilization in both areas. In general, PDE5 inhibition decreased the ratio between blood flow and glucose utilization, indicative of general oligaemia; whereas PDE4 inhibition increased this ratio, indicative of general hyperemia. Both oligaemic and hyperemic conditions are detrimental for brain function and do not explain memory enhancement. These results underscore the specific effects of cAMP and cGMP on memory consolidation (object and spatial memory) and provide evidence that the underlying mechanisms of PDE inhibition on cognition are independent of cerebrovascular effects.

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.

Partial lesion of the serotonergic system by a single dose of MDMA results in behavioural disinhibition and enhances acute MDMA-induced social behaviour on the social interaction test

The acute effects of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) on anxiety-related behaviours were studied using indices of social interaction in Dark Agouti (DA) both drug naive rats and those pretreated with MDMA (15 mg/kg i.p.) 3 weeks earlier. The functional neuroanatomy of these MDMA effects was visualised using 2-deoxyglucose imaging of local cerebral glucose use (LCMRglu), whilst MDMA-induced serotonergic neurotoxicity was measured by radioligand binding with [3H]paroxetine. Acute MDMA alone markedly decreased most typical elements of social interaction but increased adjacent lying, a behaviour that also contains social elements. In animals pre-exposed to MDMA, decreased [3H]paroxetine binding indicated serotonergic terminal depletion, and in these animals significant increases in locomotor activity, exploratory behaviour and aggressive behaviour were found. Both behavioural effects and also the metabolic activation induced by acute MDMA were potentiated in rats previously exposed to the drug. In conclusion, a single dose of MDMA caused marked changes in social behaviour acutely that might be interpreted either as a decrease or increase in anxiety. Three weeks after MDMA a behavioural disinhibition similar to psychomotor agitation, a symptom connected to depression or mania, and a sensitization to the acute effects of MDMA are apparent in both the behavioural and brain metabolic effects of the drug.

Novel analysis for improved validity in semi-quantitative 2-deoxyglucose autoradiographic imaging

The original [(14)C]-2-deoxyglucose autoradiographic imaging technique allows for the quantitative determination of local cerebral glucose utilisation (LCMRglu) [Sokoloff L, Reivich, M, Kennedy C, Desrosiers M, Patlak C, Pettigrew K, et al. The 2-deoxyglucose-C-14 method for measurement of local cerebral glucose utilisation-theory, procedure and normal values in conscious and anestherized albino rats. J Neurochem 1977;28:897-916]. The range of applications to which the quantitative method can be readily applied is limited, however, by the requirement for the intermittent measurement of arterial radiotracer and glucose concentrations throughout the experiment, via intravascular cannulation. Some studies have applied a modified, semi-quantitative approach to estimate LCMRglu while circumventing the requirement for intravascular cannulation [Kelly S, Bieneman A, Uney J, McCulloch J. Cerebral glucose utilization in transgenic mice over-expressing heat shock protein 70 is altered by dizocilpine. Eur J Neurosci 2002;15(6):945-52; Jordan GR, McCulloch J, Shahid M, Hill DR, Henry B, Horsburgh K. Regionally selective and dose-dependent effects of the ampakines Org 26576 and Org 24448 on local cerebral glucose utilisation in the mouse as assessed by C-14-2-deoxyglucose autoradiography. Neuropharmacology 2005;49(2):254-64]. In this method only a terminal blood sample is collected for the determination of plasma [(14)C] and [glucose] and the rate of LCMRglu in each brain region of interest (RoI) is estimated by comparing the [(14)C] concentration in each region relative to a selected control region, which is proposed to demonstrate metabolic stability between the experimental groups. Here we show that the semi-quantitative method has reduced validity in the measurement of LCMRglu as compared to the quantitative method and that the validity of this technique is further compromised by the inability of the methods applied within the analysis to appropriately determine metabolic stability in the selected standard region. To address these issues we have developed a novel form of analysis that provides an index of LCMRglu (iLCMRglu) for application when using the semi-quantitative approach. Provided that the methodological constraints inherent in 2-deoxyglucose autoradiography (e.g. normoglycaemia) are met this analytical technique both increases the validity of LCMRglu estimation by the semi-quantitative method and also allows for its broader experimental application.

