Sexually dimorphic role of circadian clock genes in alcohol drinking behavior.

Sex differences in alcohol use and abuse are pervasive and carry important implications for the prevention and treatment of alcohol use disorder (AUD), yet insight into underlying sexually dimorphic mechanisms is limited. Growing experimental and clinical evidence points to an important influence of circadian rhythms and circadian clock genes in the control of alcohol drinking behavior and AUD. Sex differences in the expression of circadian rhythms and in the molecular circadian clock that drive these rhythms have been reported in humans and animals. While studying the role of striatal circadian clock gene expression in the control of affective and goal-directed behaviors, we uncovered a novel sexually dimorphic function of the clock genes Bmal1 and Per2 in the control of voluntary alcohol consumption in mice, which may contribute to sex differences in alcohol drinking behavior. In this mini review, we briefly discuss relevant literature on AUD, circadian rhythms and clock genes, and on sex differences in these domains, and describe our own findings on clock genes as sexually dimorphic regulators of alcohol drinking behavior in mice.

In utero Exposure to Valproic-Acid Alters Circadian Organisation and Clock-Gene Expression: Implications for Autism Spectrum Disorders.

Autism Spectrum Disorder (ASD) is a pervasive neurodevelopmental disorder characterised by restrictive patterns of behaviour and alterations in social interaction and communication. Up to 80% of children with ASD exhibit sleep-wake cycle disturbances, emphasising the pressing need for novel approaches in the treatment of ASD-associated comorbidities. While sleep disturbances have been identified in ASD individuals, little has been done to assess the contribution of the circadian system to these findings. The objective of this study is to characterise circadian behaviour and clock-gene expression in a valproic acid (VPA)-induced animal model of autism to highlight perturbations potentially contributing to these disturbances. Male and female VPA-exposed offspring underwent circadian challenges, including baseline light-dark cycles, constant dark/light and light pulse protocols. Baseline analysis showed that VPA-exposed males, but not females, had a greater distribution of wheel-running behaviour across light-dark phases and a later activity offset (p < 0.0001), while controls showed greater activity confinement to the dark phase (p = 0.0256). Constant light analysis indicated an attenuated masking response and an increase in the number of days to reach arrhythmicity (p < 0.0001). A 1-h light pulse (150 lux) at CT 15 after 6 days of constant dark showed that both sexes exposed to VPA exhibited a lesser phase-shift when compared to controls (p = 0.0043). Immunohistochemical and western-blot assays reveal no alterations in retinal organisation or function. However, immunohistochemical assay of the SCN revealed altered expression of BMAL1 expression in VPA-exposed males (p = 0.0016), and in females (p = 0.0053). These findings suggest alterations within the core clockwork of the SCN and reduced photic-entrainment capacity, independent of retinal dysfunction. The results of this study shed light on the nature of circadian dysregulation in VPA-exposed animals and highlights the urgent need for novel perspectives in the treatment of ASD-associated comorbidities.

Bmal1 in the striatum influences alcohol intake in a sexually dimorphic manner.

Alcohol consumption has been strongly associated with circadian clock gene expression in mammals. Analysis of clock genes revealed a potential role of Bmal1 in the control of alcohol drinking behavior. However, a causal role of Bmal1 and neural pathways through which it may influence alcohol intake have not yet been established. Here we show that selective ablation of Bmal1 (Cre/loxP system) from medium spiny neurons of the striatum induces sexual dimorphic alterations in alcohol consumption in mice, resulting in augmentation of voluntary alcohol intake in males and repression of intake in females. Per2mRNA expression, quantified by qPCR, decreases in the striatum after the deletion of Bmal1. To address the possibility that the effect of striatal Bmal1 deletion on alcohol intake and preference involves changes in the local expression of Per2, voluntary alcohol intake (two-bottle, free-choice paradigm) was studied in mice with a selective ablation of Per2 from medium spiny neurons of the striatum. Striatal ablation of Per2 increases voluntary alcohol intake in males but has no effect in females. Striatal Bmal1 and Per2 expression thus may contribute to the propensity to consume alcohol in a sex -specific manner in mice.

