Immunoreactive peptides related to dynorphin B (=rimorphin) in the rat brain

We have developed a radioimmunoassay for synthetic dynorphin B, a novel opioid tridecapeptide, which shares a common precursor molecule with dynorphin_1–17 (=dynorphin A) and the neo-endorphins. The levels of immunoreactivity towards this peptide in rat brain and pituitary show a pattern quantitatively and qualitatively similar to those found for dynorphin A and -neo-endorphin in earlier studies. The antiserum used was highly specific with only dynorphin-32 and dynorphin B-29, both of which contain the dynorphin B sequence, showing substantial cross-reactivity. Gel filtration of whole rat brain extracts in combination with HPLC analysis provide strong evidence for the existence of these latter two peptides in rat brain.     

Responsiveness of nigral neurons to the stimulation of striatal dopaminergic receptors in the rat

The microinfusion of low doses of apomorphine into the striatum of anesthetized rats depressed the electrical activity of the neurons of the substantia nigra pars compacta while the infusion of bromocriptine had an excitatory or inhibitory effect. These data suggest that:1) the action of the two dopamine agonists on the striato-nigral pathway is different; 2) the striatum might contain dopaminergic receptors located on cells projecting to the substantia nigra with different roles in the feedback regulation of the latter; 3) the inhibitory action of systemically injected apomorphine is not simply due to a stimulation of dopamine “autoreceptors” but also to an action mediated by fibers descending from the striatum to the substantia nigra.     

Central dopaminergic and serotoninergic systems in the regulation of adrenal tyrosine hydroxylase.

Administration of the dopamine receptor agonists apomorphine, piribedil and bromocryptine caused an increase in adrenal tyrosine hydroxylase (TH; tyrosine-3-monooxygenase, EC 1.14.16.2) which could be partially abolished by prior injection of the dopamine blocker haloperidol. Injection of L-dihydroxyphenylalanine, along with the decarboxylase inhibitor carbidopa, also led to a highly significant increase in adrenal TH activity. Intraventricular injection of 5,7-dihydroxytryptamine (DHT), which destroys serotonin neurons, doubled adrenal TH activity in both normal and hypophysectomized rats. Splanchnicotomy abolished this effect of DHT. The increase in enzyme activity mediated by DHT could be partially prevented by peripheral administration of L-5-hydroxytryptophan together with carbidopa. Blockade of serotoninergic functions with the antagonist methiothepin also increased adrenal TH activity. The interrelationship between the dopamine and the presumed serotonin system was investigated. Intraventricular injection of 6-hydroxydopamine partially prevented the DHT-induced increase in adrenal TH activity. Administration of haloperidol to DHT-treated rats had the same effect. This suggests that an intact dopaminergic system is required. When DHT and either apomorphine or piribedil were adminstered simultancously the dopamine agonist-induced increase was potentiated. An intact serotoninergic system is therefore not required for dopamine function. Thus, the increase in adrenal TH activity is associated with either stimulation of central dopamine receptors or destruction of serotonin neurons. It is suggested that dopaminergic and serotoninergic systems are involved in the regulation of adrenal TH and that these systems have net excitatory and inhibitory roles, respectively. Furthermore, the present evidence favors the view that the interaction between the two systems is sequential, with the serotonin system preceding the dopamine one.     

5-HT7 receptors are involved in mediating 5-HT-induced activation of rat primary afferent neurons

The purpose of this study was to determine whether the 5-hydroxytryptamine7 (5-HT7) receptor is expressed by nociceptor-like neurons in the rat PNS and whether 5-HT activates these nociceptors via the 5-HT7 receptor subtype. Using a polyclonal antibody and the method of immunofluorescence staining, we demonstrated that the 5-HT7 receptor appears predominately on "nociceptor-like" neurons of the rat lumbar dorsal root ganglia. Using immunocytochemical methods, we showed that the immunoreactivity of the 5-HT7 receptor antibody complex is localized in the superficial layers of the spinal cord dorsal horn, which corresponds with laminae I, IIouter and IIinner. Furthermore, we demonstrated that noxious stimulation produced by knee injection of 5-HT or a 5-HT7 agonist dose-dependently increases c-Fos production of the rat spinal cord dorsal horn. This effect was significantly inhibited by the preinjection of a 5-HT7 antagonist. We conclude that the 5-HT7 receptor is expressed by rat primary afferent nociceptors which terminate in the superficial layers of the spinal cord dorsal horn and that the 5-HT7 receptor subtype is involved in nociceptor activation by 5-HT.     