Acute and long-term effects of a single dose of MDMA on aggression in Dark Agouti rats.

MDMA causes selective depletion of serotonergic terminals in experimental animals and the consequent decrease in synaptic 5-HT may, inter alia, increase impulsivity. To study the effects of MDMA upon brain function, the behaviour of male Dark Agouti rats exposed to MDMA (15 mg/kg i.p.), two 5-HT1B agonists (CGS-12066A and CP-94,253, both 5 mg/kg i.p.) or saline were investigated in the resident-intruder test. Studies were performed in drug-naive rats and also in rats exposed to MDMA (15 mg/kg i.p.) 21 d earlier. In parallel experiments the functional neuroanatomy of MDMA effects were assessed using 2-deoxyglucose imaging of local cerebral metabolic rate of glucose utilization (LCMRGlu) and neurotoxicity was assessed by measuring [3H]paroxetine binding. There was no significant difference in aggressive behaviour (biting, boxing, wrestling and their latencies) between drug-naive rats and rats previously exposed to MDMA 21 d earlier, despite reduced social behaviour, decreased LCMRGlu in several brain areas involved in aggression, and reductions in paroxetine binding by 30-60% in the forebrain. CGS-12066A, CP-94,253 and acute MDMA produced marked decreases in aggressive behaviours, especially in biting, boxing and kicking found in drug-naive rats. In animals previously exposed to the drug, acute anti-aggressive effects of MDMA were, in general, preserved as were MDMA-induced increases in LCMRGlu. Our studies provide evidence that in the resident-intruder test, where social isolation is a requirement, aggressive behaviour and acute anti-aggressive effects of MDMA and 5-HT1B receptor agonists remain intact 3 wk after a single dose of the drug despite significant damage to the serotonergic system.

Acute tryptophan depletion in C57BL/6 mice does not induce central serotonin reduction or affective behavioural changes

Acute tryptophan depletion is extensively used to investigate the implication of serotonin in the onset of depressive disorders. In rats, it lowers peripheral tryptophan and decreases central serotonin concentrations. We aimed to establish the rat model of acute tryptophan depletion in the mouse for potential application as serotonin challenge tool in genetic mouse models of depression. Pharmacokinetic and behavioural effects of a tryptophan-free diet were examined in Swiss and C57BL/6 mice. Peripheral amino acids were measured and central tryptophan and serotonin concentrations were compared with anxiety and depression-like behaviour in the elevated zero-maze, forced swimming test or tail suspension test. While acute tryptophan depletion resulted in a 74% reduction of the plasma ratio tryptophan to the sum of other large neutral amino acids in Swiss mice 1h after administration (2x10 ml/kg, 30 min interval), there was only a 40% reduction in C57BL/6 mice. The latter did not show anxiety in the elevated zero-maze or increased immobility in the forced swimming test or tail suspension test. A higher dose (2x20 ml/kg) with a longer interval (60 min) reduced the ratio with 68% in C57BL/6 mice, lowered hippocampal serotonin turnover and had no functional effect when tested in the elevated zero-maze and forced swimming test. These findings have important implications for the use of acute tryptophan depletion in general and in particular for its application in mice. Although in healthy mice no clear central serotonin or functional effects were observed, further research is indicated using mice with pre-existing serotonin dysfunction, as they might be more vulnerable to acute tryptophan depletion.