The eIF2α Kinase GCN2 Modulates Period and Rhythmicity of the Circadian Clock by Translational Control of Atf4.

The integrated stress response (ISR) is activated in response to diverse stress stimuli to maintain homeostasis in neurons. Central to this process is the phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α). Here, we report a critical role for ISR in regulating the mammalian circadian clock. The eIF2α kinase GCN2 rhythmically phosphorylates eIF2α in the suprachiasmatic circadian clock. Increased eIF2α phosphorylation shortens the circadian period in both fibroblasts and mice, whereas reduced eIF2α phosphorylation lengthens the circadian period and impairs circadian rhythmicity in animals. Mechanistically, phosphorylation of eIF2α promotes mRNA translation of Atf4. ATF4 binding motifs are identified in multiple clock genes, including Per2, Per3, Cry1, Cry2, and Clock. ATF4 binds to the TTGCAGCA motif in the Per2 promoter and activates its transcription. Together, these results demonstrate a significant role for ISR in circadian physiology and provide a potential link between dysregulated ISR and circadian dysfunction in brain diseases.

mTOR signaling in VIP neurons regulates circadian clock synchrony and olfaction.

Mammalian/mechanistic target of rapamycin (mTOR) signaling controls cell growth, proliferation, and metabolism in dividing cells. Less is known regarding its function in postmitotic neurons in the adult brain. Here we created a conditional mTOR knockout mouse model to address this question. Using the Cre-LoxP system, the mTOR gene was specifically knocked out in cells expressing Vip (vasoactive intestinal peptide), which represent a major population of interneurons widely distributed in the neocortex, suprachiasmatic nucleus (SCN), olfactory bulb (OB), and other brain regions. Using a combination of biochemical, behavioral, and imaging approaches, we found that mice lacking mTOR in VIP neurons displayed erratic circadian behavior and weakened synchronization among cells in the SCN, the master circadian pacemaker in mammals. Furthermore, we have discovered a critical role for mTOR signaling in mediating olfaction. Odor stimulated mTOR activation in the OB, anterior olfactory nucleus, as well as piriform cortex. Odor-evoked c-Fos responses along the olfactory pathway were abolished in mice lacking mTOR in VIP neurons, which is consistent with reduced olfactory sensitivity in these animals. Together, these results demonstrate that mTOR is a key regulator of SCN circadian clock synchrony and olfaction.

Mapping the co-localization of the circadian proteins PER2 and BMAL1 with enkephalin and substance P throughout the rodent forebrain.

Despite rhythmic expression of clock genes being found throughout the central nervous system, very little is known about their function outside of the suprachiasmatic nucleus. Determining the pattern of clock gene expression across neuronal subpopulations is a key step in understanding their regulation and how they may influence the functions of various brain structures. Using immunofluorescence and confocal microscopy, we quantified the co-expression of the clock proteins BMAL1 and PER2 with two neuropeptides, Substance P (SubP) and Enkephalin (Enk), expressed in distinct neuronal populations throughout the forebrain. Regions examined included the limbic forebrain (dorsal striatum, nucleus accumbens, amygdala, stria terminalis), thalamus medial habenula of the thalamus, paraventricular nucleus and arcuate nucleus of the hypothalamus and the olfactory bulb. In most regions examined, BMAL1 was homogeneously expressed in nearly all neurons (~90%), and PER2 was expressed in a slightly lower proportion of cells. There was no specific correlation to SubP- or Enk- expressing subpopulations. The olfactory bulb was unique in that PER2 and BMAL1 were expressed in a much smaller percentage of cells, and Enk was rarely found in the same cells that expressed the clock proteins (SubP was undetectable). These results indicate that clock genes are not unique to specific cell types, and further studies will be required to determine the factors that contribute to the regulation of clock gene expression throughout the brain.

Exploring the role of locomotor sensitization in the circadian food entrainment pathway.

