Elvire Vaucher

Activation of the mouse primary visual cortex by medial prefrontal subregion stimulation is not mediated by cholinergic basalo-cortical projections

The medial prefrontal cortex (mPFC) exerts top-down control of primary visual cortex (V1) activity. As there is no direct neuronal projection from mPFC to V1, this functional connection may use an indirect route, i.e., via basalo-cortical cholinergic projections. The cholinergic projections to V1 originate from neurons in the horizontal limb of the diagonal band of Broca (HDB), which receive neuronal projections from the ventral part of the mPFC, composed of prelimbic (PrL) and infralimbic cortices (IL). Therefore, the objective of this study was to determine whether electrical stimulation of mice mPFC subregions activate (1) V1 neurons; and (2) HDB cholinergic neurons, suggesting that the HDB serves as a relay point in the mPFC-V1 interaction. Neuronal activation was quantified using c-Fos immunocytochemistry or thallium autometallography for each V1 layer using automated particle analysis tools and optical density measurement. Stimulation of IL and PrL induced significantly higher c-Fos expression or thallium labeling in layers II/III and V of V1 in the stimulated hemisphere only. A HDB cholinergic neuron-specific lesion by saporin administration reduced IL-induced c-Fos expression in layers II/III of V1 but not in layer V. However, there was no c-Fos expression or thallium labeling in the HDB neurons, suggesting that this area was not activated by IL stimulation. Stimulation of another mPFC subarea, the anterior cingulate cortex (AC), which is involved in attention and receives input from V1, activated neither V1 nor HDB. The present results indicate that IL and PrL, but not AC, stimulation activates V1 with the minor involvement of the HDB cholinergic projections. These results suggest a functional link between the ventral mPFC and V1, but this function is only marginally supported by HDB cholinergic neurons and may involve other brain regions.

Specific Subtypes of Cortical GABA Interneurons Contribute to the Neurovascular Coupling Response to Basal Forebrain Stimulation

Neurovascular coupling, or the tight coupling between neuronal activity and regional cerebral blood flow (CBF), seems largely driven by the local processing of incoming afferent signals within the activated area. To test if cortical γ-aminobutyric acid (GABA) interneurons—the local integrators of cortical activity—are involved in this coupling, we stimulated the basalocortical pathway in vivo, monitored cortical CBF, and identified the activated interneurons (c-Fos-immunopositive) and the neuromediators involved in this response. Basal forebrain (BF) stimulation induced ipsilateral increases in CBF and selective activation of layers II to VI somatostatin- and/or neuropeptide Y-containing, as well as layer I GABA interneurons. Nitric oxide synthase interneurons displayed weak bilateral activation, whereas vasoactive intestinal polypeptide- or acetylcholine (ACh)-containing GABA interneurons were not activated. Selective cholinergic deafferentation indicated that ACh released from stimulated BF afferents triggered the CBF response, but the latter was mediated, in part, by the local release of GABA from cholinoceptive cortical interneurons, and through GABA-A receptor-mediated transmission. These data show that activation of specific subsets of GABA interneurons and their GABA-A-mediated effects on neuronal, vascular, and/or astroglial targets are necessary for the full expression of the hemodynamic response to BF stimulation. Further, these findings highlight the importance of understanding the cellular networks and circuitry that underlie hemodynamic signals, as only specific subsets of neurons may be activated by a given stimulus, depending on the afferent inputs they receive and integrate.

Estrogen effects on object memory and cholinergic receptors in young and old female mice

We investigated whether object recognition memory is modulated by estrogen in young (5 month) and aged (24 month) female C57Bl/6J mice, and if cholinergic muscarinic receptors might contribute to this response. Mice that were ovariectomized, or ovariectomized plus estradiol-treated three weeks before behavioral testing or quantitative autoradiography were compared to intact mice. Memory for a previously encountered object deteriorated significantly between 3 and 6h after initial exposure, regardless of animal age. In both young and aged mice, estradiol-treated mice showed significantly greater recall than did ovariectomized mice. In both age groups, the apparent number of [(3)H]pirenzepine/M(1)-like and [(3)H]AFDX384/M(2)-like muscarinic receptor binding sites was reduced in the basal forebrain as well as its projection areas following ovariectomy, but this decrease was not alleviated by estrogen. Aging poorly affected object memory, but reduced muscarinic binding in some cortical subregions and in the caudate nucleus. These findings suggest that estrogen effects on memory in C57Bl/6J mice are not due to changes in the number of muscarinic receptors.

