The effect of general anaesthetics on brain lactate release

The effects of anaesthetic agents on brain energy metabolism may explain their shared neurophysiological actions but remain poorly understood. The brain lactate shuttle hypothesis proposes that lactate, provided by astrocytes, is an important neuronal energy substrate. Here we tested the hypothesis that anaesthetic agents impair the brain lactate shuttle by interfering with astrocytic glycolysis. Lactate biosensors were used to record changes in lactate release by adult rat brainstem and cortical slices in response to thiopental, propofol and etomidate. Changes in cytosolic nicotinamide adenine dinucleotide reduced (NADH) and oxidized (NAD+) ratio as a measure of glycolytic rate were recorded in cultured astrocytes. It was found that in brainstem slices thiopental, propofol and etomidate reduced lactate release by 7.4 ± 3.6% (P < 0.001), 9.7 ± 6.6% (P < 0.001) and 8.0 ± 7.8% (P = 0.04), respectively. In cortical slices, thiopental reduced lactate release by 8.2 ± 5.6% (P = 0.002) and propofol by 6.0 ± 4.5% (P = 0.009). Lactate release in cortical slices measured during the light phase (period of sleep/low activity) was ~25% lower than that measured during the dark phase (period of wakefulness) (326 ± 83 μM vs 430 ± 118 μM, n = 10; P = 0.04). Thiopental and etomidate induced proportionally similar decreases in cytosolic [NADH]:[NAD+] ratio in astrocytes, indicative of a reduction in glycolytic rate. These data suggest that anaesthetic agents inhibit astrocytic glycolysis and reduce the level of extracellular lactate in the brain. Similar reductions in brain lactate release occur during natural state of sleep, suggesting that general anaesthesia may recapitulate some of the effects of sleep on brain energy metabolism

Role of Tachykinin-1 peptides in the regulation of habenular kisspeptin neurons in the zebrafish

Fear is a conserved emotion which serves as the root cause of psychiatric disorders such as phobias and depression. Hence understanding the neural pathway underlying fear is important to find a cure. Previously it was reported that the odorant-evoked fear response is regulated by habenular Kiss1 neurons through the serotonergic system. However the afferent signalling pathway upon exposure to the odorant cue remains unknown. Substance P (SP), a member of the tachykinin family of neuropeptides, is known to be involved in fear-related functions. Mammalian studies have demonstrated the role of SP in the habenula in depression-related behaviours. <i>Tac1</i> gene, encodes for SP and neurokinin A (NKA). SP and NKA are co-localized and co-released from the same neurons and nerves. The role of these peptides depends on its receptor activation and binding selectivity. The present study was designed to investigate the role of tachykinin1 (Tac1) peptides in the habenular Kiss1-regulating fear pathway in the zebrafish. <br>        In chapter 2, zebrafish-specific Tac1 antibody was generated and Tac1 cells were localized. The distribution of Tac1 fibers and their association to habenular Kiss1 neurons were examined. Immonohistochemical labelling showed Tac1 cell distribution in several regions of the brain such as in the anterior part of the parvocellular preoptic nucleus, the ventral region of the periventricular hypothalamus, the nucleus of medial longitudinal fascicle, the oculomotor nucleus and the central gray while the fiber distributions were noted throughout the brain. Alternate section labelling of Tac1 immunoreactivity and Kiss1 showed Tac1-immunoreactive fibers innervating the Kiss1-expressing region. Detection of SP receptor <i>NK1a</i> mRNA expression in the Kiss1 neurons suggests the presence of a possible direct regulation of Kiss1 by Tac1 through its cognate receptor. <br>        In chapter 3, the potential brain regions involved in the zebrafish AS-evoked fear pathway and the connectivity of AS with Tac1 was examined. Mapping of neural activity upon AS exposure revealed that several brain regions such as telencephalon, diencephalic region, cerebellum and spinal cord are involved in the fear pathway. Double labelling of Tac1 cells with a neuronal activity marker, <i>npas4a</i> showed increased activity in Tac1 cells, suggesting that the AS activates the Tac1 cells in the preoptic area. <br>     In chapter 4, to identify which Tac1-encoding peptides (SP or NKA) is involved in the habenular kisspeptin-modulating fear pathway, we examined the effect of exogenous SP and NKA on <i>kiss1</i> expression. Administration of NKA peptide had no effect on <i>kiss1 </i>mRNA expression, whereas administration of SP significantly reduced kiss1 mRNA expression. Blockade of SP receptor using a SP receptor antagonist (sendide), eliminated the effect of SP on <i>kiss1</i> and also blocked the effect of AS on <i>kiss1</i> mRNA expression, suggesting that SP but not NKA may regulate Kiss1 in AS-evoked fear response. <br>        In conclusion, the present study presents the g of a zebrafish-specific Tac1 antibody and the possible role of tachykinin peptides in the regulation of habenular Kiss1 neurons in the zebrafish. Innervation of Tac1 fibers into the Kiss1-expressing ventral habenula and the expression of a SP receptor, NK1a, in Kiss1 neurons suggest the presence of a possible direct action of Tac1 on Kiss1 neurons. Neural activity in the POA of Tac1 cells suggests that the AS-driven olfactory signalling may involve the activation of Tac1 cells in the preoptic area followed by transmission of signals to habenula Kiss1 neurons. Finally, the effects of SP and NKA on Kiss1 neurons suggest that SP exclusively regulate Kiss1 in AS-evoked fear response

