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.

Cholinergic and vasoactive intestinal polypeptidergic innervation of the cerebral arteries.

Acetylcholine and vasoactive intestinal polypeptide are not only two vasoactive agonists that predominantly induce a vasodilatation of the cerebral arteries, but also correspond to neurotransmitters that innervate the various anatomical segments of the cerebral vasculature. The distinct patterns of the cerebrovascular cholinergic and vasoactive intestinal polypeptidergic innervation, their neurochemistry, in vitro and in vivo pharmacology, as well as the putative pathophysiological implications of these neurotransmission systems are critically summarized on the basis of the most recently published literature.

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.

Acetylcholine levels and choline acetyltransferase activity in rat cerebrovascular bed after uni- or bilateral sphenopalatine ganglionectomy.

Endogenous acetylcholine (ACh) levels and choline acetyltransferase (ChAT) activity were measured in several vascular segments (major cerebral arteries, cortical pial vessels, and peripheral arteries) and nervous tissues [including the sphenopalatine ganglion (SPG)] in the rat. The effects of uni- or bilateral surgical ablation of the SPG, a putative origin of the cholinergic cerebrovascular innervation, were investigated on these two specific cholinergic markers at various postoperative times. ChAT activity and ACh levels were enriched in the cerebral as compared to the peripheral arteries. Among the cerebrovascular tissues tested, ACh levels were particularly high in the circle of Willis and the vertebrobasilar segments and, to a lesser extent, in the middle cerebral artery. Lower levels were found in the small pial vessels and choroid plexus. Overall, ChAT activity measured in different arterial beds paralleled the distribution of ACh. Following uni- or bilateral removal of the SPG, slight reductions (18-36%, statistically not significant) were observed in ChAT activity in rostral cerebral arteries and pial vessels overlying the frontal cortex. Similarly, bilateral ganglionectomy resulted in minor decreases (11-22%, not significant) in the cerebrovascular contents of ACh in these same vascular segments. These results clearly show that the SPG does not or only partly contributes to the cholinergic fibers that supply the cerebrovascular bed.

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.

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.

Galanin inhibits the vasodilatatory basalocortical cholinergic system in the anaesthetized rat.

In order to test the putative interaction between galanin and the vasodilatatory basalocortical cholinergic system, anaesthetized ventilated rats received a microinjection into the substantia innominata of 0.9% NaCl, 50 nmol carbachol, 50 nmol carbachol and 200 ng galanin, or 200 ng galanin. Cerebral blood flow (CBF) was measured with [14C]iodoantipyrine by the tissue sampling technique immediately following the intracerebral infusions. Under coinjection conditions, the flow increases observed after carbachol microinjection in the ipsilateral temporal and frontoparietal cortices were found to be significantly reduced (-37%, p < or = 0.02 and -25%, p < or = 0.05 respectively) compared with carbachol stimulated rats. The infusion of galanin by itself had no effect on CBF. These results demonstrate that galanin inhibits the vasodilatatory basalocortical cholinergic system and thus may possibly influence CBF by indirect mechanisms.

Unilateral eyeball enucleation differentially alters AMPA-, NMDA- and kainate glutamate receptor binding in the newborn rat brain.

