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The kallikrein-kinin system (KKS) contributes to vascular inflammation and neovascularization in age-related macular degeneration (AMD), particularly via the kinin B 1 receptor (B 1 R). The aim of the present study was to determine the protective effects of the topical administration of the B 1 R antagonist (R-954) on inflammation, neovascularization, and retinal dysfunction in a murine model of neovascular AMD. Choroidal neovascularization (CNV) was induced in C57BL6 mice using an argon laser. A treatment with ocular drops of R-954 (100 μg/15 μL, twice daily in both eyes), or vehicle, was started immediately on day 0, for 7, 14, or 21 days. CNV, invasive microglia, and B 1 R immunoreactive glial cells, as well as electroretinography alterations, were observed within the retina and choroid of the CNV group but not in the control group. The staining of B 1 R was abolished by R-954 treatment as well as the proliferation of microglia. R-954 treatment prevented the CNV development (volume: 20 ± 2 vs. 152 ± 5 × 10 4 µm 3 in R-954 vs. saline treatment). R-954 also significantly decreased photoreceptor and bipolar cell dysfunction (a-wave amplitude: -47 ± 20 vs. -34 ± 14 µV and b-wave amplitude: 101 ± 27 vs. 64 ± 17 µV in R-954 vs. saline treatment, day 7) as well as angiogenesis tufts in the retina. These results suggest that self-administration of R-954 by eye-drop treatment could be a promising therapy in AMD to preserve retinal health and vision.

The kallikrein-kinin system (KKS) contributes to retinal inflammation and neovascularization, notably in diabetic retinopathy (DR) and neovascular age-related macular degeneration (AMD). Bradykinin type 1 (B1R) and type 2 (B2R) receptors are G-protein-coupled receptors that sense and mediate the effects of kinins. While B2R is constitutively expressed and regulates a plethora of physiological processes, B1R is almost undetectable under physiological conditions and contributes to pathological inflammation. Several KKS components (kininogens, tissue and plasma kallikreins, and kinin receptors) are overexpressed in human and animal models of retinal diseases, and their inhibition, particularly B1R, reduces inflammation and pathological neovascularization. In this review, we provide an overview of the KKS with emphasis on kinin receptors in the healthy retina and their detrimental roles in DR and AMD. We highlight the crosstalk between the KKS and the renin-angiotensin system (RAS), which is known to be detrimental in ocular pathologies. Targeting the KKS, particularly the B1R, is a promising therapy in retinal diseases, and B1R may represent an effector of the detrimental effects of RAS (Ang II-AT1R).

As the residual vision following a traumatic optic nerve injury can spontaneously recover over time, we explored the spontaneous plasticity of cortical networks during the early post-optic nerve crush (ONC) phase. Using in vivo wide-field calcium imaging on awake Thy1-GCaMP6s mice, we characterized resting state and evoked cortical activity before, during, and 31 days after ONC. The recovery of monocular visual acuity and depth perception was evaluated in parallel. Cortical responses to an LED flash decreased in the contralateral hemisphere in the primary visual cortex and in the secondary visual areas following the ONC, but was partially rescued between 3 and 5 days post-ONC, remaining stable thereafter. The connectivity between visual and non-visual regions was disorganized after the crush, as shown by a decorrelation, but correlated activity was restored 31 days after the injury. The number of surviving retinal ganglion cells dramatically dropped and remained low. At the behavioral level, the ONC resulted in visual acuity loss on the injured side and an increase in visual acuity with the non-injured eye. In conclusion, our results show a reorganization of connectivity between visual and associative cortical areas after an ONC, which is indicative of spontaneous cortical plasticity.

Purpose: Diabetic retinopathy is characterized by multiple microcirculatory dysfunctions and angiogenesis resulting from hyperglycemia, oxidative stress, and inflammation. In this study, the retina and retinal pigmented epithelium of non-insulin-dependent diabetic Goto-Kakizaki (GK) rats were examined to detect microvascular alterations, gliosis, macrophage infiltration, lipid deposits, and fibrosis. Emphasis was given to the distribution of kinin B1 receptor (B1R) and vascular endothelial growth factor (VEGF), two major factors in inflammation and angiogenesis., Materials and Methods: 30-week-old male GK rats and age-matched Wistar rats were used. The retinal vascular bed was examined using ADPase staining. The level of lipid accumulation was graded using triglyceride staining with Oil red O. Macrophage and retinal microglia activation, as well as other markers, were revealed by immunohistochemistry and studied with confocal laser scanning microscopy., Results: Abundant lipid deposits were observed in the Bruch's membrane of GK rats. Immunohistochemistry and quantitative analysis showed significantly higher B1R, VEGF, Iba1 (microglia), CD11 (macrophages), fibronectin, and collagen I labeling in the diabetic retina. B1R immunolabeling was detected in the vascular layers of the GK retina. A strong VEGF staining within different retinal cell processes was detected and a pattern of GFAP staining suggested strong Müller cells/astrocytes reactivity. Microgliosis was apparent in the GK retina. A greater tortuosity of the retinal microvessels (an index of endothelial dysfunction) and their increased number were also observed in GK retinas., Conclusions: Data suggest retinal vascular bed alterations in spontaneous type 2 diabetic retinas at 30 weeks. Lipid and collagen accumulation in the retina and choroid, in addition to retinal upregulation of VEGF and B1R, microgliosis, and Müller cell reactivity, may contribute to vascular alterations and inflammatory processes.

