Similar perisynaptic glial localization for the Na+,K+-ATPase alpha 2 subunit and the glutamate transporters GLAST and GLT-1 in the rat somatosensory cortex.

Several isoenzymes of the Na(+),K(+)-ATPase are expressed in brain but their specific roles are poorly understood. Recently, it was suggested that an isoenzyme of the Na(+),K(+)-ATPase containing the alpha(2) subunit, together with the glutamate transporters GLAST and GLT-1, participate in a coupling mechanism between neuronal activity and energy metabolism taking place in astrocytes. To substantiate this hypothesis, we compared the distribution of alpha(2), GLAST and/or GLT-1 in the rat cerebral cortex using double immunofluorescence and confocal microscopy, and immunocytochemistry at the electron microscopic level. We also investigated the relationship between alpha(2), GLAST or GLT-1 and asymmetrical synaptic junctions (largely glutamatergic) and GABAergic nerve terminals. Results show that the alpha(2) subunit has an exclusive astroglial localization, and that it is almost completely co-distributed with GLAST and GLT-1 when evaluated by confocal microscopy. This similar distribution was confirmed at the ultrastructural level, which further showed that the vast majority of the alpha(2) staining (73% of all labelled elements), like that of GLAST and GLT-1, was located in glial leaflets surrounding dendritic spines and the dendritic and/or axonal elements of asymmetrical (glutamatergic) axo-dendritic synapses. Synapses ensheathed by alpha(2), GLAST or GLT-1 virtually never included (

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.

GABA neurons provide a rich input to microvessels but not nitric oxide neurons in the rat cerebral cortex: a means for direct regulation of local cerebral blood flow.

Basal forebrain neurons project to microvessels and the somata of nitric oxide (NO) synthase-containing neurons in the cerebral cortex, and their stimulation results in increases in cortical perfusion. gamma-Aminobutyric acid (GABA) is the second major neurotransmitter synthesized by these neurons and it has also been reported to modify cerebromicrovascular tone. We thus investigated by light and electron microscopy the association of GABA neurons (labeled for glutamic acid decarboxylase [GAD]) with cortical microvessels and/or NO neurons (identified by nicotinamide adenine dinucleotide [NADPH-D] histochemistry) within the frontoparietal and perirhinal cerebral cortex in the rat. On thick and semithin sections, a high density of GAD puncta was observed, several surrounded intracortical blood vessels and neuronal perikarya. In contrast, NADPH-D cell somata and proximal dendrites were only occasionally contacted by GAD nerve terminals. Perivascular and perisomatic GAD appositions were identified at the ultrastructural level as large (0.44-0.50 microm(2)) neuronal varicosities located in the immediate vicinity of, or being directly apposed to, vessels or unstained neuronal cell bodies. In both cortical areas, perivascular GAD terminals were located at about 1 microm from the vessels and were seen to frequently establish junctional contacts (synaptic frequency of 25-40% in single thin sections) with adjacent neuronal but not vascular elements. Ibotenic or quisqualic acid lesion of the substantia innominata did not significantly affect the density of cortical and perivascular GAD terminals, suggesting that they mostly originated locally in the cortex. These results suggest that GABA terminals can interact directly with the microvascular bed and that the somata and proximal dendrites of NO neurons are not a major target for cortical GABA neurotransmission. However, based on the colocalization of GABA and NADPH-D in a subset of cortical neurons, we suggest that these interneurons could be implicated in the cortical vascular response elicited by stimulation of basal forebrain neurons.

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.

Role of P-170 glycoprotein in colchicine brain uptake.

To study the role of P-glycoprotein (P-gp) in the delivery of colchicine from blood to brain, the pharmacokinetics of colchicine in plasma and brain was studied in the rat by an in vivo method and by the in situ brain perfusion technique. Colchicine was administered intravenously at three doses (1, 2.5, and 5 mg/kg) with or without an inhibitor of P-gp, verapamil (0.5 mg/kg i.v.); blood and brain samples were taken at t = 1, 2, and 3 hr. Areas under the colchicine curve at doses from 2.5 to 5 mg/kg were proportional to dose for plasma but not for brain. At a colchicine dose of 5 mg/kg, verapamil co-treated rats showed a 1.65-fold enhancement of the colchicine concentration in plasma but a 4.5-fold enhancement in brain. During short experimental times (in situ brain perfusion technique), a comparable enhancement was found (4.26-fold): mean distribution volumes of colchicine were enhanced from 0.23 +/- 0.17 to 0.98 +/- 0.19 microl/g for the eight gray areas, and no effect was observed in the choroid plexus, which do not express P-gp. These results clearly show that P-gp, present at the luminal surface of the capillary endothelial cells, is responsible for the weak penetration of colchicine into the brain.

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.