The aim of the present work was to evaluate the neurochemical effects of early unilateral visual deprivation as a model of impaired visual maturation. For this purpose, binding to the different ionotropic glutamate receptor subtypes was quantified in vision-related and vision-unrelated brain structures of control and unilaterally deprived newborn rats. At post-natal (PN) day 10, male Sprague-Dawley rats underwent either unilateral eyeball enucleation (enucleation group, n = 12) or sham operation (control group, n = 12). In each group, brains were obtained either at post-natal day 20 (n = 6) or post-natal day 30 (n = 6) and processed for quantitative in vitro autoradiography selective for NMDA, kainate, and AMPA glutamate-binding sites, as well as for the presynaptic adenosine A1 receptor as a control of the deafferentation efficacy. In control animals, quantitative autoradiography revealed an increase in NMDA (e.g. +45% in superior colliculus) and kainate receptor binding (e.g. +55% in visual cortex, layer IV) from post-natal day 20 to post-natal day 30, associated with stable levels of AMPA receptor binding, in the vision-related structures. In the deafferented visual structures, monocular enucleation induced a marked decrease in A1 site density (e.g. -38 to -52%, in the superficial layer of superior colliculi, at PN day 20 and PN day 30, respectively) in parallel with a mild increase in both NMDA (e.g. +8 to 9%, in superior colliculi and visual cortex, layer IV at PN day 30, respectively) and AMPA (e.g. +16%, in layer IV of the visual cortex at PN day 30). Superimposed on marked bilateral decreases at PN day 30 in the enucleated rats, kainate receptor binding also revealed a slight but significant decrease (-5%) in the deafferented superior colliculus as compared to the non-deafferented side. The present findings (different time-courses of, and differential effects of deafferentation on, the NMDA, kainate and AMPA glutamate receptor subtypes throughout the visual brain structures) further support the involvement of these receptors in distinctive roles during maturation of the visual system.

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.

Autoradiographic mapping of cerebral blood flow responses to cholinergic stimulation of the rat substantia innominata: modulatory effect of galanin.

In order to analyze the precise cerebrovascular effects of a specific cholinergic stimulation of the rat substantia innominata and their modulation by galanin, cerebral blood flow was measured by the [14C]-iodoantipyrine autoradiographic method in anesthetized (urethane and alpha-chloralose), artificially ventilated male Sprague-Dawley rats that received a microinjection into the substantia innominata of saline (n = 7), or 63 pmol of galanin (n = 8), or 50 nmoles of carbachol (n = 6) or a coinjection of carbachol and galanin (n = 8). Significant carbachol-induced cerebral blood flow increases were noted in ipsilateral cortices (+36%, p < 0.01 in the cingulate to +82%, p < 0.01 in the parietal somatosensory cortices), but also in ipsilateral hippocampus and ipsilateral thalamus. These cerebral blood flow increases were abolished by the coinjection of carbachol and galanin, while infusions of galanin alone failed to affect cerebral blood flow. Cholinergic stimulation of the substantia innominata represents thus a good model for the analysis of the detailed pharmacological properties of the cholinergic vasodilatatory basalocortical system. The existence of an inhibitory galaninergic modulation of this system could be of particular interest, in terms of cerebrovascular reactivity, in various neurodegenerative states.

Hypercapnia and stimulation of the substantia innominata increase rat frontal cortical blood flow by different cholinergic mechanisms.

This study was designed to further investigate in the rat how the cerebrovascular response to excitation of the projections from the substantia innominata (SI) to the frontal cerebral cortex was mediated. Local cortical blood flow (CoBF) (by helium clearance) and tissue gas partial pressures (pO2, pCO2) (indices of energy metabolic activity) were measured in the frontal cortex in unanesthetized adult Fischer rats in response to electrical stimulation of the SI and, for comparison, in hypercapnic conditions. SI stimulation and hypercapnia increased CoBF to a similar extent (+92% and +106%, respectively). Differences between the changes in tissue gas partial pressures under hypercapnia and SI stimulation suggest that different patterns of flow-metabolism coupling prevail in the mechanisms underlying the two cerebrovascular responses. Cortical pCO2 increased under hypercapnia, but decreased during SI stimulation, indicating that a 'vascular' mechanism (i.e. independent of energy metabolism activation) is at least partly responsible for the flow increase in the latter condition. However, cortical pO2 rose more under hypercapnia than during SI stimulation, suggesting that oxygen consumption, and hence energy metabolism, was increased in the latter case. The ability of the acetylcholine esterase inhibitor physostigmine and the muscarinic receptor antagonist scopolamine to modulate the responses was quantified. In both experimental conditions, CoBF changes were potentiated by 0.15 mg/kg/h physostigmine (by factors of about 2). In contrast, 1 mg/kg scopolamine reduced by 65% the frontal CoBF response elicited by SI stimulation but was without effect on the response to hypercapnia. Thus, although a cholinergic mechanism may be implicated in both responses, activation of muscarinic receptors appears to occur when the stimulation originates from the SI but not from the hypercapnia.