The cholinergic potentiation of visual conditioning enhances visual acuity and discrimination of the trained stimulus. To determine if this also induces long-term plastic changes on cortical maps and connectivity in the visual cortex and higher associative areas, mesoscopic calcium imaging was performed in head-fixed awake GCaMP6s adult mice before and after conditioning. The conditioned stimulus (0.03 cpd, 30°, 100% contrast, 1 Hz-drifting gratings) was presented 10 min daily for a week. Saline or Donepezil (DPZ, 0.3 mg/kg, s.c.), a cholinesterase inhibitor that potentiates cholinergic transmission, were injected prior to each conditioning session and compared to a sham-conditioned group. Cortical maps of resting state and evoked response to the monocular presentation of conditioned or non-conditioned stimulus (30°, 50 and 75% contrast; 90°, 50, 75, and 100% contrast) were established. Amplitude, duration, and latency of the peak response, as well as size of activation were measured in the primary visual cortex (V1), secondary visual areas (AL, A, AM, PM, LM, RL), retrosplenial cortex (RSC), and higher cortical areas. Visual stimulation increased calcium signaling in all primary and secondary visual areas, the RSC, but no other cortices. There were no significant effects of sham-conditioning or conditioning alone, but DPZ treatment during conditioning significantly decreased the integrated neuronal activity of superficial layers evoked by the conditioned stimulus in V1, AL, PM, and LM. The activity of downstream cortical areas was not changed. The size of the activated area was decreased in V1 and PM, and the signal-to-noise ratio was decreased in AL and PM. Interestingly, signal correlation was seen only between V1, the ventral visual pathway, and the RSC, and was decreased by DPZ administration. The resting state activity was slightly correlated and rarely affected by treatments, except between binocular and monocular V1 in both hemispheres. In conclusion, cholinergic potentiation of visual conditioning induced change in visual processing in the superficial cortical layers. This effect might be a key mechanism in the establishment of the fine cortical tuning in response to the conditioned visual stimulus., (Copyright © 2020 Laliberté, Othman and Vaucher.)

The endogenous neurotransmitter acetylcholine (ACh) is known to affect the excitatory/inhibitory (E/I) balance of primate visual cortex, enhancing feedforward thalamocortical gain while suppressing corticocortical synapses. Recent advances in the study of the human visual system suggest that ACh is a likely component underlying interocular interactions. However, our understanding of its precise role in binocular processes is currently lacking. Here we use binocular rivalry as a probe of interocular dynamics to determine ACh's effects, via the acetylcholinesterase inhibitor (AChEI) donepezil, on the binocular visual system. A total of 23 subjects (13 male) completed two crossover experimental sessions where binocular rivalry measurements were obtained before and after taking either donepezil (5 mg) or a placebo (lactose) pill. We report that enhanced cholinergic potentiation attenuates perceptual suppression during binocular rivalry, reducing the overall rate of interocular competition while enhancing the visibility of superimposition mixed percepts. Considering recent evidence that perceptual suppression during binocular rivalry is causally modulated by the inhibitory neurotransmitter GABA, our results suggest that cholinergic activity counteracts the effect of GABA with regards to interocular dynamics and may modulate the inhibitory drive within the visual cortex. SIGNIFICANCE STATEMENT Our research demonstrates that the cholinergic system is implicated in modulating binocular interactions in the human visual cortex. Potentiating the transmission of acetylcholine (ACh) via the cholinergic drug donepezil reduces the extent to which the eyes compete for perceptual dominance when presented two separate, incongruent images., (Copyright © 2020 the authors.)

Kinins are vasoactive peptides and mediators of inflammation, which signal through two G protein-coupled receptors, B1 and B2 receptors (B1R, B2R). Recent pre-clinical findings suggest a primary role for B1R in a rat model of wet age-related macular degeneration (AMD). The aim of the present study was to investigate whether kinin receptors are differentially expressed in human wet and dry AMD retinae. The cellular distribution of B1R and B2R was examined by immunofluorescence and in situ hybridization in post-mortem human AMD retinae. The association of B1R with inflammatory proteins (inducible nitric oxide synthase (iNOS) and vascular endothelial growth factor A (VEGFA)), fibrosis markers and glial cells was also studied. While B2R mRNA and protein expression was not affected by AMD, a significant increase of B1R mRNA and immunoreactivity was measured in wet AMD retinae when compared to control and dry AMD retinae. B1R was expressed by Müller cells, astrocytes, microglia and endothelial/vascular smooth muscle cells, and colocalized with iNOS and fibrosis markers, but not with VEGFA. In conclusion, the induction and upregulation of the pro-inflammatory and pro-fibrotic kinin B1R in human wet AMD retinae support previous pre-clinical studies and provide a clinical proof-of-concept that B1R represents an attractive therapeutic target worth exploring in this retinal disease.

Background and Purpose: Age-related macular degeneration (AMD) is a complex neurodegenerative disease treated by anti-VEGF intravitreal injections. As inflammation is potentially involved in retinal degeneration, the pro-inflammatory kallikrein-kinin system is a possible alternative pharmacological target. Here, we investigated the effects of anti-VEGF and anti-B 1 receptor treatments on the inflammatory mechanisms in a rat model of choroidal neovascularization (CNV)., Experimental Approach: Immediately after laser-induced CNV, Long-Evans rats were treated by eye-drop application of a B 1 receptor antagonist (R-954) or by intravitreal injection of B 1 receptor siRNA or anti-VEGF antibodies. Effects of treatments on gene expression of inflammatory mediators, CNV lesion regression and integrity of the blood-retinal barrier was measured 10 days later in the retina. B 1 receptor and VEGF-R2 cellular localization was assessed., Key Results: The three treatments significantly inhibited the CNV-induced retinal changes. Anti-VEGF and R-954 decreased CNV-induced up-regulation of B 1 and B 2 receptors, TNF-α, and ICAM-1. Anti-VEGF additionally reversed up-regulation of VEGF-A, VEGF-R2, HIF-1α, CCL2 and VCAM-1, whereas R-954 inhibited gene expression of IL-1β and COX-2. Enhanced retinal vascular permeability was abolished by anti-VEGF and reduced by R-954 and B 1 receptor siRNA treatments. Leukocyte adhesion was impaired by anti-VEGF and B 1 receptor inhibition. B 1 receptors were found on astrocytes and endothelial cells., Conclusion and Implications: B 1 receptor and VEGF pathways were both involved in retinal inflammation and damage in laser-induced CNV. The non-invasive, self-administration of B 1 receptor antagonists on the surface of the cornea by eye drops might be an important asset for the treatment of AMD., (© 2019 The British Pharmacological Society.)