Food entrainment is the internal mechanism whereby the phase and period of circadian clock genes comes under the control of daily scheduled food availability. Food entrainment allows the body to efficiently realign the internal timing of behavioral and physiological functions such that they anticipate food intake. Food entrainment can occur with or without caloric restriction, as seen with daily schedules of restricted feeding (RF) or restricted treat (RT) that restrict food or treat intake to a single feeding time. However, the extent of clock gene control is more pronounced with caloric restriction, highlighting the role of energy balance in regulating clock genes. Recent studies have implicated dopamine (DA) to be involved in food entrainment and caloric restriction is known to affect dopaminergic pathways to enhance locomotor activity. Since food entrainment results in the development of a distinct behavioral component, called food anticipatory activity (FAA), we examined the role of locomotor sensitization (LS) in food entrainment by 1) observing whether amphetamine (AMPH) sensitization results in enhanced locomotor output of FAA and 2) measuring LS of circadian and non-circadian feeding paradigms to an acute injection of AMPH (AMPH cross-sensitization). Unexpectedly, AMPH sensitization did not show enhancement of FAA. On the contrary, LS did develop with sufficient exposure to RF. LS was present after 2 weeks of RF, but not after 1, 3 or 7 days into RF. When food was returned and rats regain their original body weight at 10-15 days post-RF, LS remained present. LS did not develop to RT, nor to feedings of a non-circadian schedule, e.g. variable restricted feeding (VRF) or variable RT (VRT). Further, when RF was timed to the dark period, LS was observed only when tested at night; RF timed to the light period resulted in LS that was present during day and night. Taken together our results show that LS develops with food entrainment to RF, an effect that is dependent on the chronicity and circadian phase of RF but independent of body weight. Given that LS involves reorganization of DA-regulated motor circuitry, our work provides indirect support for the role of DA in the food entrainment pathway of RF. The findings also suggest differences in neuronal pathways involved in LS from AMPH sensitization and LS from RF.

Light-regulated translational control of circadian behavior by eIF4E phosphorylation.

The circadian (∼24 h) clock is continuously entrained (reset) by ambient light so that endogenous rhythms are synchronized with daily changes in the environment. Light-induced gene expression is thought to be the molecular mechanism underlying clock entrainment. mRNA translation is a key step of gene expression, but the manner in which clock entrainment is controlled at the level of mRNA translation is not well understood. We found that a light- and circadian clock-regulated MAPK/MNK pathway led to phosphorylation of the cap-binding protein eIF4E in the mouse suprachiasmatic nucleus of the hypothalamus, the locus of the master circadian clock in mammals. Phosphorylation of eIF4E specifically promoted translation of Period 1 (Per1) and Period 2 (Per2) mRNAs and increased the abundance of basal and inducible PER proteins, which facilitated circadian clock resetting and precise timekeeping. Together, these results highlight a critical role for light-regulated translational control in the physiology of the circadian clock.

Effect of experimental diabetic retinopathy on the non-image-forming visual system.

Diabetic retinopathy is a leading cause of blindness. Intrinsically photosensitive retinal ganglion cells (ipRGCs), which express the photopigment melanopsin, are involved in non-image-forming visual responses such as photoentrainment of circadian rhythms and pupillary light reflex. Since several reports indicate that retinal ganglion cells are affected by diabetes, we investigated the non-image-forming visual system in an advanced stage of experimental diabetes in rats induced by streptozotocin. After 15 wks of diabetes induction, clear alterations in the visual function were observed and all animals developed mature cataracts. At this time point, concomitantly with a significant decrease in the number of Brn3a(+) retinal ganglion cells, no differences in the number of melanopsin-containing cells, melanopsin levels, and retinal projections to the suprachiasmatic nuclei and the olivary pretectal nucleus were observed. At high light intensity, afferent pupil light reflex appears to be conserved in diabetic animals. After 15 wks of diabetes induction, a significant decrease in light-induced c-Fos expression in the suprachiasmatic nuclei was found. In diabetic animals, the locomotor activity pattern was conserved, although a delay in the time needed for re-entrainment after a phase delay was observed. In diabetic animals, lensectomy reversed the alterations in c-Fos expression and in the locomotor activity rhythm. These results suggest that the neuronal substrate of the non-image-forming visual system remained largely unaffected at advanced stages of diabetes, and that lensectomy, a relatively easy and safe surgery, could partially restore circadian alterations induced by diabetes.