Safety assessment of a standardized polyphenolic extract of clove buds: Subchronic toxicity and mutagenicity studies

Despite the various reports on the toxicity of clove oil and its major component eugenol, systematic evaluations on the safety of polyphenolic extracts of clove buds have not been reported. Considering the health beneficial pharmacological effects and recent use of clove polyphenols as dietary supplements, the present study investigated the safety of a standardized polyphenolic extract of clove buds (Clovinol), as assessed by oral acute (5 g/kg b.wt. for 14 days) and subchronic (0.25, 0.5 and 1 g/kg b.wt. for 90 days) toxicity studies on Wistar rats and mutagenicity studies employing Salmonella typhimurium strains. Administration of Clovinol did not result in any toxicologically significant changes in clinical/behavioural observations, ophthalmic examinations, body weights, organ weights, feed consumption, urinalysis, hematology and clinical biochemistry parameters when compared to the untreated control group of animals, indicating the no observed-adverse-effect level (NOAEL) as 1000 mg/kg b.wt./day; the highest dose tested. Terminal necropsy did not reveal any treatment-related histopathology changes. Clovinol did not show genotoxicity when tested on TA-98, TA-100 and TA-102 with or without metabolic activation; rather exhibited significant antimutagenic potential against the known mutagens, sodium azide, NPD and tobacco as well as against 2-acetamidoflourene, which needed metabolic activation for mutagenicity. Keywords: Syzygium aromaticum, Clove bud extract, Polyphenol, Subchronic toxicity, Mutagenicity, Genotoxicit

Isolation and characterization of &#947;-L-glutamyl-S-(trans-1-propenyl)-L-cysteine sulfoxide from sandal (Santalum album). Interesting occurrence of sulfoxide diastereoisomers in nature

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Thyroid Hormone Upregulates Hypothalamic kiss2 Gene in the Male Nile Tilapia, Oreochromis niloticus

Kisspeptin has recently been recognized as a critical regulator of reproductive function in vertebrates. During the sexual development, kisspeptin neurons receive sex steroids feedback to trigger gonadotropin-releasing hormone (GnRH) neurons. In teleosts, a positive correlation has been found between the thyroid status and the reproductive status. However, the role of thyroid hormone in the regulation of kisspeptin system remains unknown. We cloned and characterized a gene encoding kisspeptin (kiss2) in a cichlid fish, the Nile tilapia (Oreochromis niloticus). Expression of kiss2 mRNA in the brain was analyzed by in situ hybridization. The effect of thyroid hormone (triiodothyronine, T3) and hypothyroidism with methimazole (MMI) on kiss2 and the three GnRH types (gnrh1, gnrh2 and gnrh3) mRNA expression was analyzed by real-time PCR. Expression of thyroid hormone receptor mRNAs were analyzed in laser-captured kisspeptin and GnRH neurons by RT-PCR. The kiss2 mRNA expressing cells were seen in the nucleus of the lateral recess in the hypothalamus. Intraperitoneal administration of T3 (5&#181;g/g body weight) to sexually mature male tilapia significantly increased kiss2 and gnrh1 mRNA levels at 24 hr post injection (P &lt; 0.001), while the treatment with an anti-thyroid, MMI (100 ppm for 6 days) significantly reduced kiss2 and gnrh1 mRNA levels (P &lt; 0.05). gnrh2, gnrh3 and thyrotropin-releasing hormone mRNA levels were insensitive to the thyroid hormone manipulations. Furthermore, RT-PCR showed expression of thyroid hormone receptor mRNAs in laser-captured GnRH neurons but not in kiss2 neurons. This study shows that GnRH1 may be directly regulated through thyroid hormone, while the regulation of Kiss2 by T3 is more likely to be indirect

Knockdown of <i>Ndufs4</i> in the DMT.