By virtue of their extensive axonal arborization and perisomatic synaptic targeting, cortical inhibitory parvalbumin (PV) cells strongly regulate principal cell output and plasticity and modulate experience-dependent refinement of cortical circuits during development. An interesting aspect of PV cell connectivity is its prolonged maturation time course, which is completed only by end of adolescence. The p75 neurotrophin receptor (p75NTR) regulates numerous cellular functions; however, its role on cortical circuit development and plasticity remains elusive, mainly because localizing p75NTR expression with cellular and temporal resolution has been challenging. By using RNAscope and a modified version of the proximity ligation assay, we found that p75NTR expression in PV cells decreases between the second and fourth postnatal week, at a time when PV cell synapse numbers increase dramatically. Conditional knockout of p75NTR in single PV neurons in vitro and in PV cell networks in vivo causes precocious formation of PV cell perisomatic innervation and perineural nets around PV cell somata, therefore suggesting that p75NTR expression modulates the timing of maturation of PV cell connectivity in the adolescent cortex. Remarkably, we found that PV cells still express p75NTR in adult mouse cortex of both sexes and that its activation is sufficient to destabilize PV cell connectivity and to restore cortical plasticity following monocular deprivation in vivo Together, our results show that p75NTR activation dynamically regulates PV cell connectivity, and represent a novel tool to foster brain plasticity in adults. SIGNIFICANCE STATEMENT In the cortex, inhibitory, GABA-releasing neurons control the output and plasticity of excitatory neurons. Within this diverse group, parvalbumin-expressing (PV) cells form the larger inhibitory system. PV cell connectivity develops slowly, reaching maturity only at the end of adolescence; however, the mechanisms controlling the timing of its maturation are not well understood. We discovered that the expression of the neurotrophin receptor p75NTR in PV cells inhibits the maturation of their connectivity in a cell-autonomous fashion, both in vitro and in vivo , and that p75NTR activation in adult PV cells promotes their remodeling and restores cortical plasticity. These results reveal a new p75NTR function in the regulation of the time course of PV cell maturation and in limiting cortical plasticity., (Copyright © 2019 the authors.)

Compelling evidence suggests a role for the inducible nitric oxide synthase, iNOS, and the bradykinin type 1 receptor (B1R) in diabetic retinopathy, including a possible control of the expression and activity of iNOS by B1R. In diabetic retina, both iNOS and B1R contribute to inflammation, oxidative stress, and vascular dysfunction. The present study investigated whether inhibition of iNOS has any impact on inflammatory/oxidative stress markers and on the B1R-iNOS expression, distribution, and action in a model of type I diabetes. Diabetes was induced in 6-week-old Wistar rats by streptozotocin (65 mg.kg -1 , i.p.). The selective iNOS inhibitor 1400W (150 μg.10 μl -1 ) was administered twice a day by eye-drops during the second week of diabetes. The retinae were collected 2 weeks after diabetes induction to assess the protein and gene expression of markers by Western blot and qRT-PCR, the distribution of iNOS and B1R by fluorescence immunocytochemistry, and the vascular permeability by the Evans Blue dye technique. Diabetic retinae showed enhanced expression of iNOS, B1R, carboxypeptidase M (involved in the biosynthesis of B1R agonists), IL-1β, TNF-α, vascular endothelium growth factor A (VEGF-A) and its receptor, VEGF-R2, nitrosylated proteins and increased vascular permeability. All those changes were reversed by treatment with 1400W. Moreover, the additional increase in vascular permeability in diabetic retina induced by intravitreal injection of R-838, a B1R agonist, was also prevented by 1400W. Immunofluorescence staining highlighted strong colocalization of iNOS and B1R in several layers of the diabetic retina, which was prevented by 1400W. This study suggests a critical role for iNOS and B1R in the early stage of diabetic retinopathy. B1R and iNOS appear to partake in a mutual auto-induction and amplification loop to enhance nitrogen species formation and inflammation in diabetic retina. Hence, B1R-iNOS axis deserves closer scrutiny in targeting diabetic retinopathy.

A few hours of monocular deprivation with a diffuser eye patch temporarily strengthens the contribution of the deprived eye to binocular vision. This shift in favor of the deprived eye is characterized as a form of adult visual plasticity. Studies in animal and human models suggest that neuromodulators can enhance adult brain plasticity in general. Specifically, acetylcholine has been shown to improve certain aspects of visual function and plasticity in adulthood. We investigated whether a single administration of donepezil (a cholinesterase inhibitor) could further augment the temporary shift in perceptual eye dominance that occurs after 2 h of monocular patching. Twelve healthy adults completed two experimental sessions while taking either donepezil (5 mg, oral) or a placebo (lactose) pill. We measured perceptual eye dominance using a binocular phase combination task before and after 2 h of monocular deprivation with a diffuser eye patch. Participants in both groups demonstrated a significant shift in favor of the patched eye after monocular deprivation, however our results indicate that donepezil significantly reduces the magnitude and duration of the shift. We also investigated the possibility that donepezil reduces the amount of time needed to observe a shift in perceptual eye dominance relative to placebo control. For this experiment, seven subjects completed two sessions where we reduced the duration of deprivation to 1 h. Donepezil reduces the magnitude and duration of the patching-induced shift in perceptual eye dominance in this experiment as well. To verify whether the effects we observed using the binocular phase combination task were also observable in a different measure of sensory eye dominance, six subjects completed an identical experiment using a binocular rivalry task. These results also indicate that cholinergic enhancement impedes the shift that results from short-term deprivation. In summary, our study demonstrates that enhanced cholinergic potentiation interferes with the consolidation of the perceptual eye dominance plasticity induced by several hours of monocular deprivation.