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. γ‐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 μm2) 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 μm 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. J. Comp. Neurol. 421:161–171, 2000.

Cholinergic basal forebrain projections to nitric oxide synthase-containing neurons in the rat cerebral cortex

Stimulation of basal forebrain neurons elicits regional cerebral blood flow increases which are reportedly mediated by acetylcholine and nitric oxide. However, the modality of interaction between these two mediators remains unclear. Particularly, little is known about the source, i.e. endothelial, glial and/or neuronal, of the potent gaseous vasodilator nitric oxide. In the present study, we examined, by double immunocytochemical labelling of nitric oxide synthase and choline acteyltransferase at the light and electron microscopic level, the existence of morphological relationships between cortical nitric oxide synthase-containing neurons and cholinergic cells or nerve fibres. Using anterograde tract tracing and selective basal forebrain lesions, we further investigated the origin of the cholinergic input to cortical nitric oxide synthase neurons. The results confirm that cortical nitric oxide synthase-immunoreactive neurons are often associated with the local microvascular bed, show that intracortical neurons immunostained for nitric oxide synthase and choline acetyltransferase belong to two distinct neuronal populations and, further, that a subset of nitric oxide synthase-containing cell bodies and their proximal dendrites receive a cholinergic input which originates primarily from basalocortical projections. Altogether, these findings suggest that cholinergic basal forebrain neurons could increase cortical blood flow partly via a local nitric oxide relay neuron whereby the freely diffusing gas would be the direct smooth muscle vasodilator agent. It is concluded that this interaction might contribute to the complex relationships between the basal forebrain and the cortical microcirculation, interactions which result in fine regulation of cortical perfusion.

ACETYLCHOLINE RELEASE IS ELICITED IN THE VISUAL CORTEX, BUT NOT IN THE PREFRONTAL CORTEX, BY PATTERNED VISUAL STIMULATION: A DUAL IN VIVO MICRODIALYSIS STUDY WITH FUNCTIONAL CORRELATES IN THE RAT BRAIN

By its projections to the primary visual and the prefrontal cortices, the basal forebrain cholinergic system is involved in cognitive processing of sensory stimuli. It has been suggested that visual stimulus-induced cholinergic activation of the visual cortex may exert a permissive role on thalamocortical inputs. However, it is not known if visual stimulation elicits cholinergic activation of high-order brain areas in the absence of attentional need. In the present study, we measured the effects of patterned visual stimulation (horizontal grating) on the release of acetylcholine with dual-probe in vivo microdialysis in the visual and the prefrontal cortices of anesthetized rats. We also used retrograde tracing to determine the anatomical relationships of cholinergic neurons with neurons of the visual system and the prefrontal cortex. Finally, we evaluated a functional correlate of this stimulation, namely c-fos immunolabeling. Patterned visual stimulation elicited significant increases in acetylcholine release in the visual cortex, accompanied by an increased number of c-fos immunoreactive neurons in this brain area. In contrast, in the prefrontal cortex, neither the level of acetylcholine release nor the number of c-fos immunoreactive neurons was significantly changed because of the stimulation. Cholinergic basal forebrain neurons projecting to the visual or the prefrontal cortices were both localized within the horizontal limb of the diagonal band of Broca but were not immunoreactive for c-fos during visual stimulation. No parts of the visual system were found to directly project to these basal forebrain neurons. These results suggest the differential involvement of cholinergic projections in the integration of sensory stimuli, depending on the level of activity of the targeted cortical area.

Cholinergic pairing with visual activation results in long-term enhancement of visual evoked potentials.

