Ghrelin Stimulation of Growth Hormone-Releasing Hormone Neurons Is Direct in the Arcuate Nucleus

Background

Ghrelin targets the arcuate nucleus, from where growth hormone releasing hormone (GHRH) neurones trigger GH secretion. This hypothalamic nucleus also contains neuropeptide Y (NPY) neurons which play a master role in the effect of ghrelin on feeding. Interestingly, connections between NPY and GHRH neurons have been reported, leading to the hypothesis that the GH axis and the feeding circuits might be co-regulated by ghrelin.

Principal Findings

Here, we show that ghrelin stimulates the firing rate of identified GHRH neurons, in transgenic GHRH-GFP mice. This stimulation is prevented by growth hormone secretagogue receptor-1 antagonism as well as by U-73122, a phospholipase C inhibitor and by calcium channels blockers. The effect of ghrelin does not require synaptic transmission, as it is not antagonized by γ-aminobutyric acid, glutamate and NPY receptor antagonists. In addition, this hypothalamic effect of ghrelin is independent of somatostatin, the inhibitor of the GH axis, since it is also found in somatostatin knockout mice. Indeed, ghrelin does not modify synaptic currents of GHRH neurons. However, ghrelin exerts a strong and direct depolarizing effect on GHRH neurons, which supports their increased firing rate.

Conclusion

Thus, GHRH neurons are a specific target for ghrelin within the brain, and not activated secondary to altered activity in feeding circuits. These results support the view that ghrelin related therapeutic approaches could be directed separately towards GH deficiency or feeding disorders.     

MCT2 Expression and Lactate Influx in Anorexigenic and Orexigenic Neurons of the Arcuate Nucleus

Hypothalamic neurons of the arcuate nucleus control food intake, releasing orexigenic and anorexigenic neuropeptides in response to changes in glucose concentration. Several studies have suggested that the glucosensing mechanism is governed by a metabolic interaction between neurons and glial cells via lactate flux through monocarboxylate transporters (MCTs). Hypothalamic glial cells (tanycytes) release lactate through MCT1 and MCT4; however, similar analyses in neuroendocrine neurons have yet to be undertaken. Using primary rat hypothalamic cell cultures and fluorimetric assays, lactate incorporation was detected. Furthermore, the expression and function of MCT2 was demonstrated in the hypothalamic neuronal cell line, GT1-7, using kinetic and inhibition assays. Moreover, MCT2 expression and localization in the Sprague Dawley rat hypothalamus was analyzed using RT-PCR, in situ hybridization and Western blot analyses. Confocal immunohistochemistry analyses revealed MCT2 localization in neuronal but not glial cells. Moreover, MCT2 was localized to ∼90% of orexigenic and ∼60% of anorexigenic neurons as determined by immunolocalization analysis of AgRP and POMC with MCT2-positives neurons. Thus, MCT2 distribution coupled with lactate uptake by hypothalamic neurons suggests that hypothalamic neurons control food intake using lactate to reflect changes in glucose levels.     

Ghrelin对糖尿病大鼠下丘脑弓状核胃扩张敏感神经元和胃运动的影响

目的:探讨Ghrelin对糖尿病大鼠下丘脑弓状核胃扩张敏感神经元和胃运动的影响。方法:逆行追踪结合免疫组化观察ARC中GHSR-1的表达,细胞外放电记录,观察ghrelin对GD神经元放电活动的影响及电刺激ARC对GD神经元放电活动和胃运动的影响。结果:电生理实验结果表明,在ARC Ghrelin能够能激发GD兴奋性神经元(GD-E)和GD抑制性神经元(GD-I)。然而,ghrelin可以兴奋更少的GD-E神经元,在正常大鼠中ghrelin对于GD-E的兴奋作用比在DM大鼠中的作用弱。在体胃运动研究表明,在ARC中微量注射ghrelin可以明显的增强胃运动,并且呈现剂量依赖关系。Ghrelin在糖尿病大鼠促胃动力作用低于正常大鼠。Ghrelin诱导的效应可被生长激素促分泌素受体(GHSR)拮抗剂阻断[d-lys-3]-GHRP-6或bim28163。放射免疫法和实时荧光定量PCR数据表明胃血浆ghrelin水平,在ARC ghrelin mRNA的表达水平先上升后下降,糖尿病大鼠(DM)中,在ARC中GHSR-1a mRNA表达保持在一个比较低的水平。结论:ghrelin可以调节GD敏感神经元以及胃运动,通过ARC中ghrelin受体。在糖尿病大鼠中,Ghrelin促进胃运动作用减弱可能与ARC中ghrelin受体表达减少有关。     

