Role of glutamate transporters in corticostriatal synaptic transmission.

High-affinity glutamate transporters (GTs) play a major role in controlling the extracellular level of this excitatory neurotransmitter in the CNS. Here we have characterized, by means of in vitro patch-clamp recordings from medium spiny neurons (MSNs), the role of GTs in regulating corticostriatal glutamatergic synaptic transmission in the adult rat. Charge transfer and decay-time, but not amplitude, of excitatory postsynaptic currents (EPSCs) were enhanced by dl-threo-beta-benzyloxyaspartate (TBOA), a broad inhibitor of GTs. Moreover, TBOA also potentiated currents induced by high-frequency stimulation (HFS) protocols. Interestingly, the effect of TBOA on EPSCs was lost when MSNs were clamped at +40 mV, a condition in which neuronal GTs, that are voltage-dependent, are blocked. However, in this condition TBOA was still able to enhance HFS-induced currents, suggesting that glial GT's role is to regulate synaptic transmission when glutamate release is massive. These data suggest that neuronal GTs, rather than glial, shape EPSCs' kinetics and modulate glutamate transmission at corticostriatal synapse. Moreover, the control of glutamate concentration in the synaptic cleft by GTs may play a role in a number of degenerative disorders characterized by the hyperactivity of corticostriatal pathway, as well as in synaptic plasticity.

siRNA targeted against amyloid precursor protein impairs synaptic activity in vivo.

The amyloid precursor protein (APP) plays a central role in Alzheimer's disease (AD) pathogenesis through its cleavage leading to the accumulation of the peptide betaA4. Diffusible oligomeric assemblies of amyloid beta peptide are thought to induce synaptic dysfunction, an early change in AD. We tested the hypothesis that a reduction in presynaptic APP could itself lead to a decrease in synaptic efficacy in vivo. Twenty-four hours after intraocular injection, siRNA targeted against APP accumulated in retinal cells and the APP in retinal terminals in the superior colliculus was significantly reduced. Surprisingly, the amyloid precursor-like protein 2 (APLP2) was reduced as well. Functional imaging experiments in rats during visual stimulation showed that knockdown of presynaptic APP/APLP2 significantly reduced the stimulation-induced glucose utilization in the superior colliculus. Our results suggest that perturbations in the amount of APP/APLP2 axonally transported to, and/or in their turnover in the nerve terminal alter synaptic function and could be a pathogenic mechanism in AD.

Local injection of antisense oligonucleotides targeted to the glial glutamate transporter GLAST decreases the metabolic response to somatosensory activation.

The mechanisms responsible for the local increase in brain glucose utilization during functional activation remain unknown. Recent in vitro studies have identified a new signaling pathway involving an activation of glial glutamate transporters and enhancement of neuron-astrocyte metabolic interactions that suggest a putative coupling mechanism. The aim of the present study was to determine whether one of the glutamate transporters exclusively expressed in astrocytes, GLAST, is involved in the neurometabolic coupling in vivo. For this purpose, rats were microinjected into the posteromedial barrel subfield (PMBSF) of the somatosensory cortex with GLAST antisense or random phosphorothioate oligonucleotides. The physiologic activation was performed by stimulating the whisker-to-barrel pathway in anesthetized rats while measuring local cerebral glucose utilization by quantitative autoradiography in the PMBSF. Twenty-four hours after injection of two different antisense GLAST oligonucleotide sequences, and despite the presence of normal whisker-related neuronal activity in the PMBSF, the metabolic response to whisker stimulation was decreased by more than 50%. Injection of the corresponding random sequences still allowed a significant increase in glucose utilization in the activated area. The present study highlights the contribution of astrocytes to neurometabolic coupling in vivo. It provides evidence that glial glutamate transporters are key molecular components of this coupling and that neuronal glutamatergic activity is an important determinant of energy utilization. Results indicate that astrocytes should also be considered as possible sources of altered brain metabolism that could explain the distinct imaging signals observed in some pathologic situations.

Sustained attenuation of the cerebrovascular response to a 10 min whisker stimulation following neuronal nitric oxide synthase inhibition.