Alterations of locomotor activity rhythm and sleep parameters in patients with advanced glaucoma.

The aim of this study was to evaluate the effect of advanced glaucoma on locomotor activity rhythms and related sleep parameters. Nine normal subjects and nine age-matched patients with bilateral advanced primary open-angle glaucoma, >10 yrs since diagnosis, were included in this observational, prospective, case-control study. Patients were required to record the timing and duration of their sleep and daily activities, and wore an actigraph on the wrist of the nondominant arm for 20 d. Activity rhythm period, MESOR (24-h time-series mean), amplitude (one-half peak-to-trough variation), and acrophase (peak time), plus long sleep episodes during the wake state, sleep duration, efficiency, and latency, as well as mean activity score, wake minutes, and mean wake episodes during the sleep interval were assessed in controls and glaucomatous patients. Glaucomatous patients exhibited significant decrease in nighttime sleep efficiency, and significant increase in the mean activity score, wake minutes, and mean wake episode during the night. These results suggest that alterations of circadian physiology could be a risk to the quality of life of patients with glaucoma.

Retinal changes in an experimental model of early type 2 diabetes in rats characterized by non-fasting hyperglycemia.

Diabetic retinopathy is a leading cause of acquired blindness in young, but also in elder adults, mostly affected by type 2 diabetes mellitus (T2DM). The aim of this work was to develop an experimental model of early human T2DM in adult rats, and to analyze retinal functional, morphological, and biochemical changes arising during the early stages of the moderate metabolic derangement. For this purpose, animals were divided in four groups: adult male Wistar rats receiving: tap water and citrate buffer i.p. (group 1), tap water with 30% sucrose and citrate buffer i.p. (group 2), tap water and 25mg/kg i.p streptozotocin (STZ, group 3), or 30% sucrose and STZ (group 4). Fasting and postprandial glycemia, fructosamine and serum insulin levels were assessed. In addition, i.p. glucose and insulin tolerance tests were performed. Retinal function (electroretinogram, ERG) and morphology (optical microscopy), retinal nitric oxide synthase (NOS) activity (using (3)H-arginine), lipid peroxidation (thiobarbituric acid reactive substances, TBARS), and TNFα levels (ELISA) were evaluated. At 6 and 12 weeks of treatment, animals which received a sucrose-enriched diet and STZ showed significant differences in most metabolic tests, as compared with the other groups. At 12 weeks of treatment, a significant decrease in the ERG a- and b- wave and oscillatory potential amplitudes, and a significant increase in retinal NOS activity, TBARS, TNFα, glial fibrillary acidic protein in Müller cells, and vascular endothelial growth factor levels were observed. These results indicate that the combination of diet-induced insulin resistance and a slight secretory impairment resulting from a low-dose STZ treatment mimics some features of human T2DM at its initial stages, and provokes significant retinal alterations.

Therapeutic benefit of radial optic neurotomy in a rat model of glaucoma.