Interaction of the serotoninergic and GABA-ergic systems in "psychogenic" amnesia

The results are analyzed of the influence of blockade of separate components of BD-GABA-iontophoretic complex on activation of memory-trace amnesia under blockade of serotonine PGA synthesis in experiments with conditioned reaction (CR) of passive avoidance in mice. It has been shown that the blockade of serotonine PGA synthesis did not change the behavioral manifestations of amnesia at all terms of testing. The blockade of chlorine channel by picrotoxin and of BD receptors RO15-1788 and RO15-3505 restored the reproduction of the memory trace disturbed against the PGA background; the blockade of GABAA-receptors by bicuculline lost its effectiveness. Activation of serotonin system by sertraline against the PGA background before learning and amnestic influence or before testing of CR of passive avoidance contributed to restoration of CR activating action of bicuculline. It was found that preliminary blockade of PGA serotonine synthesis changed the amnesia development and the character of subsequent restoration of CR of passive avoidance reproduction by blockaders of separate components of BD-GABA-iontophoretic complex.     

缺氧/复氧对培养的大鼠海马神经元Fos、Jun表达和神经元凋亡的影响

目的：观察缺氧／复氧对体外培养的海马神经元Fos和Jun表达和神经元凋亡的影响。方法：取培养12d的海马神经元,置2000cm^3的恒温（36℃）密闭容器内,连续充以无氧气体（90％N2、10％CO2）,在缺氧条件下继续培养2、4h后取出,置含10％CO2和空气的培养箱内复氧培养24h和72h。于不同时间取出,观察神经元存活数,分别用抗Fos和抗Jun抗血清进行免疫组织化学染色,计数Fos和Jun表达阳性神经元百分率,并用原位末端标记（TUNEL）法和流式细胞术分别观察和测定缺氧／复氧对体外培养海马神经元凋亡的影响。结果：缺氧／复氧后Fos和Jun表达阳性神经元百分率和凋亡神经元百分率的显著增加。结论：缺氧／复氧后即早反应基因fos在神经元中的持续表达可引起神经元凋亡,原癌基因jun的表达与神经元凋亡的发生有关。     

Psychomotor stimulant- and opiate-induced c-fos mRNA expression patterns in the rat forebrain: Comparisons between acute drug treatment and a drug challenge in sensitized animals

Amphetamine-, cocaine-, and morphine-induced c-fos expression patterns were examined following an injection protocol that has previously been shown to produce behavioral sensitization and enhanced dopamine release in the striatal complex. Drug-specific c-fos patterns were observed in both acute and sensitization injection paradigms. A sensitization pretreatment schedule did, however, alter the c-fos expression patterns induced by all the drugs in the caudate putamen, nucleus accumbens, and the cerebral cortex. In some striatal and cortical regions, there was an increase or recruitment of cells expressing c-fos whereas in others there was an apparent decrease or inhibition. The somatosensory cortex was one area where pretreatment with all three drugs increased c-fos expression. The results suggest that the neuronal networks that are modulated by systemic drug injections in the sensitized animal differ from those affected by the initial drug exposure; areas of overlap may indicate common ‘sensitization’ circuits. Special issue dedicated to Dr. Eric J. Simon.     

Further contribution to the study of corticostriatal glutamatergic and nigrostriatal dopaminergic interactions within the striatal network: an in vivo voltammetric investigation

Summary In vivo voltammetry was used in freely moving rats to study the processes whereby striatal dopamine (DA) release is regulated by corticostriatal glutamatergic neurons. Electrical stimulation of the cerebral cortex was found to markedly increase the striatal DA-related voltammetric signal amplitude. Similar enhancements have been observed after intracerebroventricular administration of 10nmoles glutamate, quisqualate and AMPA, whereas NMDA was found to decrease the amplitude of the striatal signals. The NMDA receptor antagonist APV did not significantly affect the voltammetric signal but prevented the NMDA-induced depression of the DA-related signals. These data are in agreement with those obtained in numerous previous studies suggesting that the glutamatergic corticostriatal neurons exert activatory effects on the striatal DA release via non-NMDA receptors. The mechanism involved might be of a presynaptic nature. The role of the NMDA receptors may however consist of modulating the dopaminergic transmission phasically and in a depressive way, which would be consistent with behavioural data suggesting the existence of a functional antagonism between the activity of the corticostriatal glutamatergic and nigrostriatal dopaminergic systems.Abbreviations Glu glutamate - DA dopamine - NMDA N-methyl-D-aspartate - CPP 3-(2-carboxypiperazin-4µl)propyl-1-phosphonic acid - AMPA -amino-3-hydroxy-5-metylisoxazole-4-propionic acid - APV aminophosphonovaleric acid - DOPAC dihydroxyphenylacetic acid - HVA homovanillic acid - DARPP 32 dopamine-cAMP-regulated phosphoprotein 32 - CSF cerebrospinal fluid Laboratory associated with the University of Aix-Marseille II     

Interrelationships between tyrosine hydroxylase-immunoreactive dopaminergic afferents and somatostatinergic neurons in the rat central amygdaloid nucleus