We examined the effect of type I nitric oxide synthase (neuronal isoform of NOS, nNOS) inhibition on the temporal profile of the cortical blood flow (CoBF) changes induced by a relatively long period (10 min) of whisker stimulation. To address this issue, we used laser-Doppler flowmetry (LDF) to continuously monitor the CoBF in rats anesthetized with alpha-chloralose, in a control condition, and 30 and 60 min following 7-nitroindazole (25 mg/kg, i.p.). Mechanical stimulation of all whiskers for 10 min led to a continuous and sustained CoBF increase with a mean integral response of 4030+/-764%. After 30 and 60 min nNOS inhibition the CoBF response was significantly reduced by 52 and 68%, respectively (P<0. 05) with no evidence of any compensatory mechanism during the whole stimulation period. These data show that regulation of the cerebral blood flow in response to an increased neuronal activity is a dynamic and tonic process in which nNOS plays an essential role.

Structural organization of the perivascular astrocyte endfeet and their relationship with the endothelial glucose transporter: a confocal microscopy study.

Despite the increasing evidence for a prominent role played by the perivascular endfeet of astrocytes in the functional metabolic coupling between astrocytes and neurons, a clear picture of their spatial organization is still lacking. To examine the three-dimensional structure of the astrocyte endfeet and their relationships with the endothelial cells, coronal rat brain sections immunolabeled for the two astroglial markers [glial fibrillary acidic protein (GFAP)/S-100beta] and the endothelial glucose transporter (GLUT1) were analyzed under the confocal microscope. Double immunolabeling of GFAP and S-100beta showed numerous well-defined astrocytes sending one or more endfeet to the vasculature. Examination of GFAP immunolabeling at higher magnification showed that these endfeet consist of well-defined rosette-like structures lying on the vessel wall. Double immunostaining of GFAP and GLUT1 showed that the endothelial cells were the main targets of these repeated geometrical units formed by the astrocyte endfeet. When three-dimensional images were reconstructed, obvious privileged anatomical relationships were observed between endfeet and individual endothelial cells. These anatomical data provide strong support for the involvement of astrocytes in cerebral metabolic coupling. The finger-like appearance of astrocyte endfeet could allow direct metabolic exchanges between intracerebral vessels and non-glial elements such as nerve terminals.

Local uncoupling of the cerebrovascular and metabolic responses to somatosensory stimulation after neuronal nitric oxide synthase inhibition.

It has recently been shown, using either genetically engineered mutant mice (nitric oxide synthase [NOS] knockout) or specific pharmacological tools, that type I NOS (neuronal isoform of NOS, [nNOS]) participates in coupling cerebral blood flow to functional activation. However, it has not been clearly established whether the associated metabolic response was preserved under nNOS inhibition and whether this action was exerted homogeneously within the brain. To address these issues, we analyzed the combined circulatory and metabolic consequences of inhibiting the nNOS both at rest and during functional activation in the rat anesthetized with alpha-chloralose. Cerebral blood flow and cerebral glucose use (CGU) were measured autoradiographically using [14C]iodoantipyrine and [14C]2-deoxyglucose during trigeminal activation induced by unilateral whiskers stimulation in vehicle- and 7-nitroindazole-treated rats. Our data show that inhibition of nNOS globally decreased CBF without altering CGU, indicating that NO-releasing neurons play a significant role in maintaining a resting cerebrovascular tone in the whole brain. During whisker stimulation, nNOS inhibition totally abolished the cerebrovascular response only in the second order relay stations (thalamus and somatosensory cortex) of the trigeminal relay without altering the metabolic response. These findings provide evidence that the involvement of neurally-derived NO in coupling flow to somatosensory activation is region-dependent, and that under nNOS inhibition, CBF and CGU may vary independently during neuronal activation.

Cerebrovascular consequences of altering serotonergic transmission in conscious rat.