Conversion of Neuropeptide K to Neurokinin A and Vesicular Colocalization of Neurokinin A and Substance P in Neurons of the Guinea Pig Small Intestine

The highest concentration of neurokinin A-like immunoreactivity and substance P-like immunoreactivity in the guinea pig small intestine was associated with the myenteric plexus-containing longitudinal muscle layer. Chromatographic analysis of extracts of this tissue demonstrated the presence of neurokinin A and neuropeptide K but the probable absence of neurokinin B. A fraction of synaptic vesicles of density 1.133 +/- 0.003 g/ml was prepared from the myenteric plexus-containing tissue by density gradient centrifugation in a zonal rotor and was enriched 29 +/- 12-fold in the concentration of neurokinin A-like immunoreactivity and 43 +/- 13-fold in the concentration of substance P-like immunoreactivity. This fraction was separated from the fraction of vasoactive intestinal peptide-containing vesicles (density, 1.154 +/- 0.009 g/ml). Chromatographic analysis of lysates of the vesicles indicated the presence of neurokinin A but not neuropeptide K. It is postulated that beta-pre-protachykinin is processed to substance P, neurokinin A, and neuropeptide K in the cell bodies of myenteric plexus neurons but that conversion of neuropeptide K to neurokinin A takes place during packaging into storage vesicles for axonal transport. The data are consistent with the proposal that neurokinin A and substance P are stored in the same synaptic vesicle, but the possibility of cosedimentation of different vesicles of very similar density cannot be excluded.     

Induction of Fos Immunoreactivity in Oxytocin Neurons in the Paraventricular Nucleus After Female Odor Exposure in Male Rats: Effects of Sexual Experience

1. We examined whether oxytocin (OT) neurons in the paraventricular nucleus of the hypothalamus (PVN) were activated by estrus female odor and sexual contact in sexually naïve and experienced Long-Evans rats. 2. Male rats were not presented to anesthetized estrus females (control) or presented to the females without (exposure to the female odor without sexual contact) or with direct contact (exposure to the female odor with sexual contact). 3. Exposure to the female odor with sexual contact significantly increased OT neurons with Fos-ir in both males. Exposure to the female odor without contact increased OT neurons with Fos-immunoreactive cells (Fos-ir) in sexually experienced males but not in naïve males, suggesting that the female odor without sexual contact activated the oxytocinergic neuronal system in the PVN in the experienced males. 4. Therefore, exposure to the estrus female odor itself may exert different effects on sexually naïve and experienced males.     

The Distribution of Afferent Neurons in the Trigeminal Mesencephalic Nucleus and the Central Projection of Afferent Fibers of the Mylohyoid Nerve in the Rat

Horseradish peroxidase conjugated to wheatgerm agglutinin (HRP:WGA) was injected into the proximal cut ends of three branches of the mylohyoid nerve in rats: the branch to the mylohyoid muscle (BrMh), the branch to the anterior belly of the digastricus muscle (BrDg), and the cutaneous branch (BrCu). HRP-labeled cells were detected in the ipsilateral caudal portion of the trigeminal mesencephalic nucleus (Vmes) and the ipsilateral ventromedial division of the trigeminal motor nucleus, except when HRP:WGA was applied to the BrCu. Morphologically, all labeled Vmes cells were of the pseudounipolar type.

Projections of the primary afferents of the BrMh were observed in the ipsilateral trigeminal nucleus caudalis, the upper cervical dorsal horns of laminae I -III, and the dorsolateral recticular formation (Rf), whereas the primary afferents of the BrDg terminated in the ipsilateral trigeminal nucleus principalis and Rf. These observations suggest that the role of the afferent inputs of the mylohyoid muscle differs from that of those of the anterior belly of the digastricus muscle in terms of several functions associated with jaw-closing and infrahyoid muscles.     