Central acetylcholine synthesis and catabolism activities in the cuttlefish during aging.

Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities, measured in 10 central regions of young, middle-aged and old cuttlefish, showed a regional heterogeneity but with different age-related distribution patterns. Maximal acetylcholine synthesis and catabolism were observed in the inferior frontal and the optic lobes. Important age-related decreases in ChAT activities were evidenced in most regions, while only moderate variations were found for AChE. Since the superior frontal lobe is involved in visual learning, the dramatic decrease in ChAT activity observed in this lobe (-77%) could be implicated in the learning deficits reported in senescent Sepia.

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.

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.

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.

The quantification of brain lesions with an omega 3 site ligand: a critical analysis of animal models of cerebral ischaemia and neurodegeneration.

Previous investigations have indicated that the detection and quantification of omega 3 (peripheral type benzodiazepine) binding site densities that are associated with reactive astroglia and macrophages could be of widespread applicability in the localization and indirect assessment of neural tissue damage in the central nervous system. In the present study, we analyze the usefulness of this approach in a number of experimental models that are characterized by (or putatively involve) neuronal degeneration. One week after the systemic administration of the excitotoxin, kainate, a marked increase in omega 3 site densities (as assessed by [3H]PK 11195 binding) was noted, an increase that was most prominent in known regions of selective vulnerability (hippocampus and septum). However, the kainate-induced omega 3 site proliferation was not a function of the dose administered, a marked interstudy variation was observed, and the binding increase was prevented by the administration of the anticonvulsant, clonazepam. The densities of omega 3 sites were studied, by autoradiography (using [3H]PK 11195 or [3H]PK 14105 as ligands), in 4 groups of Fischer 344 rats aged 3, 12, 22 and 30 months. No age-related changes were noted except in the 30-month-old group in which discrete and focal increases (reflecting tumoral processes) were observed in various brain regions. In spontaneously hypertensive, stroke-prone rats, omega 3 binding increases were observed concomitant with the development of stroke-related neurological signs. With autoradiography, the omega 3 site increase was localized to focal increases in the boundary zones between major cerebral arteries (and corresponding to regions of ischaemic or haemorrhagic infarction). Focal cerebral ischaemia was studied in rats and mice. Subsequent to middle cerebral artery occlusion in normotensive (Wistar/Kyoto) and spontaneously hypertensive rats, the density of omega 3 sites in the ipsilateral hemisphere was markedly elevated, the increase being greater in the spontaneously hypertensive rats. The increases in omega 3 labelling in these two strains matched the absolute volumes of infarctions, determined previously. Middle cerebral artery occlusion in the mouse also increased hemispheric levels of omega 3 sites; the maximum values were obtained between 4 and 8 days following the induction of focal ischaemia. These results demonstrate the feasibility of using omega 3 sites as a marker of excitotoxic, ischaemic and proliferative damage in the rodent brain. Binding measurement in tissue homogenates is an economic and time-efficient approach, whereas the autoradiographic detection of omega 3 sites allows the localization of brain lesions with a macroscopic or microscopic level of anatomical resolution.(ABSTRACT TRUNCATED AT 400 WORDS)

Muscarinic receptor subtype mediating vasodilation feline middle cerebral artery exhibits M3 pharmacology.