Background: The cholinergic system is a potent neuromodulator system that plays a critical role in cortical plasticity, attention, and learning. Recently, it was found that boosting this system during perceptual learning robustly enhances sensory perception in rodents. In particular, pairing cholinergic activation with visual stimulation increases neuronal responses, cue detection ability, and long-term facilitation in the primary visual cortex. The mechanisms of cholinergic enhancement are closely linked to attentional processes, long-term potentiation, and modulation of the excitatory/inhibitory balance. Some studies currently examine this effect in humans., Objective: The present article reviews the research from our laboratory, examining whether potentiating the central cholinergic system could help visual perception and restoration., Methods: Electrophysiological or pharmacological enhancement of the cholinergic system are administered during a visual training. Electrophysiological responses and perceptual learning performance are investigated before and after the training in rats and humans. This approach's ability to restore visual capacities following a visual deficit induced by a partial optic nerve crush is also investigated in rats., Results: The coupling of visual training to cholinergic stimulation improved visual discrimination and visual acuity in rats, and improved residual vision after a deficit. These changes were due to muscarinic and nicotinic transmissions and were associated with a functional improvement of evoked potentials. In humans, potentiation of cholinergic transmission with 5 mg of donepezil showed improved learning and ocular dominance plasticity, although this treatment was ineffective in augmenting the perceptual threshold and electroencephalography., Conclusions: Potential therapeutic outcomes ought to facilitate vision restoration using commercially available cholinergic agents combined with visual stimulation in order to prevent irreversible vision loss in patients. This approach has the potential to help a large population of visually impaired individuals.

Cholinergic stimulation coupled with visual conditioning enhances the visual acuity and cortical responses in the primary visual cortex. To determine which cholinergic receptors are involved in these processes, qRT-PCR was used. Two modes of cholinergic enhancement were tested: a phasic increase of acetylcholine release by an electrical stimulation of the basal forebrain cholinergic nucleus projecting to the visual cortex, or a tonic pharmacological potentiation of the cholinergic transmission by the acetylcholine esterase inhibitor, donepezil. A daily visual exposure to sine-wave gratings (training) was paired with the cholinergic enhancement, up to 14 days. qRT-PCR was performed at rest, 10 min, one week or two weeks of visual/cholinergic training with samples of the visual and somatosensory cortices, and the BF for determining mRNA expression of muscarinic receptor subtypes (m1, m2, m3, m4, m5), nicotinic receptor subunits (α3, α4, α7, β2, β4), and NMDA receptors, GAD65 and ChAT, as indexes of cortical plasticity. A Kruskal-Wallis test showed a modulation of the expression in the visual cortex of m2, m3, m4, m5, α7, β4, NMDA and GAD65, but only β4 within the basal forebrain and none of these mRNA within the somatosensory cortex. The two modes of cholinergic enhancement induced different effects on mRNA expression, related to the number of visual conditioning sessions and receptor specificity. This study suggests that the combination of cholinergic enhancement and visual conditioning is specific to the visual cortex and varies between phasic or tonic manipulation of acetylcholine levels., (Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.)

Acetylcholine is an important neurotransmitter for the regulation of visual attention, plasticity, and perceptual learning. It is released in the visual cortex predominantly by cholinergic projections from the basal forebrain, where stimulation may produce potentiation of visual processes. However, little is known about the fine organization of these corticopetal projections, such as whether basal forebrain neurons projecting to the primary and secondary visual cortical areas (V1 and V2, respectively) are organized retinotopically. The aim of this study was to map these basal forebrain-V1/V2 projections. Microinjections of the fluorescent retrograde tracer cholera toxin b fragment in different sites within V1 and V2 in Long-Evans rats were performed. Retrogradely labeled cell bodies in the horizontal and vertical limbs of the diagonal band of Broca (HDB and VDB, respectively), nucleus basalis magnocellularis, and substantia innominata (SI), were mapped ex vivo with a computer-assisted microscope stage controlled by stereological software. Choline acetyltranferase immunohistochemistry was used to identify cholinergic cells. Our results showed a predominance of cholinergic projections coming from the HDB. These projections were not retinotopically organized but projections to V1 arised from neurons located in the anterior HDB/SI whereas projections to V2 arised from neurons located throughout the whole extent of HDB/SI. The absence of a clear topography of these projections suggests that BF activation can stimulate visual cortices broadly.

Background and Purpose: The kinin B 1 receptor contributes to vascular inflammation and blood-retinal barrier breakdown in diabetic retinopathy (DR). We investigated the changes in expression, cellular localization and vascular inflammatory effect of B 1 receptors in retina of streptozotocin diabetic rats., Experimental Approach: The distribution of B 1 receptors on retinal cell types was investigated by immunocytochemistry. Effects of B 1 receptor agonist, R-838, and antagonist, R-954, on retinal leukocyte adhesion, gene expression of kinin and VEGF systems, B 1 receptor immunoreactivity, microgliosis and capillary leakage were measured. Effect of B 1 receptor siRNA on gene expression was also assessed., Key Results: mRNA levels of the kinin and VEGF systems were significantly enhanced at 2 weeks in streptozotocin (STZ)-retina compared to control-retina and were further increased at 6 weeks. B 1 receptor mRNA levels remained increased at 6 months. B 1 receptor immunolabelling was detected in vascular layers of the retina, on glial and ganglion cells. Intravitreal R-838 amplified B 1 and B 2 receptor gene expression, B 1 receptor levels (immunodetection), leukostasis and vascular permeability at 2 weeks in STZ-retina. Topical application (eye drops) of R-954 reversed these increases in B 1 receptors, leukostasis and vascular permeability. Intravitreal B 1 receptor siRNA inhibited gene expression of kinin and VEGF systems in STZ-retina. Microgliosis was unaffected by R-838 or R-954 in STZ-retina., Conclusion and Implications: Our results support the detrimental role of B 1 receptors on endothelial and glial cells in acute and advanced phases of DR. Topical application of the B 1 receptor antagonist R-954 seems a feasible therapeutic approach for the treatment of DR., (© 2017 The British Pharmacological Society.)

A large body of literature supports cognitive enhancement as an effect of cholinergic potentiation. However, it remains elusive whether pharmacological manipulations of cholinergic neurotransmission enhance complex visual processing in healthy individuals. To test this hypothesis, we randomly administered either the cholinergic transmission enhancer donepezil (DPZ; 5 mg P.O.) or placebo (lactose) to young adults ( n = 17) 3 h before each session of the three-dimensional (3D) multiple object tracking (3D-MOT) task. This multi-focal attention task evaluates perceptual-cognitive learning over five sessions conducted 7 days apart. A significant amount of learning was observed in the DPZ group but not the placebo group in the fourth session. In the fifth session, this learning effect was observed in both groups. Furthermore, preliminary results for a subgroup of participants ( n = 9) 4-14 months later suggested the cholinergic enhancement effect was long lasting. On the other hand, DPZ had no effect on basic visual processing as measured by a motion and orientation discrimination task performed as an independent one-time, pre-post drug study without placebo control ( n = 10). The results support the construct that cholinergic enhancement facilitates the encoding of a highly demanding perceptual-cognitive task although there were no significant drug effects on the performance levels compared to placebo.