A Pair of Dopamine Neurons Target the D1-Like Dopamine Receptor DopR in the Central Complex to Promote Ethanol-Stimulated Locomotion in Drosophila

Dopamine is a mediator of the stimulant properties of drugs of abuse, including ethanol, in mammals and in the fruit fly Drosophila. The neural substrates for the stimulant actions of ethanol in flies are not known. We show that a subset of dopamine neurons and their targets, through the action of the D1-like dopamine receptor DopR, promote locomotor activation in response to acute ethanol exposure. A bilateral pair of dopaminergic neurons in the fly brain mediates the enhanced locomotor activity induced by ethanol exposure, and promotes locomotion when directly activated. These neurons project to the central complex ellipsoid body, a structure implicated in regulating motor behaviors. Ellipsoid body neurons are required for ethanol-induced locomotor activity and they express DopR. Elimination of DopR blunts the locomotor activating effects of ethanol, and this behavior can be restored by selective expression of DopR in the ellipsoid body. These data tie the activity of defined dopamine neurons to D1-like DopR-expressing neurons to form a neural circuit that governs acute responding to ethanol.     

MiR-124对先天性甲低新生鼠神经元凋亡的影响

目的:探讨mi R-124在甲状腺功能低下新生大鼠海马神经元凋亡中的作用。方法:将16只孕SD雌鼠随机分为实验组和对照组,分别于两组仔鼠1、5、10、15日龄称重取血,用化学发光法测定血清游离三碘甲状腺原氨酸(FT3)、血清游离甲状腺素(FT4)及促甲状腺激素(TSH)水平。将大鼠用2%戊巴比妥钠腹腔注射麻醉后,固定在脑立体定位仪,处死后收集其海马,应用荧光定量PCR和免疫荧光技术检测agomir的干预效率,确定干预有效后进行建模。观察注射后大鼠行为学改变,NeuN染色法检测海马区脑组织神经元细胞凋亡情况。结果:实验组血清TSH水平明显高于对照组,而FT3、FT4水平明显低于对照组。同正常对照组相比,甲状腺功能减退组中阳性神经元数量明显减少、细胞轮廓模糊、不清晰。经mir-124模拟处理后,神经细胞的数量和形态明显改善。结论:mi R-124对先天性甲状腺功能低下新生鼠神经元凋亡起到保护作用。     

Population genetic structure of the noxious weed Amaranthus retroflexus in Central Europe

Genetic variation was assessed in a range of populations of Amaranthus retroflexus using isoenzyme analysis. Population genetic diversity was measured by evaluating patterns of variation at six putatively neutral isoenzyme loci (comprising 24 putative alleles) within and among 20 populations of A. retroflexus collected in different habitats: ruderal habitats, cereal fields and hop gardens. Amaranthus retroflexus is a noxious weed of North American origin that infests various crops. Overall, A. retroflexus displayed moderate levels of genetic diversity in comparison with other herbaceous plants. The percentage of polymorphic loci was 50.0%, with mean values of 2.01, 0.142 and 0.227 for the average number of alleles per polymorphic locus (A), observed heterozygosity (H_o) and expected heterozygosity (H_e), respectively. A discrepancy between observed and expected heterozygosity and significant differences from H-W expectation indicate that there is an excess of homozygotes in many populations. As a result, there is strong evidence of inbreeding within populations (F_IS = 0.382) and significant population differentiation (F_ST = 0.270). Even though the species is partly autogamous, inbreeding does not lead to strong inbreeding depression resulting from self-pollination, as inbreeding has no effect on the success of the species in today's countryside. Moreover, allele frequencies detected in agricultural habitats (i.e., cereal fields and hop gardens) differed from those detected in populations collected from ruderal habitats, which is probably caused by systematic application of herbicides in agricultural ecosystems.     

A Model for the Fast Synchronous Oscillations of Firing Rate in Rat Suprachiasmatic Nucleus Neurons Cultured in a Multielectrode Array Dish