The nature of the muscarinic receptor subtype mediating the endothelium-dependent relaxation of the cat middle cerebral artery was investigated in vitro by recording the smooth muscle isometric tension of precontracted arterial segments. Relaxation induced by several agonists (acetylcholine (ACh), acetyl-beta-methylcholine, oxotremorine, carbachol and McN-A-343) was recorded. The ability of selective (pirenzepine, dicyclomine, adiphenine, AF-DX 116, methoctramine, gallamine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and hexahydro-sila-difenidol (HHSiD] and non-selective antagonists (atropine, scopolamine and quinuclidinyl benzilate (QNB] to block the relaxation induced by ACh was also estimated. The weak activity of the poorly selective M1 muscarinic receptor as together with the intermediate affinity of pirenzepine and adiphenine tend to exclude the M1 muscarinic receptor as the primary mediator of the cholinergic relaxation. The low affinity of AF-DX 116 and methoctramine further suggested that the cerebrovascular muscarinic receptor does not correspond to the M2 cardiac subtype. In contrast, 4-DAMP and HHSiD potently inhibited the ACh-induced relaxation with affinities similar to those reported at the M3 glandular receptor. We conclude that a similar to the pharmacological M3 muscarinic receptor subtype is responsible for the cholinergic relaxation of the cat middle cerebral artery.

Vasocontractile muscarinic M1 receptors in cat cerebral arteries: pharmacological identification and detection of mRNA.

The nature of the muscarinic receptor subtype mediating the acetylcholine (ACh)-induced constriction of the cat middle cerebral artery was investigated in vitro by recording the smooth muscle isometric tension of precontracted endothelium-denuded arterial segments. The ability of selective (pirenzepine, UH-AH 371, AF-DX 116, methoctramine, AQ-RA 741, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and hexahydro-sila-difenidol (HHSiD)) and non-selective (atropine) antagonists to inhibit the constriction elicited by ACh was estimated. In addition, using a subtype-specific ribonucleotide probe directed against mRNA encoding the human m1 (Hm1) muscarinic receptor, identification of the corresponding vascular receptor was undertaken in total RNA extracts from cat cerebral blood vessels. The potent inhibition of the ACh-induced constriction by M1 antagonists (pirenzepine and UH-AH 371; pA2 values respectively of 8.08 and 8.64), together with lower affinities of M2 (AF-DX 116; pA2 = 6.50, methoctramine; pA2 = 6.27 and AQ-RA 741; pA2 = 7.60) and M3 compounds (4-DAMP and HHSiD; with pA2 values of 8.85 and 7.76, respectively) strongly suggested the involvement of a pharmacological M1 receptor in this vasomotor response. Furthermore, Northern blot hybridization with the selective Hm1 ribonucleotide probe showed the presence of mRNA transcripts for this muscarinic receptor subtype in the cat cerebrovascular bed. The results indicate that muscarinic constriction in the feline cerebrovascular bed is mediated by a pharmacological M1 receptor subtype and that the corresponding m1 receptor mRNA is present in cat cerebral blood vessels. These findings clearly point to a role of M1 muscarinic receptors in cerebrovascular function.

Regional cerebral blood flow responses to neurochemical stimulation of the substantia innominata in the anaesthetized rat.

Since electrical stimulation of neurones may activate not only cell bodies but also neuronal fibres, this study aimed to test a selectively cholinergic neurochemical stimulation of the rat substantia innominata (SI) by the local microinjection of carbachol; the effects of this acetylcholine agonist were compared with glutamate. Cortical and subcortical cerebral blood flow (CBF) were measured in anaesthetized rats with the [14C]iodoantipyrine method by the tissue sampling technique immediately following the intracerebral (SI) microinjection of saline, 50 nmol of carbachol or glutamate. Carbachol microinjection into the SI induced a transient but significant vasodilation in frontoparietal motor (+28%) and temporal (+41%) cortices, that lasted for less than 10 min. Glutamate did not elicit any significant CBF modifications when compared to control rats although a significant interhemispheric asymmetry after microinjection was observed in the frontoparietal motor cortex. This latter observation would suggest that the glutamate-induced cortical response is less pronounced than that elicited by carbachol. Overall, these results demonstrate that a selective cholinergic stimulation of the SI can induce a transient cortical vasodilation and further confirms the hypothesis of a muscarinic modulation of CBF via this basal structure.