Subtype-selective GABAergic drugs facilitate extinction of mouse operant behaviour

Several recent studies have shown that reducing gamma-aminobutyric acid (GABA)-mediated neurotransmission retards extinction of aversive conditioning. However, relatively little is known about the effect of GABA on extinction of appetitively motivated tasks. We examined the effect of chlordiazepoxide (CDP), a classical benzodiazepine (BZ) and two novel subtype-selective BZs when administered to male C57Bl/6 mice during extinction following training on a discrete-trial fixed-ratio 5 (FR5) food reinforced lever-press procedure. Initially CDP had no effect, but after several extinction sessions CDP significantly facilitated extinction, i.e. slowed responding, compared with vehicle-treated mice. This effect was not due to drug accumulation because mice switched from vehicle treatment to CDP late in extinction showed facilitation immediately. Likewise, this effect could not be attributed to sedation because the dose of CDP used (15 mg/kg i.p.) did not suppress locomotor activity. The two novel subtype-selective BZ partial agonists, L-838417 and TP13, selectively facilitated extinction in similar fashion to CDP. The non-GABAergic anxiolytic buspirone was also tested and found to have similar effects when administered at a non-sedating dose. These studies demonstrate that GABA-mediated processes are important during extinction of an appetitively motivated task, but only after the animals have experienced several extinction sessions.

Fat composition of organic and conventional retail milk in northeast England

This study of UK retail milk identified highly significant variations in fat composition. The survey, conducted over 2 yr replicating summer and winter, sampled 22 brands, 10 of which indicated organic production systems. Results corroborate earlier farm-based findings considering fat composition of milk produced under conventional and organic management. Organic milk had higher concentrations of beneficial fatty acids (FA) than conventional milk, including total polyunsaturated fatty acids (PUFA; 39.4 vs. 31.8 g/kg of total FA), conjugated linoleic acid cis-9,trans-11 (CLA9; 7.4 v 5.6 g/kg of FA), and α-linolenic acid (α-LN; 6.9 vs. 4.4 g/kg of FA). As expected, purchase season had a strong effect on fat composition: compared with milk purchased in winter, summer milk had a lower concentration of saturated fatty acids (682 vs. 725 g/kg of FA) and higher concentrations of PUFA (37.6 vs. 32.8 g/kg of FA), CLA9 (8.1 vs. 4.7 g/kg of FA), and α-LN (6.5 vs. 4.6 g/kg of FA). Differences identified between sampling years were more surprising: compared with that in yr 2, milk purchased in year 1 had higher concentrations of PUFA (37.5 vs. 32.9 g/kg of FA), α-LN (6.0 vs. 5.1 g/kg of FA), and linoleic acid (19.9 vs. 17.5 g/kg of FA) and lower concentrations of C16:0 and C14:0 (332 vs. 357 and 110 vs. 118 g/kg of FA, respectively). Strong interactions were identified between management and season as well as between season and year of the study. As in the earlier farm studies, differences in fat composition between systems were greater for summer compared with winter milk. Large between-year differences may be due to changes in weather influencing milk composition through forage availability, quality, and intake. If climate change predictions materialize, both forage and dairy management may have to adapt to maintain current milk quality. Considerable variation existed in milk fat composition between brands.

The effects of dairy management and processing on quality characteristics of milk and dairy products

Studies within the QLIF project reviewed in this article suggest that organic or low-input management is more likely to result in milk with fatty acid profiles that are higher in α-linolenic acid and/or beneficial isomers of conjugated linoleic acid and antioxidants with up to a 2.5-fold increase in some cases, relative to milk from conventional production. These advantages are preserved during processing, resulting in elevated contents or concentrations of these constituents in processed dairy products of organic or low input origin. Much of the literature suggests that these benefits are very likely to be a result of a greater reliance on forages in the dairy diets (especially grazed grass). Since the adoption of alternative breeds or crosses is often an integral part sustaining these low-input systems, it is not possible to rule out an interaction with genotype in these monitored herds. The results suggest that milk fat composition with respect to human health can be optimized by exploiting grazing in the diet of dairy cows. However, in many European regions this may not be possible due to extremes in temperature, soil moisture levels or both. In such cases milk quality can be maintained by the inclusion of oil seeds in the dairy diets.