When dispersed and cultured in a multielectrode dish (MED), suprachiasmatic nucleus (SCN) neurons express fast oscillations of firing rate (FOFR; fast relative to the circadian cycle), with burst duration ∼10 min, and interburst interval varying from 20 to 60 min in different cells but remaining nevertheless rather regular in individual cells. In many cases, separate neurons in distant parts of the 1 mm recording area of a MED exhibited correlated FOFR. Neither the mechanism of FOFR nor the mechanism of their synchronization among neurons is known. Based on recent data implicating vasoactive intestinal polypeptide (VIP) as a key intercellular synchronizing agent, we built a model in which VIP acts as both a feedback regulator to generate FOFR in individual neurons, and a diffusible synchronizing agent to produce coherent electrical output of a neuronal network. In our model, VIP binding to its (VPAC₂) receptors acts through G_s G-proteins to activate adenylyl cyclase (AC), increase intracellular cAMP, and open cyclic-nucleotide-gated (CNG) cation channels, thus depolarizing the cell and generating neuronal firing to release VIP. In parallel, slowly developing homologous desensitization and internalization of VPAC₂ receptors terminates elevation of cAMP and thereby provides an interpulse silent interval. Through mathematical modeling, we show that this VIP/VPAC₂/AC/cAMP/CNG-channel mechanism is sufficient for generating reliable FOFR in single neurons. When our model for FOFR is combined with a published model of synchronization of circadian rhythms based on VIP/VPAC₂ and Per gene regulation synchronization of circadian rhythms is significantly accelerated. These results suggest that (a) auto/paracrine regulation by VIP/VPAC₂ and intracellular AC/cAMP/CNG-channels are sufficient to provide robust FOFR and synchrony among neurons in a heterogeneous network, and (b) this system may also participate in synchronization of circadian rhythms.     

银杏内酯B对豚鼠腹腔神经节神经元Fast-EPSP的抑制效应

应用细胞内记录技术,观察并分析了银杏内酯B(GB)对豚鼠(Cavia porcellus)离体腹腔神经节(CG)神经元快兴奋性突触后电位(fast-EPSP)的影响及可能机制.用4×10-6mol/L的GB灌流CG,fast-EPSP的幅值均明显降低,与对照组相比有显著性差异(n=12,P<0.05);用低Ca2 /高Mg2 Krebs液灌流,fastEPSP被完全抑制(n=3);用高Ca2 Krebs液灌流,fast-EPSP幅度则增大(n=12,P<0.05),而用4×10-6mol/L GB与高Ca2 Krebs液联合灌流,fast-EPSP幅度则减小(n=12,P<0.05).结果提示,GB对CG神经元fast-EPSP的抑制效应可能与减少或抑制CG神经元的外Ca2 内流有关.     

Consistency and Diversity of Spike Dynamics in the Neurons of Bed Nucleus of Stria Terminalis of the Rat: A Dynamic Clamp Study

Neurons display a high degree of variability and diversity in the expression and regulation of their voltage-dependent ionic channels. Under low level of synaptic background a number of physiologically distinct cell types can be identified in most brain areas that display different responses to standard forms of intracellular current stimulation. Nevertheless, it is not well understood how biophysically different neurons process synaptic inputs in natural conditions, i.e., when experiencing intense synaptic bombardment in vivo. While distinct cell types might process synaptic inputs into different patterns of action potentials representing specific “motifs” of network activity, standard methods of electrophysiology are not well suited to resolve such questions. In the current paper we performed dynamic clamp experiments with simulated synaptic inputs that were presented to three types of neurons in the juxtacapsular bed nucleus of stria terminalis (jcBNST) of the rat. Our analysis on the temporal structure of firing showed that the three types of jcBNST neurons did not produce qualitatively different spike responses under identical patterns of input. However, we observed consistent, cell type dependent variations in the fine structure of firing, at the level of single spikes. At the millisecond resolution structure of firing we found high degree of diversity across the entire spectrum of neurons irrespective of their type. Additionally, we identified a new cell type with intrinsic oscillatory properties that produced a rhythmic and regular firing under synaptic stimulation that distinguishes it from the previously described jcBNST cell types. Our findings suggest a sophisticated, cell type dependent regulation of spike dynamics of neurons when experiencing a complex synaptic background. The high degree of their dynamical diversity has implications to their cooperative dynamics and synchronization.     

NPs/NPRs Signaling Pathways May Be Involved in Depression-Induced Loss of Gastric ICC by Decreasing the Production of mSCF