Many therapeutic strategies aim at altering serotonin brain levels. However, serotonin (5-HT) is known to influence the cerebral circulation. The purpose of this study was to determine the effects of acutely decreasing intracerebral serotonin release upon cerebral blood flow and cerebrovascular reactivity to hypercapnia in conscious rats. To this end, (1) we analyzed the time-course of cortical blood flow changes measured with laser-Doppler flowmetry following injection of 0.1 mg kg(-1) 8-OHDPAT (5-HT1A agonist), and (2) we evaluated the cerebrovascular reactivity to hypercapnia using a quantitative multiregional diffusible tracer technique 5 and 60 min following 8-OHDPAT administration. 8-OHDPAT induced a rapid and transient increase in cortical blood flow (+34%) that was prevented totally by WAY100135 (5-HT1A antagonist) pre-treatment. Five min following 8-OHDPAT administration, the cerebrovascular responsiveness to hypercapnia was increased significantly in striatum (+27%) and fronto-parietal cortex (+61%). This result is consistent with a vasoconstrictor role of the serotonergic system that becomes manifest during hyperemic conditions.

Autoradiographic evidence for flow-metabolism uncoupling during stimulation of the nucleus basalis of Meynert in the conscious rat.

We earlier reported that electrical stimulation of the rat nucleus basalis of Meynert (NBM) induces large cerebral blood flow increases, particularly in frontal cortical areas but also in some subcortical regions. The present study was designed to address the issue of blood flow control exerted by NBM projections. To this aim, we have determined whether these flow increases were associated with proportionate changes in metabolic activity as evaluated by cerebral glucose utilization (CGU) strictly under the same experimental conditions in the conscious rat. An electrode was chronically implanted in a reactive site of the NBM as determined by laser-Doppler flowmetry (LDF) of the cortical circulation. One to two weeks later, while the cortical blood flow was monitored by LDF, we measured CGU using the [14C]2-deoxyglucose autoradiographic technique during unilateral electrical stimulation of the NBM, and analyzed the local flow-metabolism relationship. The large increases in cortical blood flow induced by NBM stimulation, exceeding 300% in various frontal areas, were associated with at most 24% increases in CGU (as compared with the control group) in one frontal area. By contrast, strong increases in CGU exceeding 150% were observed in subcortical regions ipsilateral to the stimulation, especially in extrapyramidal structures, associated with proportionate CBF changes. Thus, none of the blood flow changes observed in the cortex can be ascribed to an increased metabolic activity, whereas CBF and CGU were coupled in many subcortical areas. This result indicates that different mechanisms, which do not necessarily involve any metabolic factor, contribute to the regulation of the cerebral circulation at the cortical and subcortical level. Because the distribution of the uncoupling is coincident with that of cholinergic NBM projections directly reaching cortical microvessels, these data strongly support the hypothesis that NBM neurons are capable of exerting a neurogenic control of the cortical microcirculation.

Effect of sympathectomy on the phenotype of smooth muscle cells of middle cerebral and ear arteries of hyperlipidaemic rabbits.

This study was carried out to determine whether sympathectomy influences the phenotypic modulation of smooth muscle cells in the peripheral and cerebral arteries of heritable hyperlipidaemic rabbits. Unilateral superior cervical ganglionectomy (common origin of innervation to the middle cerebral artery and the central ear artery) was performed on four Watanabe heritable hyperlipidaemic rabbits. Cross-sections of the ipsi- (sympathectomized) and the contralateral (intact) cerebral and ear arteries were prepared 2 months later and labelled with monoclonal antibodies against vimentin and desmin, two markers of the differentiation of smooth muscle cells, and alpha-smooth muscle actin, a marker of these cells. Sections from control and sympathectomized arteries were analysed with a confocal laser scanning microscope. Compared with contralateral intact ear arteries, the sympathectomized ear artery developed a thickened intima with dedifferentiated smooth muscle cells, expressing alpha-smooth muscle actin but no desmin, whereas the middle cerebral artery remained unchanged. These results suggest that sympathectomy may favour the progression of atherosclerosis in peripheral but not in cerebral arteries of Watanabe heritable hyperlipidaemic rabbits.

Serotonin in the regulation of brain microcirculation.