Enhancing cortical plasticity and brain connectivity may improve residual vision following a visual impairment. Since acetylcholine plays an important role in attention and neuronal plasticity, we explored whether potentiation of the cholinergic transmission has an effect on the visual function restoration. To this end, we evaluated for 4 weeks the effect of the acetylcholinesterase inhibitor donepezil on brightness discrimination, visually evoked potentials, and visual cortex reactivity after a bilateral and partial optic nerve crush in adult rats. Donepezil administration enhanced brightness discrimination capacity after optic nerve crush compared to nontreated animals. The visually evoked activation of the primary visual cortex was not restored, as measured by evoked potentials, but the cortical neuronal activity measured by thallium autometallography was not significantly affected four weeks after the optic nerve crush. Altogether, the results suggest a role of the cholinergic system in postlesion cortical plasticity. This finding agrees with the view that restoration of visual function may involve mechanisms beyond the area of primary damage and opens a new perspective for improving visual rehabilitation in humans.

Pathological choroidal neovascularization (CNV) is the common cause of vision loss in patients with age-related macular degeneration (AMD). Macrophages possess potential angiogenic function in CNV. We have demonstrated that human T lymphocyte-derived microparticles (LMPs) exert a potent antiangiogenic effect in several pathological neovascularization models. In this study, we investigated the alteration of proangiogenic properties of macrophages by LMPs treatment in vitro and in vivo models. LMPs regulated the expression of several angiogenesis-related factors in macrophages and consequently stimulated their antiangiogenic effects evidenced by the suppression of the proliferation of human retinal endothelial cells in co-culture experiments. The involvement of CD36 receptor in LMPs uptake by macrophages was demonstrated by in vitro assays and by immunostaining of choroidal flat mounts. In addition, ex vivo experiments showed that CD36 mediates the antiangiogenic effect of LMPs in murine and human choroidal explants. Furthermore, intravitreal injection of LMPs in the mouse model of laser-induced CNV significantly suppressed CNV in CD36 dependent manner. The results of this study suggested an ability of LMPs to alter the gene expression pattern of angiogenesis-related factors in macrophages, which provide important information for a new therapeutic approach for efficiently interfering with both vascular and extravascular components of CNV.

Stimulation of the cholinergic system tightly coupled with periods of visual stimulation boosts the processing of specific visual stimuli via muscarinic and nicotinic receptors in terms of intensity, priority and long-term effect. However, it is not known whether more diffuse pharmacological stimulation with donepezil, a cholinesterase inhibitor, is an efficient tool for enhancing visual processing and perception. The goal of the present study was to potentiate cholinergic transmission with donepezil treatment (0.5 and 1mg/kg) during a 2-week visual training to examine the effect on visually evoked potentials and to profile the expression of cholinergic receptor subtypes. The visual training was performed daily, 10min a day, for 2weeks. One week after the last training session, visual evoked potentials were recorded, or the mRNA expression level of muscarinic (M1-5) and nicotinic (α/β) receptors subunits was determined by quantitative RT-PCR. The visual stimulation coupled with any of the two doses of donepezil produced significant amplitude enhancement of cortical evoked potentials compared to pre-training values. The enhancement induced by the 1mg/kg dose of donepezil was spread to neighboring spatial frequencies, suggesting a better sensitivity near the visual detection threshold. The M3, M4, M5 and α7 receptors mRNA were upregulated in the visual cortex for the higher dose of donepezil but not the lower one, and the receptors expression was stable in the somatosensory (non-visual control) cortex. Therefore, higher levels of acetylcholine within the cortex sustain the increased intensity of the cortical response and trigger the upregulation of cholinergic receptors., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Repetitive visual training paired with electrical activation of cholinergic projections to the primary visual cortex (V1) induces long-term enhancement of cortical processing in response to the visual training stimulus. To better determine the receptor subtypes mediating this effect the selective pharmacological blockade of V1 nicotinic (nAChR), M1 and M2 muscarinic (mAChR) or GABAergic A (GABAAR) receptors was performed during the training session and visual evoked potentials (VEPs) were recorded before and after training. The training session consisted of the exposure of awake, adult rats to an orientation-specific 0.12 CPD grating paired with an electrical stimulation of the basal forebrain for a duration of 1 week for 10 minutes per day. Pharmacological agents were infused intracortically during this period. The post-training VEP amplitude was significantly increased compared to the pre-training values for the trained spatial frequency and to adjacent spatial frequencies up to 0.3 CPD, suggesting a long-term increase of V1 sensitivity. This increase was totally blocked by the nAChR antagonist as well as by an M2 mAChR subtype and GABAAR antagonist. Moreover, administration of the M2 mAChR antagonist also significantly decreased the amplitude of the control VEPs, suggesting a suppressive effect on cortical responsiveness. However, the M1 mAChR antagonist blocked the increase of the VEP amplitude only for the high spatial frequency (0.3 CPD), suggesting that M1 role was limited to the spread of the enhancement effect to a higher spatial frequency. More generally, all the drugs used did block the VEP increase at 0.3 CPD. Further, use of each of the aforementioned receptor antagonists blocked training-induced changes in gamma and beta band oscillations. These findings demonstrate that visual training coupled with cholinergic stimulation improved perceptual sensitivity by enhancing cortical responsiveness in V1. This enhancement is mainly mediated by nAChRs, M2 mAChRs and GABAARs. The M1 mAChR subtype appears to be involved in spreading the enhancement of V1 cortical responsiveness to adjacent neurons.