It is well known that natriuretic peptides (NPs) are involved in the regulation of gastrointestinal motility. Interstitial cells of Cajal (ICC) are the pacemaker cells of gastrointestinal motility and gastrointestinal dyskinesia is one of the important digestive tract symptoms of depression. However, it is unclear whether they are involved in depression-induced loss of ICC. The aim of the present study was to investigate the relationship between the natriuretic peptide signaling pathway and depression-induced loss of gastric ICC in depressed rats. These results showed that the expression of c-kit and stem cell factor (SCF) in smooth muscle layers of stomach were down-regulated in depressed rats at the mRNA and protein levels. The expression of natriuretic peptide receptor (NPR)-A, B and C were up-regulated in the stomach of depressed rats at the mRNA and protein levels. NPR-A, B and C can significantly decrease the expression of SCF to treat cultured gastric smooth muscle cells (GSMCs) obtained from normal rats with different concentrations of C-type natriuretic peptide (CNP). Pretreatment of cultured GSMCs with 8-Brom-cGMP (8-Br-cGMP, a membrane permeable cGMP analog), cANF (a specific NPR-C agonist) and CNP (10⁻⁶ mol/L) demonstrated that 8-Br-cGMP had a similar effect as CNP, but treatment with cANF did not. The results of the methyl thiazolyl tetrazolium bromide (MTT) assay indicated that high concentrations of cANF (10⁻⁶ mol/L) restrained the proliferation of cultured GSMCs. Taken together, these results indicate that the up-regulation of the NPs/NPR-C and NPs/NPR-A, B/cGMP signaling pathways may be involved in depression-induced loss of gastric ICC.     

Quantitative Mass Spectrometry Analysis Reveals that Deletion of the TRPV1 Receptor in Mice Alters Substance P and Neurokinin A Expression in the Central Nervous System

Vanilloid receptors have a central role in the processing of nociceptive stimuli. TRPV1 null mice showed significant decrease in response to heat noxious stimuli. However, thermal sensitivity is still present suggesting that the TRPV1 is not an exclusive transducer of thermal stimuli. Additionally, tachykinin peptides play a central role in pain processing and expression levels may also contribute in modifying the pain threshold. The LC?CMS/MS analysis revealed that SP and NKA were significantly down-regulated in TRPV1^?/? in spinal cord and brain tissues. In spinal cord, SP concentrations were 23.4?% lower (p?<?0.0049) and NKA concentrations were 22.0?% lower (p?<?0.0022) in TRPV1 null mice. Additionally, brain SP concentrations were 26.9?% lower (p?<?0.0260) and brain NKA concentrations were 31.9?% lower (p?<?0.0063) in TRPV1 null mice. These results clearly demonstrate that TPRV1 null mice exhibit lower SP and NKA concentrations in the central nervous system. The deficit of thermal responses may also be related to the down-regulations of SP and NKA.     

A Common Structural Motif in Elongation Factor Ts and Ribosomal Protein L7/12 May Be Involved in the Interaction with Elongation Factor Tu

Elongation factor (EF) Tu alternates between two interaction partners, EF-Ts and the ribosome, during its functional cycle. On the ribosome, the interaction involves, among others, ribosomal protein L7/12. Here we compare EF-Ts and L7/12 with respect to the conservation of sequence and structure. There is significant conservation of functionally important residues in the N-terminal domain of EF-Ts and in the C-terminal domain of L7/12. The structure alignment based on the crystal structures of the two domains suggests a high degree of similarity between the αA–βD–αB motif in L7/12 and the h1–turn–h2 motif in EF-Ts which defines a common structural motif. The motif is remarkably similar with respect to fold, bulkiness, and charge distribution of the solution surface, suggesting that it has a common function in binding EF-Tu. Received: 12 June 2000 / Accepted: 10 October 2000     

Specific Involvement of Central 5-HT1A Receptors in the Mediation of Male Rat Ejaculatory Behavior

The aminotetralin 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), pharmacologically characterized as a 5-HT_1A receptor agonist, produces a pronounced decrease in ejaculation latency in the male rat. Stimulation of 5-HT receptors by a pharmacologically induced increase in the synaptic availability of 5-HT has been shown to produce the opposite effect. The 8-OH-DPAT-induced decrease in ejaculation latency is specific for this compound, and some chemically related ergot derivatives. In this paper we review the evidence in support for stimulation of serotonergic auto-receptors of the 5-HT_1A receptor subtype as a mechanism of action for effects by 8-OH-DPAT on male rat ejaculatory behavior. We also present the questions posed by the fact that quinpirole and lisuride both produce 8-OH-DPAT-like effects on male rat ejaculatory behavior. The effects by quinpirole, lisuride or 8-OH-DPAT are not sensitive to pretreatment with the DA D_2/3 receptor antagonist raclopride. Continued studies will show whether the effects of quinpirole and lisuride can be related to stimulation of 5-HT_1A receptors, or if all these compounds have as yet undefined common properties.