Higher PUFA and omega-3 PUFA, CLA, α-tocopherol and iron, but lower iodine and selenium concentrations in organic milk: a systematic literature review and meta- and redundancy analyses

Demand for organic milk is partially driven by consumer perceptions that it is more nutritious. However, there is still considerable uncertainty over whether the use of organic production standards affects milk quality. Here we report results of meta-analyses based on 170 published studies comparing the nutrient content of organic and conventional bovine milk. There were no significant differences in total SFA and MUFA concentrations between organic and conventional milk. However, concentrations of total PUFA and n-3 PUFA were significantly higher in organic milk, by an estimated 7 (95 % CI −1, 15) % and 56 (95 % CI 38, 74) %, respectively. Concentrations of α-linolenic acid (ALA), very long-chain n-3 fatty acids (EPA+DPA+DHA) and conjugated linoleic acid were also significantly higher in organic milk, by an 69 (95 % CI 53, 84) %, 57 (95 % CI 27, 87) % and 41 (95 % CI 14, 68) %, respectively. As there were no significant differences in total n-6 PUFA and linoleic acid (LA) concentrations, the n-6:n-3 and LA:ALA ratios were lower in organic milk, by an estimated 71 (95 % CI −122, −20) % and 93 (95 % CI −116, −70) %. It is concluded that organic bovine milk has a more desirable fatty acid composition than conventional milk. Meta-analyses also showed that organic milk has significantly higher α-tocopherol and Fe, but lower I and Se concentrations. Redundancy analysis of data from a large cross-European milk quality survey indicates that the higher grazing/conserved forage intakes in organic systems were the main reason for milk composition differences.

Melanin-concentrating hormone projections to areas involved in somatomotor responses

The lateral hypothalamic area (LHA) participates in the integration of sensory information and somatomotor responses associated with hunger and thirst. Although the LHA is neurochemically heterogeneous, a particularly high number of cells express melanin-concentrating hormone (MCH), which has been reported to play a role in energy homeostasis. Treatment with MCH increases food intake, and MCH mRNA is overexpressed in leptin-deficient (ob/ob) mice. Mice lacking both MCH and leptin present reduced body fat, mainly due to increased resting energy expenditure and locomotor activity. Dense MCH innervation of the cerebral motor cortex (MCx) and the pedunculopontine tegmental nucleus (PPT), both related to motor function, has been reported. Therefore, we postulated that a specific group of MCH neurons project to these areas. To investigate our hypothesis, we injected retrograde tracers into the MCx and the PPT of rats, combined with immunohistochemistry. We found that 25% of the LHA neurons projecting to the PPT were immunoreactive for MCH, and that 75% of the LHA neurons projecting to the MCx also contained MCH. Few MCH neurons were found to send collaterals to both areas. We also found that 15% of the incerto-hypothalamic neurons projecting to the PPT expressed MCH immunoreactivity. Those neurons preferentially innervated the rostral PPT. In addition, we observed that the MCH neurons express glutamic acid decarboxylase mRNA, a gamma-aminobutyric acid (GABA) synthesizing enzyme. We postulate that MCH/GABA neurons are involved in the inhibitory modulation of the innervated areas, decreasing motor activity in states of negative energy balance. (C) 2007 Published by Elsevier B.V.

