Core body temperature varies according to the time of exercise without affecting orexin-A production in the dorsal hypothalamus in male rats.

Physical exercise differentially increases body temperature according to the time of day, which shows the importance of circadian rhythm in thermal regulation. Given its contribution in central pathways involved in thermoregulation, orexin A could play a role in the regulation of core body temperature during and after exercise. To test this hypothesis, we assessed the effect of exercise, performed at two times of day, on core temperature and on the amount of orexin A in the production zone, i.e., the dorsal hypothalamus. Forty-nine male Wistar rats underwent forced treadmill exercise during the HG phase and HL phase of core temperature. Basal core temperature was recorded continuously for 48 h by implanted telemetric sensors in 11 rats. Regulation of core temperature during exercise (20 min) and after each exercise (60 min) was modeled with a modified logistic-type function. During HG exercise, core temperature curve reached a significantly higher maximum (asymptote: +0.70 ± 0.10 °C) and took longer to attain the strongest inclination of the core temperature regulation curve (Xmid: 3.46 ± 0.72 min). After HG exercise, time of recovery was significantly longer than after HL exercise. In male rats, thermoregulatory response to acute physical exercise was influenced by the time of day. There was no effect of either physical activity or time of day on the level of orexin A in the dorsal hypothalamus. Our results suggest that orexin A in the dorsal hypothalamus is not involved in the effects of physical exercise on thermoregulation.

Influence of cell confluence on the cAMP signalling pathway in vascular smooth muscle cells.

The influence of cell confluence on the ß-adrenoceptor (ß-AR)/cAMP/phosphodiesterase (PDE) pathway was investigated in cultured rat aortic smooth muscle cells (RASMCs). Cells were plated either at low density (LD: 3·103cells/cm2) or high density (HD: 3·104cells/cm2) corresponding to non-confluent or confluent cells, respectively, on the day of experiment. ß-AR-stimulated cAMP was monitored in real-time using the fluorescence resonance energy transfer (FRET)-based cAMP sensor, Epac2-camps. A brief application (15s) of the ß-AR agonist isoprenaline (Iso) induced a typical transient FRET signal, reflecting cAMP production followed by its rapid degradation. The amplitude of this response, which increased with the concentration of Iso (10 or 100nM), was higher in HD than in LD cells, whatever the Iso concentration used. However, activation of adenylyl cyclase by L-858051 (100µM) induced a similar saturating response in both LD and HD cells. A ß1-AR antagonist (CGP 20712A, 100nM) reduced the Iso (100nM) response in HD but not LD cells, whereas a ß2-AR antagonist (ICI 118,551, 5nM) reduced this response in HD cells and almost abolished it in LD cells. Competitive [125I]-ICYP binding experiments with betaxolol, a ß-AR ligand, identified two binding sites in HD cells, corresponding to ß1- and ß2-ARs with a proportion of 11% and 89%, respectively, but only one binding site in LD cells, corresponding to ß2-ARs. Total cAMP-PDE activity (assessed by a radioenzymatic assay) was increased in HD cells compared to LD cells. This increase was associated with a rise in mRNA expression of five cAMP-PDEs subtypes (PDE1A, 3A, 4A, 4B and 7B) in HD cells, and a decrease in basal [cAMP]i (assessed by an EIA assay). PDE4 inhibition with Ro-20-1724 (10µM) strongly prolonged the Iso response in LD and HD cells, whereas PDE3 inhibition with cilostamide (1µM) slightly prolonged Iso response only in LD cells. Interestingly, inhibition of PDE4 unmasked an effect of PDE3 in HD cells. Our results show that in cultured RASMCs, the ß-AR/cAMP/PDE signalling pathway is substantially modulated by the cell density. In HD cells, Iso response involves both ß1- and ß2-AR stimulation and is mainly controlled by PDE4, PDE3 being recruited only after PDE4 inhibition. In LD cells, Iso response involves only ß2-AR stimulation and is controlled by PDE4 and to a lower degree by PDE3. This low density state is associated with an absence of membrane expression of the ß1-AR, a lower cAMP-PDE activity and a higher basal [cAMP]i. This study highlights the critical role of the cellular environment in controlling the vascular ß-AR signalling.