Muscarinic cholinergic receptors modulate the activity and plasticity of the visual cortex. Muscarinic receptors are divided into five subtypes that are not homogeneously distributed throughout the cortical layers and cells types. This distribution results in complex action of the muscarinic receptors in the integration of visual stimuli. Selective activation of the different subtypes can either strengthen or weaken cortical connectivity (e.g., thalamocortical vs. corticocortical), i.e., it can influence the processing of certain stimuli over others. Moreover, muscarinic receptors differentially modulate some functional properties of neurons during experience-dependent activity and cognitive processes and they contribute to the fine-tuning of visual processing. These functions are involved in the mechanisms of attention, maturation and learning in the visual cortex. This minireview describes the anatomo-functional aspects of muscarinic modulation of the primary visual cortex's (V1) microcircuitry.

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.

The cholinergic system is a potent neuromodulatory system that plays critical roles in cortical plasticity, attention and learning. In this review, we propose that the cellular effects of acetylcholine (ACh) in the primary visual cortex during the processing of visual inputs might induce perceptual learning; i.e., long-term changes in visual perception. Specifically, the pairing of cholinergic activation with visual stimulation increases the signal-to-noise ratio, cue detection ability and long-term facilitation in the primary visual cortex. This cholinergic enhancement would increase the strength of thalamocortical afferents to facilitate the treatment of a novel stimulus while decreasing the cortico-cortical signaling to reduce recurrent or top-down modulation. This balance would be mediated by different cholinergic receptor subtypes that are located on both glutamatergic and GABAergic neurons of the different cortical layers. The mechanisms of cholinergic enhancement are closely linked to attentional processes, long-term potentiation (LTP) and modulation of the excitatory/inhibitory balance. Recently, it was found that boosting the cholinergic system during visual training robustly enhances sensory perception in a long-term manner. Our hypothesis is that repetitive pairing of cholinergic and sensory stimulation over a long period of time induces long-term changes in the processing of trained stimuli that might improve perceptual ability. Various non-invasive approaches to the activation of the cholinergic neurons have strong potential to improve visual perception.

Acetylcholine modulates maturation and neuronal activity through muscarinic and nicotinic receptors in the primary visual cortex. However, the specific contribution of different muscarinic receptor subtypes in these neuromodulatory mechanisms is not fully understood. The present study evaluates in vivo the functional organization and the properties of the visual cortex of different groups of muscarinic receptor knock-out (KO) mice. Optical imaging of intrinsic signals coupled to continuous and episodic visual stimulation paradigms was used. Retinotopic maps along elevation and azimuth were preserved among the different groups of mice. However, compared to their wild-type counterparts, the apparent visual field along elevation was larger in M2/M4-KO mice but smaller in M1-KO. There was a reduction in the estimated relative receptive field size of V1 neurons in M1/M3-KO and M1-KO mice. Spatial frequency and contrast selectivity of V1 neuronal populations were affected only in M1/M3-KO and M1-KO mice. Finally, the neuronal connectivity was altered by the absence of M2/M4 muscarinic receptors. All these effects suggest the distinct roles of different subtypes of muscarinic receptors in the intrinsic organization of V1 and a strong involvement of the muscarinic transmission in the detectability of visual stimuli., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The cholinergic afferents from the basal forebrain to the primary visual cortex play a key role in visual attention and cortical plasticity. These afferent fibers modulate acute and long-term responses of visual neurons to specific stimuli. The present study evaluates whether this cholinergic modulation of visual neurons results in cortical activity and visual perception changes. Awake adult rats were exposed repeatedly for 2 weeks to an orientation-specific grating with or without coupling this visual stimulation to an electrical stimulation of the basal forebrain. The visual acuity, as measured using a visual water maze before and after the exposure to the orientation-specific grating, was increased in the group of trained rats with simultaneous basal forebrain/visual stimulation. The increase in visual acuity was not observed when visual training or basal forebrain stimulation was performed separately or when cholinergic fibers were selectively lesioned prior to the visual stimulation. The visual evoked potentials show a long-lasting increase in cortical reactivity of the primary visual cortex after coupled visual/cholinergic stimulation, as well as c-Fos immunoreactivity of both pyramidal and GABAergic interneuron. These findings demonstrate that when coupled with visual training, the cholinergic system improves visual performance for the trained orientation probably through enhancement of attentional processes and cortical plasticity in V1 related to the ratio of excitatory/inhibitory inputs. This study opens the possibility of establishing efficient rehabilitation strategies for facilitating visual capacity.

Diabetic retinopathy (DR) is a major microvascular complication associated with type 1 and type 2 diabetes mellitus, which can lead to visual impairment and blindness. Current treatment strategies for DR are mostly limited to laser therapies, steroids, and anti-VEGF agents, which are often associated with unwanted side effects leading to further complications. Recent evidence suggests that kinins play a primary role in the development of DR through enhanced vascular permeability, leukocytes infiltration, and other inflammatory mechanisms. These deleterious effects are mediated by kinin B1 and B2 receptors, which are expressed in diabetic human and rodent retina. Importantly, kinin B1 receptor is virtually absent in sane tissue, yet it is induced and upregulated in diabetic retina. These peptides belong to the kallikrein-kinin system (KKS), which contains two separate and independent pathways of regulated serine proteases, namely plasma kallikrein (PK) and tissue kallikrein (TK) that are involved in the biosynthesis of bradykinin (BK) and kallidin (Lys-BK), respectively. Hence, ocular inhibition of kallikreins or antagonism of kinin receptors offers new therapeutic avenues in the treatment and management of DR. Herein, we present an overview of the principal features and known inflammatory mechanisms associated with DR along with the current therapeutic approaches and put special emphasis on the KKS as a new and promising therapeutic target due to its link with key pathways directly associated with the development of DR.

Purpose: A clear correlation between vascular deficits and retinal ganglion cell (RGC) loss in glaucoma has not yet been established. The question arose as to whether there is loss of inner retinal vessels following intraocular pressure (IOP) increase and, if so, whether it occurs prior to, concomitantly with, or after RGC death. We also sought to establish whether galantamine, an acetylcholinesterase inhibitor that promotes RGC survival, can protect the retinal microvasculature and enhance blood flow in experimental glaucoma., Methods: Ocular hypertension was induced in Brown Norway rats by injection of hypertonic saline into an episcleral vein. Retinas were processed for simultaneous visualization of the retinal microvasculature and RGCs in glaucomatous and control eyes. Retinal blood flow was examined by quantitative autoradiography using N-isopropyl-p-[(14)C]-iodoamphetamine. Vascular reactivity was further assessed using an in vitro retinal microvasculature preparation., Results: Substantial loss of retinal capillaries was observed after induction of ocular hypertension. The onset of both microvasculature and RGC loss occurred early and proceeded at a similar rate for at least 5 weeks after the initial damage. Systemic administration of galantamine preserved microvasculature density and improved retinal blood flow in glaucomatous retinas. The vasoactive effects of galantamine on retinal microvessels occurred through activation of muscarinic acetylcholine receptors both in vitro and in vivo., Conclusions: The onset and progression of microvessel and RGC loss are concomitant in experimental glaucoma, suggesting a tight codependence between these cellular compartments. Early interventions aimed to protect the retinal microvasculature and improve blood supply are likely to be beneficial for the treatment of glaucoma.