Radial optic neurotomy (RON) has been proposed as a surgical treatment to alleviate the neurovascular compression and to improve the venous outflow in patients with central retinal vein occlusion. Glaucoma is characterized by specific visual field defects due to the loss of retinal ganglion cells and damage to the optic nerve head (ONH). One of the clinical hallmarks of glaucomatous neuropathy is the excavation of the ONH. The aim of this work was to analyze the effect of RON in an experimental model of glaucoma in rats induced by intracameral injections of chondroitin sulfate (CS). For this purpose, Wistar rats were bilaterally injected with vehicle or CS in the eye anterior chamber, once a week, for 10 weeks. At 3 or 6 weeks of a treatment with vehicle or CS, RON was performed by a single incision in the edge of the neuro-retinal ring at the nasal hemisphere of the optic disk in one eye, while the contralateral eye was submitted to a sham procedure. Electroretinograms (ERGs) were registered under scotopic conditions and visual evoked potentials (VEPs) were registered with skull-implanted electrodes. Retinal and optic nerve morphology was examined by optical microscopy. RON did not affect the ocular hypertension induced by CS. In eyes injected with CS, a significant decrease of retinal (ERG a- and b-wave amplitude) and visual pathway (VEP N2-P2 component amplitude) function was observed, whereas RON reduced these functional alterations in hypertensive eyes. Moreover, a significant loss of cells in the ganglion cell layer, and Thy-1-, NeuN- and Brn3a- positive cells was observed in eyes injected with CS, whereas RON significantly preserved these parameters. In addition, RON preserved the optic nerve structure in eyes with chronic ocular hypertension. These results indicate that RON reduces functional and histological alterations induced by experimental chronic ocular hypertension.

Ischemic tolerance protects the rat retina from glaucomatous damage.

Glaucoma is a leading cause of acquired blindness which may involve an ischemic-like insult to retinal ganglion cells and optic nerve head. We investigated the effect of a weekly application of brief ischemia pulses (ischemic conditioning) on the rat retinal damage induced by experimental glaucoma. Glaucoma was induced by weekly injections of chondroitin sulfate (CS) in the rat eye anterior chamber. Retinal ischemia was induced by increasing intraocular pressure to 120 mmHg for 5 min; this maneuver started after 6 weekly injections of vehicle or CS and was weekly repeated in one eye, while the contralateral eye was submitted to a sham procedure. Glaucoma was evaluated in terms of: i) intraocular pressure (IOP), ii) retinal function (electroretinogram (ERG)), iii) visual pathway function (visual evoked potentials, (VEPs)) iv) histology of the retina and optic nerve head. Retinal thiobarbituric acid substances levels were assessed as an index of lipid peroxidation. Ischemic conditioning significantly preserved ERG, VEPs, as well as retinal and optic nerve head structure from glaucomatous damage, without changes in IOP. Moreover, ischemia pulses abrogated the increase in lipid peroxidation induced by experimental glaucoma. These results indicate that induction of ischemic tolerance could constitute a fertile avenue for the development of new therapeutic strategies in glaucoma treatment.

Effect of experimental glaucoma on the non-image forming visual system.

Glaucoma is a leading cause of blindness worldwide, characterized by retinal ganglion cell degeneration and damage to the optic nerve. We investigated the non-image forming visual system in an experimental model of glaucoma in rats induced by weekly injections of chondroitin sulphate (CS) in the eye anterior chamber. Animals were unilaterally or bilaterally injected with CS or vehicle for 6 or 10 weeks. In the retinas from eyes injected with CS, a similar decrease in melanopsin and Thy-1 levels was observed. CS injections induced a similar decrease in the number of melanopsin-containing cells and superior collicular retinal ganglion cells. Experimental glaucoma induced a significant decrease in the afferent pupil light reflex. White light significantly decreased nocturnal pineal melatonin content in control and glaucomatous animals, whereas blue light decreased this parameter in vehicle- but not in CS-injected animals. A significant decrease in light-induced c-Fos expression in the suprachiasmatic nuclei was observed in glaucomatous animals. General rhythmicity and gross entrainment appear to be conserved, but glaucomatous animals exhibited a delayed phase angle with respect to lights off and a significant increase in the percentage of diurnal activity. These results indicate the glaucoma induced significant alterations in the non-image forming visual system.

Inner retinal circadian clocks and non-visual photoreceptors: novel players in the circadian system.