Important GABAergic mechanism within the NTS and the control of sympathetic baroreflex in SHR

Inhibitory neurotransmission has an important role in the processing of sensory afferent signals in the nucleus of the solitary tract (NTS), particularly in spontaneously hypertensive rats (SHR). In the present study, we tested the hypothesis that gamma-aminobutyric acid (GABA) mediated neurotransmission within the NTS produces an inhibition of the baroreflex response of splanchnic sympathetic nerve discharge (sSND). In urethane-anesthetized, artificially ventilated and vagotomized male SHR and Wistar Kyoto (WKY) rats we compared baroreflex-response curves evoked after bilateral injections into the NTS of the GABA-A antagonist bicuculline (25 pmol/50 nl) or the GABA-B antagonist CGP 35348 (5 nmol/50 nl). Baseline MAP in SHR was higher than the WKY rats (SHR: 153+/-5, vs. WKY: 112+/-6 mm Hg, p<0.05). Bilateral injection of bicuculline or CGP 35348 into the NTS induced a transient (5 min) reduction in MAP (Delta = -26+/-4 and -41+/-6 mm Hg, respectively vs. saline Delta = +4+/-3 mm Hg, p<0.05) and sSND (Delta = -21+/-13 and -78+/-7%, respectively vs. saline: Delta = +6+/-4% p<0.05). Analysis of the baroreceptor curve revealed a decrease in the lower plateau (43+/-11 and 15+/-5%, respectively vs. saline: 78+/-6%, p<0.05) and an increase in the sympathetic gain of baroreflex (6.3+/-0.3, 7.2+/-0.8% respectively vs. saline: 4.2+/-0.4%, p<0.05). Bicuculline or CGP35348 into the NTS in WKY rats did not change MAP, sSND and sympathetic baroreflex gain. These data indicate that GABAergic mechanisms within the NTS act tonically reducing sympathetic baroreflex gain in SHR. Crown Copyright (C) 2010 Published by Elsevier By. All rights reserved.

Long-Term Culture of Mouse Embryonic Stem Cell-Derived Adherent Neurospheres and Functional Neurons

Innumerous protocols, using the mouse embryonic stem (ES) cells as model for in vitro study of neurons functional properties and features, have been developed. Most of these protocols are short lasting, which, therefore, does not allow a careful analysis of the neurons maturation, aging, and death processes. We describe here a novel and efficient long-lasting protocol for in vitro ES cells differentiation into neuronal cells. It consists of obtaining embryoid bodies, followed by induction of neuronal differentiation with retinoic acid of nonadherent embryoid bodies (three-dimensional model), which further allows their adherence and formation of adherent neurospheres (AN, bi-dimensional model). The AN can be maintained for at least 12 weeks in culture under repetitive mechanical splitting, providing a constant microenvironment (in vitro niche) for the neuronal progenitor cells avoiding mechanical dissociation of AN. The expression of neuron-specific proteins, such as nestin, sox1, beta III-tubulin, microtubule-associated protein 2, neurofilament medium protein, Tau, neuronal nuclei marker, gamma-aminobutyric acid, and 5-hydroxytryptamine, were confirmed in these cells maintained during 3 months under several splitting. Additionally, expression pattern of microtubule-associated proteins, such as lissencephaly (Lis1) and nuclear distribution element-like (Ndel1), which were shown to be essential for differentiation and migration of neurons during embryogenesis, was also studied. As expected, both proteins were expressed in undifferentiated ES cells, AN, and nonrosette neurons, although presenting different spatial distribution in AN. In contrast to previous studies, using cultured neuronal cells derived from embryonic and adult tissues, only Ndel1 expression was observed in the centrosome region of early neuroblasts from AN. Mature neurons, obtained from ES cells in this work, display ionic channels and oscillations of membrane electrical potential typical of electrically excitable cells, which is a characteristic feature of the functional central nervous system (CNS) neurons. Taken together, our study demonstrated that AN are a long-term culture of neuronal cells that can be used to analyze the process of neuronal differentiation dynamics. Thus, the protocol described here provides a new experimental model for studying neurological diseases associated with neuronal differentiation during early development, as well as it represents a novel source of functional cells that can be used as tools for testing the effects of toxins and/or drugs on neuronal cells.