Purpose: A new noninvasive laser Doppler flowmetry (LDF) probe (one emitting fiber surrounded by a ring of eight collecting fibers, 1-mm interaxis distance) was tested for its sensitivity to assess the retinal/choroidal blood flow variations in response to hypercapnia, hyperoxia, diverse vasoactive agents and following retinal arteries photocoagulation in the rat., Materials and Methods: After pupil dilation, a LDF probe was placed in contact to the cornea of anesthetized rats in the optic axis. Hypercapnia and hyperoxia were induced by inhalation of CO(2) (8% in medical air) and O(2) (100%) while pharmacological agents were injected intravitreously. The relative contribution of the choroidal circulation to the LDF signal was estimated after retinal artery occlusion by photocoagulation., Results: Blood flow was significantly increased by hypercapnia (18%), adenosine (14%) and sodium nitroprusside (16%) as compared to baseline values while it was decreased by hyperoxia (-8%) and endothelin-1 (-11%). Photocoagulation of retinal arteries significantly decreased blood flow level (-45%)., Conclusions: Although choroidal circulation most likely contributes to the LDF signal in this setting, the results demonstrate that LDF represents a suitable in vivo noninvasive technique to monitor online relative reactivity of retinal perfusion to metabolic or pharmacological challenge. This technique could be used for repeatedly assessing blood flow reactivity in rodent models of ocular diseases.

In rats, selective depletion of the cholinergic interneurons in the ventral striatum (nucleus accumbens or N.Acc.) results in heightened behavioural sensitivity to amphetamine and impaired sensorimotor gating processes, suggesting a hyper-responsiveness to dopamine (DA) activity in the N.Acc. We hypothesized that local cholinergic depletion may also trigger distal functional alterations, particularly in prefrontal cortex (PFC). Adult male Sprague-Dawley rats were injected bilaterally in the N.Acc. with an immunotoxin targeting choline acetyltransferase. Two weeks later, cognitive function was assessed using the delayed alternation paradigm in the T-maze. The rats were then implanted with voltammetric recording electrodes in the ventromedial PFC to measure in vivo extracellular DA release in response to mild tail pinch stress. The PFC was also examined for density of tyrosine hydroxylase (TH)-labelled varicosities. In another cohort of control and lesioned rats, we measured post mortem tissue content of DA. Depletion of cholinergic neurons (restricted to N.Acc.) significantly impaired delayed alternation performance across delay intervals. While (basal) post mortem indices of PFC DA function were unaffected by N.Acc. lesions, in vivo mesocortical DA activation was markedly reduced; this deficit correlated significantly with cognitive impairments. TH-labelled varicosities however, were unaffected in cortical layer V relative to controls. These data suggest that selective depletion of cholinergic interneurons in N.Acc. triggers widespread functional impairments in mesocorticolimbic DA function and cognition. The possible relevance of these findings is also discussed in relation to schizophrenia, where reduced density of cholinergic neurons in ventral striatum has been reported., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Purpose: Kinin B(1) receptor (B(1)R) 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 B(1)R antagonists reversed the increased retinal plasma extravasation in STZ rats. The present study aims at determining whether ocular application of a water soluble B(1)R 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 (B(1)R, iNOS, COX-2, ICAM-1, VEGF-A, VEGF receptor type 2, IL-1β and HIF-1α) and anti-inflammatory (B(2)R, eNOS) markers and retinal level of superoxide anion (dihydroethidium staining)., Results: Retinal plasma extravasation, leukostasis and mRNA levels of B(1)R, 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. B(1)R antagonist also significantly inhibited the increased production of superoxide anion in diabetic retinae., Conclusion: B(1)R 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 B(1)R antagonist appears a highly promising novel approach for the treatment of diabetic retinopathy.

Purpose: Intravitreal (ITV) injection of anti-VEGFs like bevacizumab are widely used to treat neovascular AMD. However, VEGF is essential for biologic functions such as blood pressure regulation. Indeed, anti-VEGF intravenous administration is associated with hypertension. Therefore, the effect of ITV bevacizumab on retinal circulation was examined., Methods: Twenty-three patients with neovascular AMD treated with three repeat ITV injections of bevacizumab were recruited. Blood arteriolar diameter and flow measurements were performed with a bidirectional laser Doppler flowmeter at baseline, 1 week after the first injection, just before the second injection, and 5 weeks after the third injection. Scanning laser Doppler flowmetry was used to assess the effect of bevacizumab on tissue perfusion at the first and fourth visits., Results: Arteriolar diameter significantly decreased from 122.5 ± 14.5 μm to 118.9 ± 14.0 μm (P = 0.03) during the first week to reach a mean value of 117.2 ± 13.7 μm at the end of the study (P < 0.01). Arterial blood flow did not change significantly. Neuroretinal rim perfusion decreased from 181.1 ± 84.1 arbitrary flow units to 167.7 ± 76.5 arbitrary flow units, which was borderline significant (P = 0.06). No significant change was observed in the peripapillary retina., Conclusions: Arteriolar diameter decreased significantly after the first injection and persisted until the end of the study suggesting a long-term effect of bevacizumab on vascular tone. However, the blood flow change is not significant. A borderline significant decrease in neuroretinal rim perfusion was observed and suggests that the neuroretinal rim may be more sensitive than the peripapillary retina to the effects of bevacizumab.