Effects of long-term methylphenidate treatment in adolescent and adult rats on hippocampal shape, functional connectivity and adult neurogenesis.

Methylphenidate (MPH) is a widely prescribed stimulant drug for the treatment of attention deficit hyperactivity disorder (ADHD) in children and adolescents. Its use in this age group raises concerns regarding the potential interference with ongoing neurodevelopmental processes. Particularly the hippocampus is a highly plastic brain region that continues to develop postnatally and is involved in cognition and emotional behavior, functions known to be affected by MPH. In this study, we assessed whether hippocampal structure and function were affected by chronic oral MPH treatment and whether its effects were different in adolescent or adult rats. Using behavioral testing, resting-state functional MRI, post-mortem structural magnetic resonance imaging (MRI), and immunohistochemistry, we assessed MPH's effects on recognition memory, depressive-like behavior, topological features of functional connectivity networks, hippocampal shape and markers for hippocampal neurogenesis and proliferation. Object recognition memory was transiently impaired in adolescent treated rats, while in animals treated during adulthood, increased depressive-like behavior was observed. Neurogenesis was increased in adolescent treated rats, whereas cell proliferation was decreased following adult treatment. Adolescent treated rats showed inward shape deformations adjacent to ventral parahippocampal regions known to be involved in recognition memory, whereas such deformations were not observed in adult treated animals. Irrespective of the age of treatment, MPH affected topological features of ventral hippocampal functional networks. Thus, chronic oral treatment with a therapeutically relevant dose of MPH preferentially affected the ventral part of the hippocampus and induced contrasting effects in adolescent and adult rats. The differences in behavior were paralleled by opposite effects on adult neurogenesis and granule cell proliferation.

Rescue of cognitive aging by long-lasting environmental enrichment exposure initiated before median lifespan.

The rescue of cognitive function through environmental enrichment (EE) during aging has been extensively documented. However, the age at onset, the duration of EE, and the cerebral mechanisms required to obtain the greatest benefits still remain to be determined. We have recently shown that EE applied for 3 mo after the median lifespan, i.e., the age at which 50% of the population is still alive (from 17 to 20 mo in NMRI mice), failed to prevent cognitive deficits in senescent animals. In the present study, mice were exposed to EE prior to the median lifespan, and for a longer total duration (from 14 to 20 mo), before the assessment of memory performance and the electrophysiological properties of hippocampal neuronal networks. The EE prevented memory deficits and reduced anxiety as the animal aged. Moreover, EE attenuated the age-related impairment of basal glutamatergic neurotransmission in CA1 hippocampal slices, and reversed the decrease in isolated N-methyl-D-Aspartate receptor (NMDA-R)-dependent synaptic potentials. Surprisingly, EE did not prevent the age-related alteration of theta-burst-induced long-term potentiation (LTP). This study therefore suggests that EE needs to be initiated before the age corresponding to the median lifespan and/or required long duration (> 3 mo) to have an effect on cognitive aging. In addition, we show that EE probably acts through theta-burst-independent mechanisms of synaptic plasticity.

RS 67333 and D-cycloserine accelerate learning acquisition in the rat.

Various 5-hydroxytryptamine (5-HT) central receptor subtypes have been implicated in cognitive performances. In the present investigation, we studied the effects of the selective 5-HT(4) receptor agonist RS 67333 (1-(4-amino-5-chloro-2-methoxyphenyl)-3-(1-n-butyl-4-piperidinyl)-1-propanone; 1 mg/kg, i.p.) on spatial learning in the rat, and compared them to those of a reference drug, the partial NMDA receptor agonist D-cycloserine (10 mg/kg, i.p.). The effects of these two drugs were evaluated in four protocols which employed the Morris water maze task with various numbers of daily trials and inter-trial intervals (ITI; 4 trials with 30 s ITI; 2 trials with 2 h or 12 h ITI; or one daily trial). In the 2 trial-2 h ITI protocol, rats treated with RS 67333 or D-cycloserine exhibit a reduced mean swim distance during the first days of training when compared to controls. Neither RS 67333 nor D-cycloserine modified the acquisition performances in the 2 trial-12 h ITI or the one daily trial tests or the retention score measured in each protocol. These data suggest that RS 67333 and D-cycloserine can improve the learning rate in a high demand memory task and confirm that selective 5-HT(4) receptor ligands may provide novel approaches for the development of cognitive enhancers.