Acetylcholine (ACh) contributes to learning processes by modulating cortical plasticity in terms of intensity of neuronal activity and selectivity properties of cortical neurons. However, it is not known if ACh induces long term effects within the primary visual cortex (V1) that could sustain visual learning mechanisms. In the present study we analyzed visual evoked potentials (VEPs) in V1 of rats during a 4–8 h period after coupling visual stimulation to an intracortical injection of ACh analog carbachol or stimulation of basal forebrain. To clarify the action of ACh on VEP activity in V1, we individually pre-injected muscarinic (scopolamine), nicotinic (mecamylamine), α7 (methyllycaconitine), and NMDA (CPP) receptor antagonists before carbachol infusion. Stimulation of the cholinergic system paired with visual stimulation significantly increased VEP amplitude (56%) during a 6 h period. Pre-treatment with scopolamine, mecamylamine and CPP completely abolished this long-term enhancement, while α7 inhibition induced an instant increase of VEP amplitude. This suggests a role of ACh in facilitating visual stimuli responsiveness through mechanisms comparable to LTP which involve nicotinic and muscarinic receptors with an interaction of NMDA transmission in the visual cortex.

Confocal Analysis of Cholinergic and Dopaminergic Inputs onto Pyramidal Cells in the Prefrontal Cortex of Rodents

Cholinergic and dopaminergic projections to the rat medial prefrontal cortex (mPFC) are both involved in cognitive functions including attention. These neuronal systems modulate mPFC neuronal activity mainly through diffuse transmission. In order to better understand the anatomical level of influence of these systems, confocal microscopy with triple-fluorescent immunolabeling was used in three subregions of the mPFC of rats and Drd1a-tdTomato/Drd2-EGFP transgenic mice. The zone of interaction was defined as a reciprocal microproximity between dopaminergic and cholinergic axonal segments as well as pyramidal neurons. The density of varicosities, along these segments was considered as a possible activity-dependant morphological feature. The percentage of cholinergic and dopaminergic fibers in microproximity ranged from 12 to 40% depending on the layer and mPFC subregion. The cholinergic system appeared to have more influence on dopaminergic fibers since a larger proportion of the dopaminergic fibers were within microproximity to cholinergic fibers. The density of both cholinergic and dopaminergic varicosities was significantly elevated within microproximities. The main results indicate that the cholinergic and dopaminergic systems converge on pyramidal cells in mPFC particularly in the layer V. In transgenic mice 93% of the pyramidal cells expressed the transgenic marker for Drd2 expression, but only 22% expressed the maker for Drd1ar expression. Data presented here suggest that the modulation of mPFC by dopaminergic fibers would be mostly inhibitory and localized at the output level whereas the cholinergic modulation would be exerted at the input and output level both through direct interaction with pyramidal cells and dopaminergic fibers.

Ocular Application of the Kinin B1 Receptor Antagonist LF22-0542 Inhibits Retinal Inflammation and Oxidative Stress in Streptozotocin-Diabetic Rats

Purpose Kinin B1 receptor (B1R) is upregulated in retina of Streptozotocin (STZ)-diabetic rats and contributes to vasodilation of retinal microvessels and breakdown of the blood-retinal barrier. Systemic treatment with B1R antagonists reversed the increased retinal plasma extravasation in STZ rats. The present study aims at determining whether ocular application of a water soluble B1R antagonist could reverse diabetes-induced retinal inflammation and oxidative stress. Methods Wistar rats were made diabetic with STZ (65 mg/kg, i.p.) and 7 days later, they received one eye drop application of LF22-0542 (1% in saline) twice a day for a 7 day-period. The impact was determined on retinal vascular permeability (Evans blue exudation), leukostasis (leukocyte infiltration using Fluorescein-isothiocyanate (FITC)-coupled Concanavalin A lectin), retinal mRNA levels (by qRT-PCR) of inflammatory (B1R, iNOS, COX-2, ICAM-1, VEGF-A, VEGF receptor type 2, IL-1β and HIF-1α) and anti-inflammatory (B2R, eNOS) markers and retinal level of superoxide anion (dihydroethidium staining). Results Retinal plasma extravasation, leukostasis and mRNA levels of B1R, iNOS, COX-2, VEGF receptor type 2, IL-1β and HIF-1α were significantly increased in diabetic retinae compared to control rats. All these abnormalities were reversed to control values in diabetic rats treated with LF22-0542. B1R antagonist also significantly inhibited the increased production of superoxide anion in diabetic retinae. Conclusion B1R displays a pathological role in the early stage of diabetes by increasing oxidative stress and pro-inflammatory mediators involved in retinal vascular alterations. Hence, topical application of kinin B1R antagonist appears a highly promising novel approach for the treatment of diabetic retinopathy.

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.