The vasoactive kinin B₁ receptor (B₁R) is overexpressed in the retina of diabetic rats in response to hyperglycemia and oxidative stress. The aim of the present study was to determine whether B₁R could contribute to the early retinal blood flow changes occurring in diabetes. Male Wistar rats were rendered diabetic with a single i.p. injection of Streptozotocin (STZ) and studied 4 days or 6 weeks after diabetes induction. The presence of B₁R in the retina was confirmed by Western blot. The impact of oral administration of the B₁R selective antagonist SSR240612 (10mg/kg) was measured on alteration of retinal perfusion in awake diabetic rats by quantitative autoradiography. Data showed that B₁R was upregulated in the STZ-diabetic retina at 4 days and 6 weeks. Retinal blood flow was not altered in 4-day diabetic rats compared with age-matched controls but was significantly decreased following SSR240612 treatment. In 6-week diabetic rats, retinal blood flow was markedly reduced compared to control rats and SSR240612 did not further decrease the blood flow. These results suggest that B₁R is upregulated in STZ-diabetic retina and has a protective compensatory role on retinal microcirculation at 4 days but not at 6 weeks following diabetes induction., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Diffuse transmission is an important non-synaptic communication mode in the cerebral neocortex, in which neurotransmitters released from en passant varicosities interact with surrounding cells. In a previous study we have shown that the cholinergic axonal segments which were in the microproximity with dopaminergic fibers possessed a greater density of en passant varicosities compared to more distant segments, suggesting an activity-dependent level of en passant varicosities in the axonal zone of interaction. To further evaluate this plastic relationship, the density of cholinergic varicosities was quantified on fiber segments within the microproximity of activated or non-activated pyramidal cells of the prefrontal cortex (mPFC). Repetitive 14 days patterned visual stimulation paired with an electrical stimulation of the cholinergic fibers projecting to the mPFC from the HDB was performed to induce persistent axonal plastic changes. The c-Fos early gene immunoreactivity was used as a neuronal activity marker of layer V pyramidal cells, labelled with anti-glutamate transporter EAAC1. Cholinergic fibers were labeled with anti-ChAT (choline acetyltransferase) immunostaining. The density of ChAT+ varicosities on and the length of fiber segments within the 3 µm microproximity of c-Fos positive/negative pyramidal cells were evaluated on confocal images. More than 50% of the pyramidal cells in the mPFC were c-Fos immunoreactive. Density of ChAT+ varicosities was significantly increased within 3 µm vicinity of activated pyramidal cells (0.50±0.01 per µm of ChAT+ fiber length) compared to non-activated cells in this group (0.34±0.001; p≤0.05) or control rats (0.32±0.02; p≤0.05). Different types of stimulation (visual, HDB or visual/HDB) induced similar increase of the density of ChAT+ varicosities within microproximity of activated pyramidal cells. This study demonstrated at the subcellular level an activity-dependent enrichment of ChAT+ varicosities in the axonal zone of interaction with other neuronal elements.

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.

Purpose: To investigate whether postmenopausal hormone therapy (HT) increases retinal and ONH blood flow (BF) and protects ONH topography and the function of retinal ganglion cells in postmenopausal women (PMW). The effect of estradiol (E(2)) treatment on retinal tissue perfusion was also investigated in ovariectomized rats, an animal model for menopause., Methods: Sixty-four healthy PMW were recruited, 29 of whom never used HT ( HT) and 35 of whom had used HT (+HT) continuously since the onset of menopause. Blood flow of the inferotemporal retinal artery (ITRA), peripapillary retina, and ONH rim were measured in one eye. The ONH stereometric parameters and the pattern electroretinogram (PERG) were also measured. In ovariectomized rats, the retinal tissue perfusion was assessed using the BF tracer N-isopropyl-p-[(14)C]-iodoamphetamine ([(14)C]-IMP) in rats treated with either E(2) (n = 7) or placebo (n = 5)., Results: Compared with the HT group, the +HT group presented significantly greater BF of the ITRA (P = 0.006), greater rim volume for the entire ONH region (P = 0.032), and greater rim volume (P = 0.042), height variation contour (P = 0.011), mean thickness (P = 0.033), and cross-sectional area (P = 0.020) of the retinal nerve fiber layer for the inferotemporal region of the ONH when adjusted for age, ocular perfusion pressure, and age at menarche. In ovariectomized rats, E(2) treatment significantly increased retinal perfusion in a range of 22% to 45%., Conclusions: These findings indicate that estrogens and HT increase retinal blood flow and protect the retinal nerve fiber layer.

Quantitative and regional measurement of retinal blood flow in rodents is of prime interest for the investigation of regulatory mechanisms of ocular circulation in physiological and pathological conditions. In this study, a quantitative autoradiographic method using N-isopropyl-p-(14)C-iodoamphetamine ([(14)C]-IMP), a diffusible radioactive tracer, was evaluated for its ability to detect changes in retinal blood perfusion during hypercapnia. Findings were compared to cerebral blood flow values measured simultaneously. Hypercapnia was induced in awaken Wistar rats by inhalation of 5% or 8% CO(2) in medical air for 5 min. [(14)C]-IMP (100 microCi/kg) was injected in the femoral vein over a 30 s period and the rats were sacrificed 2 min later. Blood flow was calculated from whole-mount retinae and 20 microm thick brain sections in discrete regions of interest by quantitative autoradiography or from digested samples of retina and brain by liquid scintillation counting. Retinal blood flow values measured with quantitative and regional autoradiography were higher in the central (108 +/- 20 ml/100 g/min) than in peripheral (84 +/- 15 ml/100 g/min) retina. These values were within the same range as cortical blood flow values (97 +/- 4 ml/100 g/min). The retinal blood flow values obtained on whole-mount retinae were validated by the sampling method. Hypercapnia significantly increased overall blood flow in the retina (24-53%) with a maximal augmentation in the peripheral region and in the brain (22-142%). The changes were stronger in the brain compared to retina (p = 0.016). These results demonstrate that retinal blood flow can be quantified using [(14)C]-IMP and compared with cerebral blood flow. This technique is a powerful tool to study how retinal blood flow is regulated in different regions of the rat retina.

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), alpha7 (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 alpha7 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.

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 gamma-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.

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.