<p><b>A.</b> Fluorescent images of brain slices from mice injected with active WT-Cre virus (<b>A</b>) into the DMT (Co-ordinates: ML = ± 0.32; AP = -1.2; DV = 3.7, Magnification X40). <b>B</b>, <b>C.</b> Schematic figures from the Allen mouse brain atlas [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188087#pone.0188087.ref024" target="_blank">24</a>] depicting the viral spread (Image credit: Allen Institute). Reprinted from the Allen mouse brain atlas under a CC BY license, with permission from the Allen Institute, original copyright 2008. <b>D.</b> EC₅₀s for ISO and HAL for the active (n = 6, black bars) and sham (n = 6, grey bars) virus injected mice in the LORR assay. <b>E.</b> EC₅₀s for ISO and HAL for the active (n = 6, black bars) and sham (n = 6, grey bars) virus injected mice in the TC assay. Scale bar: 1mm. *** indicates p-values <0.001.</p

Regional knockdown of NDUFS4 implicates a thalamocortical circuit mediating anesthetic sensitivity

<div><p>Knockout of the mitochondrial complex I protein, NDUFS4, profoundly increases sensitivity of mice to volatile anesthetics. In mice carrying an <i>Ndufs4</i>^lox/lox gene, adeno-associated virus expressing Cre recombinase was injected into regions of the brain postulated to affect sensitivity to volatile anesthetics. These injections generated otherwise phenotypically wild type mice with region-specific, postnatal inactivation of <i>Ndufs4</i>, minimizing developmental effects of gene loss. Sensitivities to the volatile anesthetics isoflurane and halothane were measured using loss of righting reflex (LORR) and movement in response to tail clamp (TC) as endpoints. Knockdown (KD) of <i>Ndufs4</i> in the vestibular nucleus produced resistance to both anesthetics for movement in response to TC. <i>Ndufs4</i> loss in the central and dorsal medial thalami and in the parietal association cortex increased anesthetic sensitivity to both TC and LORR. Knockdown of <i>Ndufs4</i> only in the parietal association cortex produced striking hypersensitivity for both endpoints, and accounted for half the total change seen in the global KO (<i>Ndufs4(KO)</i>). Excitatory synaptic transmission in the parietal association cortex in slices from <i>Ndufs4(KO)</i> animals was hypersensitive to isoflurane compared to control slices. We identified a direct neural circuit between the parietal association cortex and the central thalamus, consistent with a model in which isoflurane sensitivity is mediated by a thalamic signal relayed through excitatory synapses to the parietal association cortex. We postulate that the thalamocortical circuit is crucial for maintenance of consciousness and is disrupted by the inhibitory effects of isoflurane/halothane on mitochondria.</p></div

Knockdown of <i>Ndufs4</i> in the CMT.

<p><b>A.</b> Fluorescent images of brain slices from mice injected with active WT-Cre virus (<b>A</b>) into the CMT (Co-ordinates: ML = ± 0.32; AP = -1.2; DV = 3.85, Magnification X40). <b>B</b>, <b>C.</b> Schematic figures from the Allen mouse brain atlas [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188087#pone.0188087.ref024" target="_blank">24</a>] depicting the viral spread (Image credit: Allen Institute). Reprinted from the Allen mouse brain atlas under a CC BY license, with permission from the Allen Institute, original copyright 2008. <b>D.</b> EC₅₀s for ISO and HAL for the active (n = 6, black bars) and sham (n = 6, grey bars) virus injected mice in the LORR assay. <b>E.</b> EC₅₀s for ISO and HAL for the active (n = 6, black bars) and sham (n = 6, grey bars) virus injected mice in the TC assay. Scale bar: 1mm. **indicates p-values <0.005, *** indicates p-values <0.001.</p