Solid phase synthesis of sulphonamides: novel ligands of 5-HT6 receptors.

Using solid phase synthesis techniques, we have rapidly obtained a series of eight aryl sulphonamides derived from putrescine. These conjugates with various aryl groups were evaluated for their affinity towards 5-HT6 receptors in man. This evaluation revealed the interest of two compounds which present the same activity level, in the submicromolar range, as two reference derivatives. The most potent will be considered as a new lead for further investigations.

Inhibition of neuronal nitric oxide synthase by 7-methoxyindazole and related substituted indazoles.

The synthesis and pharmacological evaluation of methoxyindazoles as new inhibitors of neuronal nitric oxide synthase are presented. The 7-methoxyindazole, although less potent than 7-NI, is the most active compound of the series in an in vitro enzymatic assay of neuronal nitric oxide synthase activity. This result shows that the nitro-substitution is not indispensable to the biological activity of the indazole ring. 7-Methoxyindazole possesses in vivo NOS inhibitory as well and related antinociceptive properties.

First tricyclic oximino derivatives as 5-HT3 ligands.

The design and synthesis of a new type of 5-HT3 ligand with subnanomolar affinity are described. The O-dialkylaminoethyloximinothienopyrrolizine structure was deduced from molecular modeling studies by replacement of an amidine moiety by an oximino one.

Radiosynthesis of [18F]Lu29-024: a potential PET ligand for brain imaging of the serotonergic 5-HT2 receptor.

In a previous work, Lu29-024 (2,5-dimethyl-3-(4-fluorophenyl)-1-(1-methyl-4-piperidinyl)-1H-indole), a selective 5-HT2A receptor antagonist with nanomolar affinity and high selectivity, was labeled with carbon-11 to evaluate its behavior as a potential PET ligand for the serotonergic 5-HT2A receptor in the central nervous system. Administration of this tracer to rats was followed by a good brain uptake, no brain labeled metabolites but no specific, regio-selective, binding at 20 and 40 min post injection. Despite this, the data noted at 20 and 40 min suggest that this tracer, if associated with a radioactive emitter with a longer half-life than that of carbon-11, could be useful for the quantification of 5HT2A receptors. For these reasons, we chose to label this compound, bearing a fluorine atom, with [18F]fluoride, in order to perform rat studies over a more prolonged time-scale. The precursor for the radiosynthesis of [18F]Lu29-024 was obtained in an overall yield of 20% by a multi-step synthesis including an acetonylation reaction followed by a Fisher indole reaction. The radiotracer was prepared by an aromatic substitution with activated [18F]fluoride followed by a decarbonylation reaction that employed Wilkinson's catalyst. The radiosynthesis of [18F]Lu29-024 required approximatively 110 min with an overall radiochemical yield of 20-35% and specific activities of 37GBq/micromol. Fluorine-labeled Lu29-024 may thus be envisaged as a potentially useful PET tracer that can be applied to a wide range of neurological and psychiatric diseases.

Exofocal alterations in opioidergic receptor densities following focal cerebral ischemia in the mouse.

In previous studies of our group, we have reported differential alterations in opioidergic receptor subtypes densities in infarcted and periinfarcted brain tissue following middle cerebral artery occlusion (MCAO) in mice. Other studies have also described subcortical alterations consecutive to focal cortical ischemia. For a better understanding of ischemic processes in exofocal areas, we have investigated the evolution of opioidergic receptors following focal cortical ischemia through the quantification of relative binding densities, B(max) and K(d) values for the mu, delta, and kappa subtypes. Our results demonstrate that opioid receptor subtypes exhibit adaptations at distance from the ischemic core, mainly in the striatum, the thalamus, and the substantia nigra. Indeed, mu and delta B(max) values were increased in ventral thalamic nuclei, while kappa relative binding densities were transiently increased in nucleus medialis dorsalis and nucleus lateralis, pars posterior. Moreover, the B(max) of mu and delta receptors were transiently decreased at 6 h post-MCAO in ipsi- and contralateral patches and matrices of the striatum. Conversely, the mu B(max) values were increased in ipsi- and contralateral substantia nigra, pars compacta, and pars reticulata, 24 h following MCAO. In contralateral substantia nigra, pars compacta, kappa B(max) was found to be decreased at 24 h post-MCAO. These alterations could reflect neuronal dysfunction in exofocal brain structures, consecutively to the degeneration of defined neuroanatomical pathways. Our study indicates that opioidergic receptors could be used as markers of the neuronal reorganization that take place in subcortical areas following an ischemic insult of the brain cortex.

