Localization of neurotransmitters and calcium binding proteins to neurons of salamander and mudpuppy retinas

We wished to identify the different types of retinal neurons on the basis of their content of neuroactive substances in both larval tiger salamander and mudpuppy retinas, favored species for electrophysiological investigation. Sections and wholemounts of retinas were labeled by immunocytochemical methods to demonstrate three calcium binding protein species and the common neurotransmitters, glycine, GABA and acetylcholine. Double immunostained sections and single labeled wholemount retinas were examined by confocal microscopy. Immunostaining patterns appeared to be the same in salamander and mudpuppy. Double and single cones, horizontal cells, some amacrine cells and ganglion cells were strongly calbindin-immunoreactive (IR). Calbindin-IR horizontal cells colocalized GABA. Many bipolar cells, horizontal cells, some amacrine cells and ganglion cells were strongly calretinin-IR. One type of horizontal cell and an infrequently occurring amacrine cell were parvalbumin-IR. Acetylcholine as visualized by ChAT-immunoreactivity was seen in a mirror-symmetric pair of amacrine cells that colocalized GABA and glycine. Glycine and GABA colocalized with calretinin, calbindin and occasionally with parvalbumin in amacrine cells. (C) 2001 Elsevier Science Ltd. All rights reserved.

GABAA receptor β3 subunit gene and psychiatric morbidity in a post-traumatic stress disorder population

GABAergic systems have been implicated in the pathogenesis of anxiety, depression and insomnia. These symptoms are part of the core and comorbid psychiatric disturbances in post-traumatic stress disorder (PTSD) In a sample of Caucasian male PTSD patients, dinucleotide repeat polymorphisms of the GABAA receptor beta3 subunit gene were compared to scores on the General Health Questionnaire-28 (GHQ). As the major allele at this gene locus (GABRB3) was GI, the alleles were divided into GI and non-GI groups. On the total score of the GHQ, which comprises the somatic symptoms, anxiety/insomnia, social dysfunction and depression subscales, patients with the GI non-GI genotype had a significantly higher score when compared to either the G1G1 genotype (alpha = 0.01) or the non-GI non-GI genotype (alpha = 0.05). No significant difference was found between the G1G1 and non-Gl non-G1 genotypes. When the GI non-G1 heterozygotes were compared to the combined G1G1 and non-GI non-GI homozygotes, a significantly higher total GHQ score was found in the heterozygotes (P = 0.002). These observations suggest a heterosis effect. Further analysis of GHQ subscale scores showed that heterozygotes compared to the combined homozygotes had higher scores on the somatic symptoms (P = 0.006), anxiety/insomnia (P = 0.003), social dysfunction (P = 0.054) and depression (P = 0.004) subscales. In conclusion, the present study indicates that in a population of PTSD patients, heterozygosity of the GABRB3 major (GI) allele confers higher levels of somatic symptoms, anxiety/insomnia, social dysfunction and depression than found in homozygosity. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

Sub-structure syntheses and relative stereochemistry in the bistramide (bistratene) series of marine metabolites

The (6R*,9S*,11S*) and (22S*,23R*,27R*,31R*) stereochemistry, respectively, of the tetrahydropyranyl and spiroacetal moieties in bistramide A (1) have been established by stereoselective syntheses and high field NMR comparisons. Routes to the gamma-amino acid moiety are outlined. (C) 2002 Elsevier Science Ltd. All rights reserved.

Light and electron microscopic analysis of aquaporin 1-like-immunoreactive amacrine cells in the rat retina

Aquaporin 1 (AQP1; also known as CHIP, a channel-forming integral membrane protein of 28 kDa) is the first protein to be shown to function as a water channel and has been recently shown to be present in the rat retina. We previously showed (Kim et al. [1998] Neurosci Lett 244:52-54) that AQP1-like immunoreactivity is present in a certain population of amacrine cells in the rat retina. This study was conducted to characterize these cells in more detail, With immunocytochemistry using specific antisera against AQP1, whole-mount preparations and 50-mum-thick vibratome sections were examined by light and electron microscopy. These cells were a class of amacrine cells, which had symmetric bistratified dendritic trees ramified in stratum 2 and in the border of strata 3 and 4 of the inner plexiform layer (IPL). Their dendritic field diameters ranged from 90 to 230 mum. Double labeling with antisera against AQP1 and gamma-aminobutyric acid or glycine demonstrated that these AQP1-like-immunoreactive amacrine cells were immunoreactive for glycine. Their most frequent synaptic input was from other amacrine cell processes in both sublaminae a and b of the IPL, followed by a few cone bipolar cells. Their primary targets were other amacrine cells and ganglion cells in both sublaminae a and b of the IPL. In addition, synaptic output Onto bipolar cells was rarely observed in sublamina b of the IPL. Thus, the AQP1 antibody labels a class of glycinergic amacrine cells with small to medium-sized dendritic fields in the rat retina. (C) 2002 Wiley-Liss, Inc.