Manipulation of brainstem serotonin (5-HT) raphe neurons induces significant alterations in local cerebral metabolism and perfusion. The vascular consequences of intracerebrally released 5-HT point to a major vasoconstrictor role, resulting in cerebral blood flow (CBF) decreases in several brain regions such as the neocortex. However, vasodilatations, as well as changes in blood-brain barrier (BBB) permeability, which are blocked by 5-HT receptor antagonists also can be observed. A lack of relationship between the changes in flow and metabolism indicates uncoupling between the two variables and is suggestive of a direct neurogenic control by brain intrinsic 5-HT neurons on the microvascular bed. In line with these functional data are the close associations that exist between 5-HT neurons and the microarterioles, capillaries and perivascular astrocytes of various regions but more intimately and/or more frequently so in those where CBF is altered significantly following manipulation of 5-HT neurons. The ability of the microvascular bed to respond directly to intracerebrally released 5-HT is underscored by the expression of distinct 5-HT receptors in the various cellular compartments of the microvascular bed. Thus, it appears that while some 5-HT-mediated microvascular functions involve directly the blood vessel wall, others would be relayed through the perivascular astrocyte. The strategic localization of perivascular astrocytes and the different 5-HT receptors that they harbor strongly emphasize their putative pivotal role in transmitting information between 5-HT neurons and microvessels. It is concluded that the cerebral circulation has full capacity to adequately and locally adapt brain perfusion to changes in central 5-HT neurotransmission either directly or indirectly via the neuronal-astrocytic-vascular tripartite functional unit. Dysfunctions in these neurovascular interactions might result in perfusion deficits and might be involved in specific pathological conditions.

Effect of neuronal NO synthase inhibition on the cerebral vasodilatory response to somatosensory stimulation.

Whether nitric oxide (NO) mediates--or not--the local cerebral blood flow (CBF) increases occurring during functional brain activation is still a controversial issue. In the present study, we sought to determine whether neuronal NO synthase is involved in the cerebrovascular response to activation of the trigeminal pathway in the rat. Local CBF was measured using the autoradiographic [14C]iodoantipyrine technique in control alpha-chloralose anesthetized rats and 30 min following administration of 7-nitroindazole (7-NI), an inhibitor of the neuronal NO synthase. Unilateral whiskers stroking increased local CBF in all six regions of the trigeminal pathway. Under 7-NI, CBF was slightly decreased and the vasodilatatory response to whisker stimulation was unaltered in the four trigeminal nuclei studied. In contrast, no significant vasodilatation was noted in the ventral posteromedial thalamic nucleus and somatosensory cortex. These results suggest that the neuronal NO synthase mediates the hyperemia associated with somatosensory activation in second order relay stations but not in the site of termination of primary afferents.

Is alpha-chloralose plus halothane induction a suitable anesthetic regimen for cerebrovascular research?

The aim of this study was to determine whether alpha-chloralose, when associated with an initial period of halothane, is a suitable anesthetic regimen for cerebrovascular studies. For this purpose, rats anesthetized with alpha-chloralose plus halothane induction were first subjected to noxious stimuli, and the behavior, EEG and systemic variables were recorded. During a second step, cortical blood flow was measured with laser-Doppler flowmetry and the time-course of the cerebrovascular reactivity to hypercapnia were measured in artificially ventilated rats anesthetized with either alpha-chloralose (40 mg.kg-1, s.c.) plus halothane induction (1.5% given during the first 45-60 min) or halothane alone (1.5%). Finally, an experimental paradigm was developed that allowed the comparison of the hypercapnic reactivity, both in awake and anesthetized conditions in the same animal. Our results show that the association of alpha-chloralose with halothane leads to stable cardiovascular parameters and immobility of ventilated rats, placed in ear bars without curare, for 3 h without any sign of discomfort. Based on EEG criteria, we found that halothane induction lengthens the duration of alpha-chloralose anesthesia (253 +/- 19 vs. 200 +/- 15 min, P < 0.01). Under alpha-chloralose alone or in association with halothane induction, the vascular reactivity to hypercapnia was considerably impaired (-85% compared to the awake state, P < 0.01), but this impairment was transient, since a control reactivity was restored 150-190 min after induction of anesthesia. Under halothane alone, the vascular reactivity remained reduced throughout the experiment. These results provide evidence that alpha-chloralose plus halothane induction is a suitable anesthetic regimen which displays a temporal window of normal cerebrovascular reactivity.