Persistent cerebrovascular effects of MDMA and acute responses to the drug

Acutely, 3,4,‐methylenedioxymethamphetamine (MDMA) induces cerebrovascular dysfunction [ Quate et al., (2004)Psychopharmacol., 173, 287–295]. In the longer term the same single dose results in depletion of 5‐hydroxytrptamine (5‐HT) nerve terminals. In this study we examined the cerebrovascular consequences of this persistent neurodegeneration, and the acute effects of subsequent MDMA exposure, upon the relationship that normally exists between local cerebral blood flow (LCBF) and local cerebral glucose utilization (LCMRglu). Dark agouti (DA) rats were pre‐treated with 15 mg/kg i.p. MDMA or saline. Three weeks later, rats from each pre‐treatment group were treated with an acute dose of MDMA (15 mg/kg i.p.) or saline. Quantitative autoradiographic imaging was used to measure LCBF or LCMRglu with [14C]‐iodoantipyrine and [14C]‐2‐deoxyglucose, respectively. Serotonergic terminal depletion was assessed using radioligand binding with [3H]‐paroxetine and immunohistochemistry. Three weeks after MDMA pre‐treatment there were significant reductions in densities of 5‐HT transporter (SERT)‐positive fibres (−46%) and [3H]‐paroxetine binding (−47%). In animals pre‐treated with MDMA there were widespread significant decreases in LCMRglu, but no change in LCBF indicating a persistent loss of cerebrovascular constrictor tone. In both pre‐treatment groups, acute MDMA produced significant increases in LCMRglu, while LCBF was significantly decreased. In 50% of MDMA‐pre‐treated rats, random areas of focal hyperaemia indicated a loss of autoregulatory capacity in response to MDMA‐induced hypertension. These results suggest that cerebrovascular regulatory dysfunction resulting from acute exposure to MDMA is not diminished by previous exposure, despite a significant depletion in 5‐HT terminals. However, there may be a sub‐population, or individual circumstances, in which this dysfunction develops into a condition that might predispose to stroke.

Acute SSRI-induced anxiogenic and brain metabolic effects are attenuated 6 months after initial MDMA-induced depletion

To assess the functional state of the serotonergic system, the acute behavioural and brain metabolic effect of SSRI antidepressants were studied during the recovery period after MDMA-induced neuronal damage. The effects of the SSRI fluoxetine and the serotonin receptor agonist meta-chloro-phenylpiperazine (m-CPP) were investigated in the social interaction test in Dark Agouti rats, 6 months after treatment with a single dose of MDMA (15 or 30 mg kg(-1), i.p.). At earlier time points these doses of MDMA have been shown to cause 30-60% loss in axonal densities in several brain regions. Densities of the serotonergic axons were assessed using serotonin-transporter and tryptophan-hydroxylase immunohistochemistry. In a parallel group of animals, brain function was examined following an acute challenge with either fluoxetine or citalopram, using 2-deoxyglucose autoradiographic imaging. Six months after MDMA treatment the densities of serotonergic axons were decreased in only a few brain areas including hippocampus and thalamus. Basal anxiety was unaltered in MDMA-treated animals. However, the acute anxiogenic effects of fluoxetine, but not m-CPP, were attenuated in animals pretreated with MDMA. The metabolic response to both citalopram and fluoxetine was normal in most of the brain areas examined with the exception of ventromedial thalamus and hippocampal sub-fields where the response was attenuated. These data provide evidence that 6 months after MDMA-induced damage serotonergic axons show recovery in most brain areas, but serotonergic functions to challenges with SSRIs including anxiety and aggression remain altered.

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.

Sex influences the effect of a lifelong increase in serotonin transporter function on cerebral metabolism

Polymorphic variation in the human serotonin transporter (SERT; 5‐HTT) gene resulting in a lifelong increase in SERT expression is associated with reduced anxiety and a reduced risk of affective disorder. Evidence also suggests that sex influences the effect of this polymorphism on affective functioning. Here we use novel transgenic mice overexpressing human SERT (hSERT OVR) to investigate the possible influence of sex on the alterations in SERT protein expression and cerebral function that occur in response to increased SERT gene transcription. SERT binding levels were significantly increased in the brain of hSERT OVR mice in a region‐dependent manner. The increased SERT binding in hSERT OVR mice was more pronounced in female than in male mice. Cerebral metabolism, as reflected by a quantitative index of local cerebral glucose utilization (iLCMRglu), was significantly decreased in many brain regions in hSERT OVR female as compared with wild‐type female mice, whereas there was no evidence for a significant effect in any region in males. The ability of hSERT overexpression to modify cerebral metabolism was significantly greater in females than in males. This effect was particularly pronounced in the medial striatum, globus pallidus, somatosensory cortex, mamillary body, and ventrolateral thalamus. Overall, these findings demonstrate that the influence of a lifelong increase in SERT gene transcription on cerebral function is greater in females than in males and may relate, in part, to the influence of sex on genetically driven increases in SERT protein expression.