Identification of cholinoceptive glycinergic neurons in the mammalian retina

The light-evoked release of acetylcholine (ACh) affects the responses of many retinal ganglion cells, in part via nicotinic acetylcholine receptors (nAChRs). nAChRs that contain beta2alpha3 neuronal nicotinic acetylcholine receptors have been identified and localized in the rabbit retina; these nAChRs are recognized by the monoclonal antibody mAb210. We have examined the expression of beta2alpha3 nAChRs by glycinergic amacrine cells in the rabbit retina and have identified different subpopulations of nicotinic cholinoceptive glycinergic cells using double and triple immunohistochemistry with quantitative analysis. Here we demonstrate that about 70% of the cholinoceptive amacrine cells in rabbit retina are glycinergic cells. At least three nonoverlapping subpopulations of mAb210 glycine-immunoreactive cells can be distinguished with antibodies against calretinin, calbindin, and gamma-aminobutyric acid (GABA)(A) receptors. The cholinergic cells in rabbit retina are thought to synapse only on other cholinergic cells and ganglion cells. Thus, the expression of beta2alpha3 nAChRs on diverse populations of glycinergic cells is puzzling. To explore this finding, the subcellular localization of beta2alpha3 was studied at the electron microscopic level. mAb210 immunoreactivity was localized on the dendrites of amacrines and ganglion cells throughout the inner plexiform layer, and much of the labeling was not associated with recognizable synapses. Thus, our findings indicate that ACh in the mammalian retina may modulate glycinergic circuits via extrasynaptic beta2alpha3 nAChRs. (C) 2002 Wiley-Liss, Inc.

Glutathione deficit during development induces anomalies in the rat anterior cingulate GABAergic neurons: Relevance to schizophrenia.

A series of studies in schizophrenic patients report a decrease of glutathione (GSH) in prefrontal cortex (PFC) and cerebrospinal fluid, a decrease in mRNA levels for two GSH synthesizing enzymes and a deficit in parvalbumin (PV) expression in a subclass of GABA neurons in PFC. GSH is an important redox regulator, and its deficit could be responsible for cortical anomalies, particularly in regions rich in dopamine innervation. We tested in an animal model if redox imbalance (GSH deficit and excess extracellular dopamine) during postnatal development would affect PV-expressing neurons. Three populations of interneurons immunolabeled for calcium-binding proteins were analyzed quantitatively in 16-day-old rat brain sections. Treated rats showed specific reduction in parvalbumin immunoreactivity in the anterior cingulate cortex, but not for calbindin and calretinin. These results provide experimental evidence for the critical role of redox regulation in cortical development and validate this animal model used in schizophrenia research.

Muscimol-induced death of GABAergic neurons in rat brain aggregating cell cultures.

During brain development, spontaneous neuronal activity has been shown to play a crucial role in the maturation of neuronal circuitries. Activity-related signals may cause selective neuronal cell death and/or rearrangement of neuronal connectivity. To study the effects of sustained inhibitory activity on developing inhibitory (GABAergic) neurons, three-dimensional primary cell cultures of fetal rat telencephalon were used. In relatively immature cultures, muscimol (10 microns), a GABAA receptor agonist, induced a transient increase in apoptotic cell death, as evidenced by a cycloheximide-sensitive increase of free nucleosomes and an increased frequency of DNA double strand breaks (TUNEL labeling). Furthermore, muscimol caused an irreversible reduction of glutamic acid decarboxylase activity, indicating a loss of GABAergic neurons. The muscimol-induced death of GABAergic neurons was attenuated by the GABAA receptor blockers bicuculline (100 microns) and picrotoxin (100 microns), by depolarizing potassium concentrations (30 mM KCl) and by the L-type calcium channel activator BAY K8644 (2 microns). As compared to the cholinergic marker (choline acetyltransferase activity), glutamic acid decarboxylase activity was significantly more affected by various agents known to inhibit neuronal activity, including tetrodotoxin (1 micron), flunarizine (5 microns), MK 801 (50 microns) and propofol (40 microns). The present results suggest that the survival of a subpopulation of immature GABAergic neurons is dependent on sustained neuronal activity and that these neurons may undergo apoptotic cell death in response to GABAA autoreceptor activation.

Syndrome des jambes sans repos chez la personne âgée: une affection méconnue [Restless legs syndrome in the elderly: an unrecognized disorder].