Actions of octocoral and tobacco cembranoids on nicotinic receptors

Nicotinic acetylcholine receptors (AChRs) are pentameric proteins that form agonist-gated cation channels through the plasma membrane. AChR agonists and antagonists are potential candidates for the treatment of neurodegenerative diseases. Cembranoids are naturally occurring diterpenoids that contain a 14-carbon ring. These diterpenoids interact with AChRs in complex ways: as irreversible inhibitors at the agonist sites, as noncompetitive inhibitors, or as positive modulators, but no cembranoid was ever shown to have agonistic activity on AChRs. The cembranoid eupalmerin acetate displays positive modulation of agonist-induced currents in the muscle-type AChR and in the related gamma-aminobutyric acid (GABA) type A receptor. Moreover, cembranoids display important biological effects, many of them mediated by nicotinic receptors. Cembranoids from tobacco are neuroprotective through a nicotinic anti-apoptotic mechanism preventing excitotoxic neuronal death which in part could result from anti-inflammatory properties of cembranoids. Moreover, tobacco cembranoids also have anti-inflammatory properties which could enhance their neuroprotective properties. Cembranoids from tobacco affect nicotine-related behavior: they increase the transient initial ataxia caused by first nicotine injection into naive rats and inhibit the expression of locomotor sensitization to repeated injections of nicotine. In addition, cembranoids are known to act as anti-tumor compounds. In conclusion, cembranoids provide a promising source of lead drugs for many clinical areas, including neuroprotection, smoking-cessation, and anti-cancer therapies. (C) 2009 Elsevier Ltd. All rights reserved.

Steroid 21-hydroxylase expression in cultured rat astrocytes

Over the past decade or so it has become widely recognised that the brain is a significant steroidogenic organ. Many publications have highlighted the ability of the brain to synthesise and interconvert a large number of steroid products including cholesterol, progesterone and testosterone. In this study, in vitro experiments were performed to determine if 21-hydroxylation of steroids is undertaken by rat brain astrocytes in culture. This is a common reaction that occurs in the adrenal gland and other organs in mammals, catalysing the conversion of pregn-4-ene-3,20-dione (progesterone) to 21-hydroxypregn-4-ene-3,20-dione (deoxycorticosterone).

Previous reports have indicated that 21-hydroxylation occurs within the rat brain, however, the precise identity of the cells expressing 21-hydroxylase has not yet been determined. Several metabolites, such as 5α-pregnan-3α-ol-20-one (tetrahydroprogesterone) and 3α,21-dihydroxy-5-pregnan-20-one (tetrahydrodeoxycorticosterone) were of particular interest because of their modulatory role in neuronal function, such as their agonist activity at γ-aminobutyric acid (GABA_A) receptors.

Evidence was obtained for the expression of peripheral 21-hydroxylase enzyme (P450c21) in cultured rat brain astrocytes by a combination of mass spectroscopy and molecular biology techniques. This is a significant finding as expression of 21-hydroxylase within astrocytes may be indicative of a wider role for these cells in modulating neuronal behaviour.

Omega 3 fatty acids and the brain : review of studies in depression

The brain is a lipid-rich organ containing mostly complex polar  phospholipids, sphingolipids, gangliosides and cholesterol. These lipids are involved in the structure and function of cell membranes in the brain. The glycerophospholipids in the brain contain a high proportion of  polyunsaturated fatty acids (PUFA) derived from the essential fatty acids, linoleic acid and alpha-linolenic acid. The main PUFA in the brain are docosahexaenoic acid (DHA, all cis 4,7,10,13,16,19-22:6) derived from the omega 3 fatty acid, alpha-linolenic acid, and arachidonic acid (AA, all cis 5,8,11,14-20:4) and docosatetraenoic acid (all cis 7,10,13,16-22:4), both derived from the omega 6 fatty acid, linoleic acid. Experimental studies in animals have shown that diets lacking omega 3 PUFA lead to substantial disturbances in neural function, which in most circumstances can be restored by the inclusion of omega 3 PUFA in the diet. In the past 10 years there has been an emerging interest in treating neuropsychological  disorders (depression and schizophrenia) with omega 3 PUFA. This paper discusses the clinical studies conducted in the area of depression and omega 3 PUFA and the possible mechanisms of action of these PUFA. It is clear from the literature that DHA is involved in a variety of processes in neural cells and that its role is far more complex than simply influencing cell membrane properties.