Syntheses of R and S isomers of AF-DX 384, a selective antagonist of muscarinic M2 receptors.

Enantiomers of 5,11-dihydro-11-[2-[2-[(N,N-dipropylaminomethyl)piperidin-1- yl]ethylamino]-carbonyl]-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one (AF-DX 384) 1, have been synthesized from (S)-(+) and (R)-(-)-2-[N,N-dipropylaminomethyl]piperidine 4. The enantiomeric excess of 1 has been determined by capillary electrophoresis by using the alpha-highly sulphated cyclodextrin (alpha-HSCD) as chiral selector within the running electrolyte. (S)-(+)-(4) was prepared from (S)-(-)-pipecolic acid in a 4-step procedure (overall yield: 30%, ee: 99%) and (R)-(-)-AF-DX 384 from (R)-(+)-pipecolic acid. The (R)-(-) isomer exhibited in vitro a 23-fold higher affinity than its enantiomer (S)-(+) towards muscarinic receptors of subtype 2.

The use of cerebral blood flow as an index of neuronal activity in functional neuroimaging: experimental and pathophysiological considerations.

Over recent years, activation studies that have been undertaken using brain imaging techniques, such as functional magnetic resonance imaging, positron emission tomography or near infrared spectroscopy, have greatly improved our knowledge of the functional anatomy of the brain. Nevertheless, activation studies do not directly quantify the variations of synaptic transmission (neuronal activity) but detect it indirectly either through the visualisation of changes in cerebral blood flow, oxidative or glycolytic metabolism (for positron emission tomography), or through the measurement of a global index that is dependent on both cerebral blood flow and oxidative metabolism (for functional magnetic resonance imaging and near infrared spectroscopy). Such approaches are based on the concept of a tight parallelism--termed coupling--between variations in neuronal activity, metabolism and cerebral blood flow. However, several "uncoupled" situations between these parameters have been reported over the last decade through experimental, pharmacological and pathophysiological studies. The aim of this review is to focus on these data that have to be taken into account for the interpretation of the results obtained in activation paradigms.

Neurochemical stimulation of the rat substantia innominata increases cerebral blood flow (but not glucose use) through the parallel activation of cholinergic and non-cholinergic pathways.

Neurochemical activation of the substantia innominata (SI) in the rat, through the direct injection of the cholinergic agonist carbachol, has been reported to induce large increases in cerebral blood flow (CBF) throughout cortical and subcortical projection regions. The present study aimed to determine whether the vasomotor responses to cholinergic stimulation of the SI were, or were not, the consequence of an increase in metabolic activity. To this end, coupled measurements of CBF and cerebral glucose use (CGU) were undertaken during carbachol-elicited stimulation of the SI. Infusion of carbachol into the basal forebrain induced significant CBF increases in several ipsilateral cortical and subcortical areas including the amygdala. In contrast, CGU increased only in the ipsilateral amygdala and SI. Thus, we tested the hypothesis of a direct neurogenic, rather than metabolic, contribution of the basalocortical system. In this respect, carbachol-elicited stimulation resulted in significant increases in extracellular acetylcholine concentrations in the ipsilateral parietal cortex; systemic pretreatment with the muscarinic receptor antagonist scopolamine completely abolished the increase in cortical CBF elicited by cholinergic stimulation of the SI in the ipsilateral frontoparietal motor cortex while it failed to affect the increase observed in the ipsilateral temporal cortex. Several conclusions can be drawn from the present study. The stimulation of the SI by carbachol induces an increase in CBF that can be dissociated from changes in underlying glucose metabolism. Secondly, these induced changes in cortical CBF are paralleled by an increase in acetylcholine release. Lastly, the failure of scopolamine to block the flow response in all cortical regions would suggest that SI stimulation will evoke the release of vasodilatatory neurotransmitter(s) as well as acetylcholine itself.