The restless legs syndrome (RLS) is a frequent, often unrecognized disorder in the elderly. The diagnosis is essentially based on the clinical history. The RLS is characterized by (1) an urge to move the limbs, usually associated with abnormal sensations in the legs; (2) symptoms are worse at rest; (3) they are relieved by movements; (4) they mainly occur in the evening or at night. Specific diagnostic criteria have been developed for cognitively impaired elderly persons. The RLS is a chronic disorder with high impact on sleep and quality of life. Treatment is symptomatic and recommended drugs are dopaminergic agents, opioids, and gabapentine.

Consequences of sleep deprivation on neurotransmitter receptor expression and function.

Several pieces of evidence suggest that sleep deprivation causes marked alterations in neurotransmitter receptor function in diverse neuronal cell types. To date, this has been studied mainly in wake- and sleep-promoting areas of the brain and in the hippocampus, which is implicated in learning and memory. This article reviews findings linking sleep deprivation to modifications in neurotransmitter receptor function, including changes in receptor subunit expression, ligand affinity and signal transduction mechanisms. We focus on studies using sleep deprivation procedures that control for side-effects such as stress. We classify the changes with respect to their functional consequences on the activity of wake-promoting and/or sleep-promoting systems. We suggest that elucidation of how sleep deprivation affects neurotransmitter receptor function will provide functional insight into the detrimental effects of sleep loss.

An international, randomized, double-blind, placebo-controlled, phase III trial of pregabalin monotherapy in treatment of patients with fibromyalgia.

OBJECTIVE: To evaluate the efficacy and safety of pregabalin monotherapy versus placebo for symptomatic pain relief and improvement of patient global assessment in patients with fibromyalgia (FM) enrolled from countries outside the United States. METHODS: This international, multicenter, double-blind, placebo-controlled trial randomly assigned 747 patients with FM to placebo or 300, 450, or 600 mg/day pregabalin twice daily for 14 weeks. Primary efficacy measures were endpoint mean pain scores and Patient Global Impression of Change (PGIC). Secondary outcomes included assessments of sleep and function. RESULTS: Patients in the 450 mg/day pregabalin group showed significant improvements versus placebo in endpoint mean pain score (-0.56; p = 0.0132), PGIC (73% improved vs 56% placebo; p = 0.0017), and function [Fibromyalgia Impact Questionnaire (FIQ) total score -5.85; p = 0.0012]. PGIC was also significant for 600 mg/day pregabalin (69% improved; p = 0.0227). Results for these endpoints were nonsignificant for pregabalin at 300 mg/day and for pain and FIQ score at 600 mg/day. Early onset of pain relief was seen, with separation from placebo detected by Week 1 in all pregabalin groups. All pregabalin doses demonstrated superiority to placebo on the Medical Outcomes Study-Sleep Scale Sleep Disturbance subscale and the Sleep Quality diary. Dizziness and somnolence were the most frequently reported adverse events. CONCLUSION: Pregabalin demonstrated modest efficacy in pain, global assessment, and function in FM at 450 mg/day, and improved sleep across all dose levels, but it did not provide consistent evidence of benefit at 300 and 600 mg/day in this study. Pregabalin was generally well tolerated for the treatment of FM. (Clinical trial registry NCT00333866).

Phasic, nonsynaptic GABA-A receptor-mediated inhibition entrains thalamocortical oscillations.

GABA-A receptors (GABA-ARs) are typically expressed at synaptic or nonsynaptic sites mediating phasic and tonic inhibition, respectively. These two forms of inhibition conjointly control various network oscillations. To disentangle their roles in thalamocortical rhythms, we focally deleted synaptic, γ2 subunit-containing GABA-ARs in the thalamus using viral intervention in mice. After successful removal of γ2 subunit clusters, spontaneous and evoked GABAergic synaptic currents disappeared in thalamocortical cells when the presynaptic, reticular thalamic (nRT) neurons fired in tonic mode. However, when nRT cells fired in burst mode, slow phasic GABA-AR-mediated events persisted, indicating a dynamic, burst-specific recruitment of nonsynaptic GABA-ARs. In vivo, removal of synaptic GABA-ARs reduced the firing of individual thalamocortical cells but did not abolish slow oscillations or sleep spindles. We conclude that nonsynaptic GABA-ARs are recruited in a phasic manner specifically during burst firing of nRT cells and provide sufficient GABA-AR activation to control major thalamocortical oscillations.