GAD2 on chromosome 10p12 is a candidate gene for human obesity

The gene GAD2 encoding the glutamic acid decarboxylase enzyme (GAD65) is a positional candidate gene for obesity on Chromosome 10p11–12, a susceptibility locus for morbid obesity in four independent ethnic populations. GAD65 catalyzes the formation of γ-aminobutyric acid (GABA), which interacts with neuropeptide Y in the paraventricular nucleus to contribute to stimulate food intake. A case-control study (575 morbidly obese and 646 control subjects) analyzing GAD2 variants identified both a protective haplotype, including the most frequent alleles of single nucleotide polymorphisms (SNPs) +61450 C>A and +83897 T>A (OR = 0.81, 95% CI [0.681–0.972], p = 0.0049) and an at-risk SNP (−243 A>G) for morbid obesity (OR = 1.3, 95% CI [1.053–1.585], p = 0.014). Furthermore, familial-based analyses confirmed the association with the obesity of SNP +61450 C>A and +83897 T>A haplotype (χ2 = 7.637, p = 0.02). In the murine insulinoma cell line βTC3, the G at-risk allele of SNP −243 A>G increased six times GAD2 promoter activity (p < 0.0001) and induced a 6-fold higher affinity for nuclear extracts. The −243 A>G SNP was associated with higher hunger scores (p = 0.007) and disinhibition scores (p = 0.028), as assessed by the Stunkard Three-Factor Eating Questionnaire. As GAD2 is highly expressed in pancreatic β cells, we analyzed GAD65 antibody level as a marker of β-cell activity and of insulin secretion. In the control group, −243 A>G, +61450 C>A, and +83897 T>A SNPs were associated with lower GAD65 autoantibody levels (p values of 0.003, 0.047, and 0.006, respectively). SNP +83897 T>A was associated with lower fasting insulin and insulin secretion, as assessed by the HOMA-B% homeostasis model of β-cell function (p = 0.009 and 0.01, respectively). These data support the hypothesis of the orexigenic effect of GABA in humans and of a contribution of genes involved in GABA metabolism in the modulation of food intake and in the development of morbid obesity.

Lipids and FA analysis of canine prostate tissue

It is widely reported that an association exists between dietary fat intake and the incidence of prostate cancer in humans. To study this association, there is a need for an animal model where prostate carcinogenesis occurs spontaneously. The canine prostate is considered a suitable experimental model for prostate cancer in humans since it is morphologically similar to the human prostate and both humans and dogs have a predisposition to benign and malignant prostate disease. In this study, the FA and lipids profiles of the normal canine prostate tissue from nine dogs were examined. The total lipid content of the canine prostate tissue was 1.7±0.5% (wet weight). The lipid composition analysis using TLC-FID showed that the two major lipid classes were phospholipids and TAG. Total FA, phospholipid, and TAG FA analysis showed that the major FA were palmitic acid (16∶0), stearic acid (18∶0), oleic acid (18∶1), linoleic acid (18∶2n−6), and arachidonic acid (20∶4n−6), The n−3 FA were present at <3% of total FA and included α-linolenic acid (18∶3n−3) (in total and TAG tissue FA), EPA (20∶5n−3) (not in TAG), and DHA (22∶6n−3) (not in TAG). The n−3/n−6 ratio was 1∶11, 1∶13, and 1∶8 in total, phospholipid, and TAG FA, respectively. This study shows the canine prostate has a low level of n−3 FA and a low n−3/n−6 ratio. This is perhaps due to low n−3 content of the diet of the dogs. FA analysis of dogfoods available in Australia showed that the n−3 content in both supermarket and premium bran dogfoods was <3% (wet weight), and the n−3/n−6 ratio was low.