Widespread attenuation of the cerebrovascular reactivity to hypercapnia following inhibition of nitric oxide synthase in the conscious rat.

Despite the increasing number of publications devoted to the cerebrovascular role of NO, its precise influence in awake animals is still poorly characterized. The effect of nitric oxide synthase (NOS) inhibition on the cerebrovascular CO2 reactivity was therefore studied in conscious rats. Regional CBF was measured using the [14C]iodoantipyrine technique and brain tissue sampling. The CO2 reactivity was determined 60 min after administration of 30 mg kg-1 N omega-nitro-L-arginine methyl ester (L-NAME). Blockade of NOS by L-NAME significantly decreased CBF in all 11 brain regions studied (-17 to -49%) and increased arterial pressure from 117 +/- 12 to 147 +/- 11 mn Hg. In control conditions, CO2 responsiveness ranged from 1.3 +/- 0.4 in the hypophysis to 6.4 +/- 0.6 ml 100 g-1 min-1 mm Hg-1 in the parietal cortex. Following L-NAME injection, the reactivity to hypercapnia was significantly attenuated in all structures, the magnitude of the reduction ranging from 57% in the medulla to 74% in the cerebellum. This result shows that NO is an important mediator of the hypercapnic vasodilation in the conscious rat.

Spreading depression reversibly impairs autoregulation of cortical blood flow.

The experiment examines whether the mechanisms responsible for the autoregulation of cerebral blood flow (CBF) in response to hypotension were affected during the initial phase of cortical spreading depression (CSD). CSD was induced by a cortical pinprick in anesthetized rabbits, and CBF was measured by laser-Doppler flowmetry through a chronically implanted Plexiglas window. The reactivity to CO2 and papaverine was also studied before and after CSD. Fifteen minutes after CSD, autoregulatory vasodilation was reduced (P < 0.01). This impairment was reversible, since the autoregulatory response was restored 35 min after CSD. The time course of the reactivity to papaverine after CSD paralleled the autoregulatory response, with a significant correlation between the two reactivities (r = 0.47; P < 0.01). Conversely, the reactivity to CO2 was significantly reduced after CSD (P < 0.001) and remained affected for at least 95 min. We conclude that the mechanisms underlying autoregulation are transiently disturbed by CSD and that these mechanisms are not mediated by an accumulation of CO2 but seem instead to be related to an increase in adenosine 3',5'-cyclic monophosphate concentration.

Effect of nimodipine on the autoregulation of cerebral blood flow studied by laser-Doppler flowmetry.

The present work examines whether nimodipine impairs autoregulation of CBF during hypotension. The CBF of 16 anesthetized rabbits was measured with a laser-Doppler flowmetry probe placed on the external surface of a plexiglas window, chronically inserted in the skull. Autoregulation was triggered by aortic bleeding. First, the effects of three doses of nimodipine (1, 3 and 10 micrograms/kg) and the solvent were studied in 10 rabbits in which MABP was maintained at 50 mmHg for one minute. Second, 10 micrograms/kg i.v. nimodipine was administered to 6 rabbits in which MABP was kept at 30 mmHg for one minute. Before bleeding, the 10 micrograms/kg dose significantly decreased MABP (from 96 +/- 11 mmHg to 81 +/- 11 mmHg, P < 0.01) and increased CBF (from 104 +/- 20% to 147 +/- 25%, P < 0.01) as compared to the solvent. In the first set of experiments, only the 10 micrograms/kg dose suppressed the autoregulatory vasodilation, but CBF was not different from control (84 +/- 17% versus 87 +/- 12%), probably because of the previous induced vasodilation. In the second set of experiments, active vasodilation occurred and the CBF during hypotension was not different from control (72 +/- 26% versus 65 +/- 11%). We conclude that under nimodipine the triggering of the active autoregulatory vasodilation is dependent on both the severity of hypotension and the previous nimodipine-induced vasodilation.