Effectiveness of Pregabalin as Monotherapy or Combination Therapy for Neuropathic Pain in Patients Unresponsive to Previous Treatments in a Spanish Primary Care Setting

BACKGROUND AND OBJECTIVE Patients from a previous study of neuropathic pain (NP) in the Spanish primary care setting still had symptoms despite treatment. Subsequently, patients were treated as prescribed by their physician and followed up for 3 months. Since pregabalin has been shown to be effective in NP, including refractory cases, the objective of this study was to assess the effectiveness of pregabalin therapy in patients with NP refractory to previous treatments. METHODS This was a post hoc analysis of pregabalin-naïve NP patients treated with pregabalin in a 3-month follow-up observational multicenter study to assess symptoms and satisfaction with treatment. Patients were evaluated with the Douleur Neuropathique en 4 questions (DN4), the Brief Pain Inventory (BPI) and the Treatment Satisfaction for Medication Questionnaire (SATMED-Q) overall satisfaction domain. RESULTS 1,670 patients (mean age 58 years, 59 % women), previously untreated or treated with ≥1 drug other than pregabalin, were treated with pregabalin (37 % on monotherapy). At 3 months, pain intensity and its interference with activities decreased by half (p < 0.0001), while the number of days with no or mild pain increased by a mean of 4.5 days (p < 0.0001). Treatment satisfaction increased twofold (p < 0.0001). Patients with a shorter history of pain and those with neuralgia and peripheral nerve compression syndrome (PCS) as etiologies had the highest proportion on monotherapy and showed the greatest improvements in pain-related parameters in their respective group categories. CONCLUSION Treatment with pregabalin (as monotherapy or combination therapy) provides benefits in pain and treatment satisfaction in patients with NP, including refractory cases. Shorter disease progression and neuralgia and PCS etiologies are favorable factors for pregabalin treatment response.

Que faire des nouveaux anti-épileptiques [Clinical utilization of new anti-epileptic agents].

Since the beginning of the 1990's, a dozen of new anti-epileptic drugs have been on the market or will be soon. This article reviews the daily clinical utilisation of new anti-epileptic drugs. It considers, without being complete, the current opinions and tendencies. The new anti-epileptic substances are generally as efficient as conventional medications. However, they are better tolerated and are more easily used in combination with conventional anti-epileptic drugs. Polytherapy is certainly the form of treatment, which is used in the most cases of resistant epilepsies. The surgical treatment can be used in only a very limited number of cases. The objective of treatment is the complete control of seizures, with minimum secondary effects. Though this objective is rarely reached, the NAE significantly improves the quality of life of patients suffering from severe epilepsy. The utilisation of NAE is not without risk. Increase in the frequency and severity of seizures may occur; we should remember that severe adverse effects appeared in the post-marketing period of the use of Vigabatrine and Felbamate. Therefore, we must remain vigilant in the clinical use of the anti-epileptic drugs.

Reduction of plasma alanine aminotransferase during vigabatrin treatment.

In 9 drug-resistant patients with partial seizures treated with vigabatrin, gamma-vinyl GABA (VGB), alanine aminotransaminase (ALAT) activity in plasma was significantly reduced. Comparison of in vitro with in vivo measurements led us to conclude that this reduction is mainly an in vivo phenomenon, perhaps due to cross-enzyme inhibition. The assessment of two biological variables linked with ALAT, glucose and alanine levels under fasting conditions, failed to show any significant metabolic alterations. VGB is an effective drug for partial epilepsy. Our observations do not suggest that reduced ALAT activity is of clinical concern.

Neurochemical profile of the mouse hypothalamus using in vivo 1H MRS at 14.1T.

The hypothalamus plays an essential role in the central nervous system of mammals by among others regulating glucose homeostasis, food intake, temperature, and to some extent blood pressure. Assessments of hypothalamic metabolism using, e.g. (1)H MRS in mouse models can provide important insights into its function. To date, direct in vivo (1)H MRS measurements of hypothalamus have not been reported. Here, we report that in vivo single voxel measurements of mouse hypothalamus are feasible using (1)H MRS at 14.1T. Localized (1)H MR spectra from hypothalamus were obtained unilaterally (2-2.2 microL, VOI) and bilaterally (4-4.4 microL) with a quality comparable to that of hippocampus (3-3.5 microL). Using LCModel, a neurochemical profile consisting of 21 metabolites was quantified for both hypothalamus and hippocampus with most of the Cramér-Rao lower bounds within 20%. Relative to the hippocampus, the hypothalamus was characterized by high gamma-aminobutryric acid and myo-inositol, and low taurine concentrations. When studying transgenic mice with no glucose transporter isoform 8 expressed, small metabolic changes were observed, yet glucose homeostasis was well maintained. We conclude that a specific neurochemical profile of mouse hypothalamus can be measured by (1)H MRS which will allow identifying and following metabolic alterations longitudinally in the hypothalamus of genetic modified models.