Cerebrovascular evidence for a GABAergic modulation of the cholinergic vasodilatatory basalocortical system in the rat.

The present work is aimed to study the functional relevance of GABAergic-cholinergic interactions on the modulation of cerebral blood flow (CBF) exerted by the basalocortical system. Injections of GABA into the substantia innominata (SI) induce increases in blood flow in several cortical areas and inhibit partly the increases in cortical blood flow induced by cholinergic activation of this structure. Blockade of local GABAergic receptors by picrotoxin induced almost similar effects. These findings suggest that local GABAergic neurones of the SI exert a complex cortical cerebrovascular modulation at a resting and an activated state.

Differential time-course decreases in nonselective, mu-, delta-, and kappa-opioid receptors after focal cerebral ischemia in mice.

BACKGROUND AND PURPOSE: Neuroprotection studies have demonstrated the involvement of opioids in ischemia, and we have previously demonstrated alterations in Bmax of opioidergic receptors after 2 post-MCAO time points in mice. METHODS: In the present study, we have investigated in a detailed manner the postischemic time course of variations in [3H]diprenorphine (nonselective), [3H]DAMGO (mu), [3H]DADLE (delta), and [3H]U69593 (kappa) relative binding densities after focal cerebral ischemia (0 to 48 hours) in mice. RESULTS: In frontoparietal cortices, our results demonstrate decreases in (1) delta receptor densities at 1 to 3 hours after MCAO, (2) mu and nonselective binding sites at 6 to 12 hours after MCAO, and (3) kappa receptor densities between 6 and 24 hours after MCAO. In the rostral part of the infarct border zone, a decrease in delta-receptors was found concomitant with the extension of the infarct core; conversely, the decrease in delta-receptors appeared before (6 to 12 hours) macroscopic histological damage, which occurred between 12 hours and 24 hours after MCAO in the caudal part of this area. In this frontier, mu- and especially kappa-binding sites were decreased later (12 to 48 hours after MCAO). CONCLUSIONS: These differential alterations in opioidergic receptors could be due to the selective sublocalization of receptors, postsynaptically on cortical interneurons for mu- and delta-receptors versus presynaptically on cortical afferent pathways for the kappa subtype. Further, our results suggest that delta- and mu-opioidergic receptors could be markers of infarct extension and neuronal death; the study of [3H]diprenorphine and selective binding sites argues in favor of the use of receptor-specific ligands. Finally, the relative preservation of kappa-receptors might be correlated with the neuroprotective role of kappa-agonists, as previously reported.

Calcitonin increases 5-HT1A binding site densities in the brain of adrenalectomized rats.

Calcitonin is a peptide which acts in the brain to modulate behavior and hormone release, possibly through an interaction with serotonergic systems. We investigated the effects of chronic systemic injections of salmon calcitonin on the [3H]-8-OHDPAT binding to 5-HT1A receptors in the frontal cortex and hippocampus in adrenalectomized and intact (non adrenalectomized) rats. The results show that salmon calcitonin increases the maximal density of 5-HT1A binding sites in both structures in adrenalectomized animals (and decreases the affinity in the frontal cortex only). Calcitonin does not alter this binding in intact rats. These results demonstrate the existence of interactions between calcitonin, serotonin and glucocorticoids, and raise the hypothesis of a neurotrophic effect of calcitonin on serotonergic neurons.

Parallel regional quantification of choline acetyltransferase and cholinesterase activity in the central nervous system of an invertebrate (Sepia officinalis).

The present study describes (i) a procedure to dissect the central nervous system of the cuttlefish (Cephalopod) into ten, functionally distinct, anatomical regions of interest and (ii) the parallel measurement of acetylcholine synthesis (choline acetyltransferase) and degradation (cholinesterase) activities. Both aspects (dissection and parallel quantification of acetylcholine synthesis and degradation) could be of great importance for quantitative regional studies in neurochemistry in this animal model, it is interesting to study the cellular and molecular mechanisms involved in learning and aging processes. The parallel quantification of acetylcholine synthesis and degradation applicable to any animal model is pivotal since both enzymes are essential for the cholinergic neurotransmission and may be differentially modulated by specific functions such as learning and aging processes. Furthermore, since choline acetyltransferase and cholinesterase show different localization into the brain, their parallel quantification may underlie the involvement of cholinesterase in non-cholinergic functions, which remain unclear throughout the animal kingdom.