Effects of dorsal raphe nucleus stimulation on cerebral blood flow and flow-metabolism coupling in the conscious rat.

In the present study, we have investigated the effects of an activation of the ascending serotonergic pathway on the cerebral blood supply to a number (63) of well-defined neuroanatomical structures. To this end, we have measured the local cerebral blood flow during electrical stimulation of the dorsal raphe nucleus. Measurement of regional blood flow was performed in the conscious rat through the use of the [14C]iodoantipyrine autoradiographic technique. Stimulation of the dorsal raphe nucleus induced increases (> 15% compared to control) in cerebral blood flow in 17 structures of which statistical significance (P < 0.05) was achieved in nine; raphe stimulation significantly decreased flow in three regions. The greatest increases (+71 and +46%) were found in the frontal sensorimotor and posterior parietal cortices. Other increases were noted in relay stations of the extrapyramidal and limbic systems. Stimulation induced a decrease in two regions of the primary auditory system and in the lateral habenular nucleus. These results show that activation of the serotonergic pathway in the conscious rat effects regional cerebral blood flow heterogeneously, differing from the widespread increase in glucose utilization that we previously observed using the same experimental paradigm. Statistical analyses indicated that activation of the dorsal raphe nucleus resulted in a global modification of the flow-metabolism ratio. Moreover, in 19 out of 31 regions analysed, this ratio is significantly altered as compared to control. The dichotomy between raphe-induced changes in flow and glucose-metabolism could be explained by one or both of two hypotheses; firstly there could be a direct serotonergic innervation of cerebral resistance vessels; secondly, during raphe stimulation it could be that glucose use is not the primary determinant of tissue perfusion.

Synthesis and regional rat brain distribution of [11C]MDL 72222: a 5HT3 receptor antagonist.

MDL 72222, an antagonist of 5HT3 receptors, was labeled with a specific radioactivity of 340-400 mCi/mumol by alkylation of the nor-precursor with [11C]CH3I. The yield of the synthesis, starting from [11C]methyliodide to the purified product and corrected for decay, was good approximately 70-75%. After i.v. injection, [11C]MDL 72222 diffuses readily in the central nervous system but is not detected as metabolites in brain and blood, during 1 h study carried out in rats. The time course and distribution of [11C]MDL 72222 was assessed in various organs (liver, lung, kidney, heart, whole brain) and in blood; the organ uptake was rapid and large; the highest accumulation was found in the lung. The regional brain distribution shows initial uptake and subsequent retention of tracer in favor of the cerebral cortex. The level of brain radioactivity was not reduced by pretreatment with a 1000-fold excess of unlabeled MDL 72222. These results suggest that [11]MDL 72222 is of limited interest for 5HT3 receptor binding studies in brain in vivo, presumably mainly because of large non-specific binding.

Effect of local injection of 8-OH-DPAT into the dorsal or median raphe nuclei on extracellular levels of serotonin in serotonergic projection areas in the rat brain.

Using in vivo microdialysis, we have examined the effects of local administration of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) into either the dorsal (DRN) or the median (MRN) raphe nucleus on extracellular levels of serotonin (5-HT) in the corresponding projection fields, namely striatum and ventral hippocampus in the anaesthetized rat. Local injection of 8-OH-DPAT (0.5 microgram/0.1 microliter) in the DRN reduced extracellular 5-HT levels in the striatum (-55%) and to a lesser extent in the hippocampus (-22%). When injected at the same dose in the MRN, 8-OH-DPAT caused a marked decrease in 5-HT output in the hippocampus (-41%) and had no effect in the striatum. Autoradiographic studies performed on brains from animals that received a local injection of [8H]8-OH-DPAT into either the DRN or MRN under similar experimental conditions indicated that the radioactivity remained localized within each midbrain raphe nucleus. These results confirm anatomical data demonstrating that the striatum and the ventral hippocampus receive their serotonergic innervation preferentially from the DRN and the MRN, respectively.