Daily and annual changes in ambient illumination serve as specific stimuli that associate light with time and regulate the physiology of the organism through the eye. The eye acts as a dual sense organ linking light and vision, and detecting light that provides specific stimuli for non-classical photoreceptors located in the inner retina. These photoreceptors convey information to the master circadian pacemaker, the hypothalamic suprachiasmatic nuclei (SCN). Responsible for sensing the light that regulates several non-visual functions (i.e. behavior, pupil reflex, sleep, and pineal melatonin production), the retina plays a key role in the temporal symphony orchestra playing the musical score of life: it is intrinsically rhythmic in its physiological and metabolic activities. We discuss here recent evidence in support of the hypothesis that retinal oscillators distributed over different cell populations may act as clocks, inducing changes in the visual and circadian system according to the time of the day. Significant progress has recently been made in identifying photoreceptors/photopigments localized in retinal ganglion cells (RGCs) that set circadian rhythms and modulate non-visual functions. Autonomous retinal and brain oscillators could have a more complex organization than previously recognized, involving a network of "RGC clock/SCN clock cross-talk". The convergence of oscillatory and photoreceptive capacities of retinal cells could deeply impact on the circadian system, which in turn may be severely impaired in different retinal pathologies. The aim of this review is to discuss the state of the art on inner retinal cell involvement in the light and temporal regulation of health and disease.

Effect of chondroitin sulfate on intraocular pressure in rats.

PURPOSE: To study the effect of intracameral injections of chondroitin sulfate (CS) on intraocular pressure (IOP), retinal function, and histology in rats. METHODS: Acute or chronic injections of CS were performed unilaterally in the rat anterior chamber, whereas the contralateral eye was injected with vehicle. IOP was daily or weekly assessed by a tonometer. Retinal function was assessed by scotopic electroretinography (ERG) and the visual pathway by flash visual evoked potentials (VEPs), whereas the retinal and optic nerve head structure were examined by histologic analysis. RESULTS: A single injection of 8 mg (but not 2 or 4 mg) CS induced a significant increase of IOP. The increase of IOP induced by a single injection of 8 mg CS lasted for 7 days, whereas chronic (weekly) administration during 10 weeks induced a significant and sustained increase in IOP compared with eyes injected with vehicle. A significant decrease of scotopic ERG a- and b- wave amplitude was observed after 6 and 10 weeks of CS administration. Moreover, a significant decrease in scotopic flash VEP N2-P2 component amplitude was observed in eyes treated with CS for 6 and 10 weeks. A significant loss of ganglion cell layer cells and optic nerve axons was observed in eyes receiving CS for 10 weeks. CONCLUSIONS: These results suggest that exogenous CS simulates the accumulation of CS in primary open-angle glaucoma and that increased amounts of CS could play a key role in the IOP dysregulation characteristic of glaucoma.

Melatonin: a novel neuroprotectant for the treatment of glaucoma.

Glaucoma is a leading cause of blindness. Although ocular hypertension is the most important risk factor, several concomitant factors such as elevation of glutamate and decrease in gamma-aminobutyric acid (GABA) levels, disorganized NO metabolism, and oxidative damage could significantly contribute to the neurodegeneration. The aim of this report was to analyze the effect of melatonin on retinal glutamate clearance, GABA concentrations, NO synthesis, and retinal redox status, as well as on functional and histological alterations provoked by chronic ocular hypertension induced by intracameral injections of hyaluronic acid (HA) in the rat eye. In normal retinas, melatonin increased glutamate uptake, glutamine synthase activity, GABA turnover rate, glutamic acid decarboxylase activity, superoxide dismutase activity, and reduced glutathione (GSH) levels, whereas it decreased NOS activity, L-arginine uptake, and lipid peroxidation. To assess the effect of melatonin on glaucomatous neuropathy, weekly injections of HA were performed in the eye anterior chamber. A pellet of melatonin was implanted subcutaneously 24 hr before the first injection or after six weekly injections of HA. Melatonin, which did not affect intraocular pressure (IOP), prevented and reversed the effect of ocular hypertension on retinal function (assessed by electroretinography) and diminished the vulnerability of retinal ganglion cells to the deleterious effects of ocular hypertension. These results indicate that melatonin could be a promissory resource in the management of glaucoma.