A Review of Factors Affecting Fat Absorption in Hot Chips

Consumption of hot chips is a convenience food in most countries. Unfortunately, these are high in fat and contribute to fat-related diseases in societies with a high fat consumption. There is substantial scope through best-practice deep-frying techniques for producing lower fat, high-quality chips. From a review of the literature, the main factors associated with a lower-fat content of chips are thick (>12 mm), straight cut chips; cryogenic freezing methods; low moisture content of potatoes (specific gravity >1.1); frying fat: chip volume ratio of 6:1; frying at optimal temperature (180 to 185°C) during cooking and turning the temperature down (∼140°C) and covering the vats during slack periods; vigorously shaking the basket and hanging it over the deep fryer to drain after frying; maintaining the quality of the frying fat by regularly skimming the cracklings, filtering the fat, and topping up the fryer with fresh fat; keeping the fat turnover <5 days; regular cleaning of frying equipment. It is important that all deep frying operators are adequately trained in these techniques. It is also important that the frying medium is low in saturated and trans fatty acids (<20%) because of their effects on blood lipids and low in linolenic acid (<3%) because it is readily degraded. The widespread implementation of best-practice deep-frying would reduce fat content of hot chips and thus lower overall fat consumption.

The presence of GABA in gastropod mucus and its role in inducing larval settlement

Chemical substances that induce larval settlement have been the focus of many gastropod studies due to the importance of wild stock recruitment and production within aquaculture facilities. Gamma-aminobutyric acid (GABA), GABA analogs, and GABA-mimetics associated with certain crustose coralline algae (CCA), are known to induce larval settlement in commercial abalone (Haliotis) species, and other gastropods. Furthermore, mucus secreted from these gastropods has been shown to induce larval settlement, but the stimulatory components of mucus have not been thoroughly investigated. We now present data confirming that GABA is the settlement-inducing effector molecule contained within abalone mucus. To do this, we initially generated anti-GABA for use in immunoenzyme and immunofluorescent microscopy. Using these techniques GABA was identified in the nerves and epithelial cells of the foot, including mucus. Dried mucus samples subject to HPLC analysis revealed a mean concentration of 0.68 mM GABA after sample rehydration. The presence of GABA in these samples was confirmed by time-of-flight mass spectroscopy (TOF-MS). In addition, GABA was detected in the mucus of several abalone species and other gastropods by immunocytochemistry. Subsequent bioassays using both dry and fresh mucus strongly promoted induction of larval settlement.

Larval settlement of the tropical abalone, Haliotis asinina Linnaeus, using natural and artificial chemical inducers

Many kinds of chemical and biological materials have been used as inducers of settlement of abalone larvae, as well as other species of marine gastropods, with responses being highly variable, even to the same chemical cue. The present study tested chemical inducers, γ-aminobutyric acid (GABA), δ-aminovaleric acid (5-AVA) and l-glutamic acid (GA) and the effects they have on larval settlement of Haliotis asinina. Additionally, a relatively inexpensive commercial substance, monosodium glutamate (MSG), was trialed. The datum provided shows all chemicals to be active inducers of settlement in this study, in order of effectiveness of 5-AVA, GABA, MSG to GA. Induction as adjudged from larval numbers settled was best at 6 h 62%, with 10⁻¹ mM 5-AVA. At 24 h, induction was the highest at 78% when exposed to 10⁻² mM 5-AVA. Larvae that were allowed to settle up to 72 h showed the highest numbers of settled larvae, and declined back to 60% when exposed to 10⁻² 5-AVA and 10⁻¹ mM GABA respectively. Monosodium glutamate, although third in settlement standings would bypass the other chemicals, with regard to cost versus yield. The assessment of settlement surface, rough or smooth proved to be irrelevant, which had no significant impact on larval settlement.