Radiosynthesis of [11C]Lu 29-024: a potential radiotracer for 5HT2 receptors PET studies.

For mapping 5-HT2 receptors in the central nervous system with positron emission tomography (PET), 2,5-dimethyl-3-(4-fluorophenyl)-1-(1-[11C]methyl-4-piperidinyl)-1H-indol e ([11C]Lu29-024) has been prepared. The precursor for the radiosynthesis of [11C]Lu29-024 was obtained in an overall yield of 53% by a convenient five-step synthesis; its reaction with [11C]methyl iodide afforded [11C]Lu29-024 in 35-50% radiochemical yield (decay corrected) in 45 to 50 min with a specific radioactivity ranging from 11 to 15 GBq/micromol. Following i.v. injections into rats, the analysis of plasma samples showed that the metabolism of [11C]Lu29-024 was rapid and extensive (60% of the original tracer was metabolized at 40 min). In contrast, only unmetabolized [11C]Lu29-024 could be detected in brain tissue. These biological results suggest that labeled metabolites have no access to brain tissue and further propose [11C]Lu29-024 as an interesting tool for PET studies of brain 5HT2 receptors.

Synthesis and biological investigations of [18F]MR18445, a 5-HT3 receptor partial agonist.

18F Labelled MR18445 (4-[4-(4-[18F]fluorobenzyl)piperazino]-7-methoxypyrrolo++ +[1,2-alpha] quinoxaline), a selective 5-HT3 receptor partial agonist with nanomolar affinity, was synthesized and examined as a potential radioligand for PET imaging of brain 5HT3 receptors. Radiotracer was prepared by N-alkylation of the MR18491 precursor with 4-[18F]fluorobenzyl iodide. This latter was synthesized in a three-step procedure from 4-[18F]fluorobenzaldehyde obtained by 18F-nucleophilic displacement of 4-nitrobenzaldehyde, subsequently reduced to 4-[18F]fluorobenzyl alcohol and converted into reactive 4-[18F]fluorobenzyl iodide. The reduction step was performed on a column filled with NaBH4/Al2O3 and 4-[18F]fluorobenzyl alcohol was obtained with high reproducible yield (82-93% from 4-[18F]fluorobenzaldehyde) if there were traces of water in the system. The radiosynthesis of [18F]MR18445 required approximately 120 min. Semi-preparative HPLC purification followed by formulation gave injectable solutions of [18F]MR18445 with a radiochemical purity > 99%. The overall yield of the synthesis was mainly dependent upon the first step efficiency of aromatic incorporation of 18F- and varied from 12% to 29%. All the synthetic procedure was realized on a ZYMARK robotic system. Biological in vivo studies in rats showed that uptake of [18F]MR18445 in brain was rapid and high. No evidence of radiolabeled metabolites could be observed in the brain as late as 40 min after injection, despite the rapid appearance of metabolites in the plasma. However, neither phosphorimaging autoradiographic studies in rats nor PET experiments in baboons revealed specific binding of the radiotracer in brain, suggesting [18F]MR18445 is not suitable for 5-HT3 receptors PET studies.

Maximal densities of mu, delta, and kappa receptors are differentially altered by focal cerebral ischaemia in the mouse.

Though opioids are known to have neuroprotective properties, little information is available on the functional state of opioidergic receptors following focal cerebral ischaemia. The present study investigated the evolution of the Bmax and Kd for [3H]DAMGO, [3H]DADLE, and [3H]U69,593, respectively, for the mu, delta, and kappa opioidergic receptors after permanent focal cerebral ischaemia in mice. While the various Kd were unchanged, mu and delta Bmax values were precociously decreased in frontoparietal cortices, earlier than kappa receptors, reflecting infarct extension with time. The Bmax values for mu and delta receptors were also altered in non-infarcted tissues, such as tissues at risk (e.g., temporal auditory cortex) and exofocal (e.g., contralateral and non-infarcted) cortices. These results suggest that, in non-infarcted areas, the observed changes reflect functional modifications to focal ischaemia.