Circadian variations of prostaglandin E2 and F2 alpha release in the golden hamster retina.

Circadian variations of prostaglandin E2 and F2alpha release were examined in the golden hamster retina. Both parameters showed significant diurnal variations with maximal values at midnight. When hamsters were placed under constant darkness for 48 h, the differences in prostaglandin release between subjective mid-day and subjective midnight persisted. Western blot analysis showed that cyclooxygenase (COX)-1 levels were significantly higher at midnight than at mid-day, and at subjective midnight than at subjective mid-day, whereas no changes in COX-2 levels were observed among these time points. Immunohistochemical studies indicated the presence of COX-1 and COX-2 in the inner (but not outer) retina. Circadian variations of retinal prostaglandin release were also assessed in suprachiasmatic nuclei (SCN)-lesioned animals. Significant differences in retinal prostaglandin release between subjective mid-day and subjective midnight were observed in SCN-lesioned animals. These results indicate that hamster retinal prostaglandin release is regulated by a retinal circadian clock independent from the SCN. Thus, the present results suggest that the prostaglandin/COX-1 system could be a retinal clock output or part of the retinal clock mechanism.

Effect of ocular hypertension on retinal GABAergic activity.

Glutamate and gamma-aminobutyric acid (GABA) are major excitatory and inhibitory retinal neurotransmitters. The balance between these signals is a key principle of organization at retinal level. Although glutamate-induced excitotoxicity could mediate retinal ganglion cell death in glaucoma, the GABAergic system was not previously examined in this disease. The aim of this work was to study the retinal GABAergic activity in eyes with ocular hypertension induced by hyaluronic acid (HA). For this purpose, weekly injections of HA were performed unilaterally in the rat anterior chamber, whereas the contralateral eye was injected with saline solution. At 3 weeks of treatment with HA, GABA turnover rate, glutamic acid decarboxylase activity, and both glutamate- and high K(+)-induced GABA release significantly decreased, whereas GABA uptake increased in HA-treated eyes. The binding of t-butylbicyclophosphorothionate (TBPS) to GABA(A)/benzodiazepine Cl(-) channels significantly increased in eyes injected with HA as compared with vehicle-injected eyes. Changes in GABA uptake and TBPS binding persisted at 6 weeks of treatment with HA. These results indicate a dysfunction of the retinal GABAergic activity in hypertensive eyes, which could suggest the involvement of GABA in glaucomatous neuropathy.

Effect of glaucoma on the retinal glutamate/glutamine cycle activity.

Glutamate-induced excitotoxicity has been proposed to mediate the death of retinal ganglion cells in glaucoma. The metabolic dependence of glutamatergic neurons upon glia via the glutamate/glutamine cycle to provide the precursor for neurotransmitter glutamate is well established. Thus, the aim of the present work was to study the retinal glutamate/glutamine activity in eyes with hypertension induced by intracameral injections of hyaluronic acid (HA). For this purpose, weekly injections of HA were performed unilaterally in the rat anterior chamber, whereas the contralateral eye was injected with saline solution. At 3 or 10 weeks of treatment, glutamate and glutamine uptake and release were assessed using [3H]-glutamate and [3H]-glutamine as radioligands, respectively. In addition, glutamine synthetase activity was assessed by a spectrophotometric assay, whereas glutaminase activity was measured through the conversion of [3H]-glutamine to [3H]-glutamate. At 3 weeks of treatment with HA, a significant decrease (P<0.01) in glutamate uptake and glutamine synthetase activity was observed. Glutamine uptake and release, as well as glutaminase activity, were significantly increased (P<0.01) in eyes injected with HA for 3 weeks compared with vehicle-injected eyes, whereas [3H]-glutamate release did not change in hypertensive eyes. Only the changes in glutamine synthetase activity persisted at 10 weeks of treatment with HA. These results indicate a significant alteration in the retinal glutamate/glutamine cycle activity in hypertensive eyes. Since these changes preceded both functional and histological alterations induced by ocular hypertension, these results support the involvement of glutamate in glaucomatous neuropathy.