 Interrelationships between dopaminergic afferents and somatostatinergic neurons of the rat central amygdaloid nucleus were studied using tyrosine hydroxy-lase/somatostatin double immunolabeling for light and electron microscopy. Additionally, morphological features of somatostatin neurons in different subnuclei of the central nucleus were studied, and the results were complemented by codistribution studies of somatostatin and D₁ and D₂ dopamine receptor mRNA expression. Dense axonal immunolabeling for tyrosine hydroxylase was colocalized with somatostatin-immunoreactive or somatostatin mRNA-reactive neurons in the medial and the central lateral part of the central nucleus. The number of somatostatinergic neurons detected was higher using in situ hybridization than using immunolabeling. Somatostatin-immunoreactive neurons of the medial central nucleus possessed deeply indented nuclei, and immunoreaction product was confined to the Golgi apparatus and its vicinity. On the other hand, those in the central lateral subnucleus possessed nuclei without indentations and showed diffuse staining of the cytoplasm and/or in large vesicles. Double labeling showed that in the central lateral central nucleus, somatostatin-immunoreactive neurons were contacted by tyrosine hydroxylase-immunoreactive terminals, and on the electron microscopic level synaptic contacts between differently labeled structures were observed. D₁ and D₂ receptor mRNA-reactive neurons were differentially distributed in central nucleus subnuclei. D₁ receptor mRNA-expressing neurons were found only in the medial subnucleus, while D₂ receptor mRNA was expressed by a number of neurons in the lateral central and a few in the medial one. Thus, the study proves that somatostatin-immunoreactive neurons of the central lateral central nucleus are directly innervated by dopaminergic afferents and may express the D₂ dopamine receptor. Accepted: 2 July 1996     

Interaction between dopaminergic and cholinergic systems under conditions of deficiency of mesencephalo-striatal dopamine in rats

In chronic behavioral experiments on rats with a unilateral deficiency of mesencephalo-striatal dopamine, we studied the effect of the blocker of M-cholinoreceptors atropine on the rotational motor activity induced by systemic injections of dopamine agonists exerting direct (apomorphine) and indirect (amphetamine) actions. We found that premedication with atropine increased significantly the intensity of the rotational movements induced by both apomorphine and amphetamine. We conclude that the mesencephalo-striatal dopaminergic system exerts inhibitory effects on cholinergic neurons of the neostriatum. Neirofiziologiya/Neurophysiology, Vol. 37, Nos. 5/6, pp. 459–462, September–December, 2005.     

Specifics of participation of dopaminergic and serotoninergic systems of the brain in latent inhibition based on pharmacological models

Single intraperitoneal injections of haloperidol (0.5 mg/kg) or sertralin (5 mg/kg) or 20 preexpositions of conditional stimulus before conditioning induced similar changes of passive avoidance reactions of rats. The combinative application of drugs (sertralin 1h and bupropion 30 min before conditioning) simultaneously enhancing activity of serotonin and dopamine in brain did not produce changes of passive avoidance reaction comparing with intact control. The results obtained showed that high selective drugs and analysis of latent inhibition of some parameters enable creation of pharmacological models and their use as instrument at experimental study of neurochemical mechanisms of attention.     

Effect of DN-1417, a thyrotropin releasing hormone analog, on dopaminergic neurons in rat brain

Acute and chronic effects of γ-butyrolactone-γ-carbonyl-histidyl-prolinamide (DN-1417) were investigated on motor activity, dopamine (DA) metabolites and DA receptors in various brain regions of rats. The motor activity, as measured with Automex recorder, was enhanced after a single injection with DN-1417 (20 mg/kg, IP), and the motor stimulating action persisted during 21 daily injections. Acute DN-1417 elevated both homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) levels in 7 brain regions, prefrontal cortex polar, medial and lateral fields, nucleus accumbens, olfactory tubercles, amygdala and striatum. After chronic treatment for 7 days, the acute effect of DN-1417 on DA metabolites disappeared in all regions except for the striatum in which DN-1417 still increased HVA and DOPAC. The response of striatal DA metabolites was also observed after chronic treatment for 21 days. Chronic DN-1417 produced no significant change in ³H-spiperone binding in the prefrontal cortex, nucleus accumbens, olfactory tubercles and striatum, while striatal ³H-DA binding displaced by 30 nM spiperone was enhanced after chronic treatment. These results indicate that DN-1417 interacts with mesocortical, mesolimbic and nigrostriatal DA systems in the different modes of action. The lack of tolerance to motor hyperactivity, however, raises the question as to whether DN-1417-induced hyperactivity may be mediated by the activation of mesolimbic DA neurons. The involvement of nigrostriatal neurons in DN-1417-induced motor hyperactivity is suggested.     

Interaction of serotonin and nitric oxide (NO) in activation of the serotoninergic system in Helix

Serotonin and the NO donors were shown to induce excitation in all serotoninergic neurones under study and to activate synchronous synaptic input in the Helix brain. The serotonin effects may be blocked by 5,7-DOT and N-monomethyl-L-arginine. The 5,7-DOT blocked activation of the NO-induced synchronous bursts but did not affect their activating effect. The data obtained suggest that serotonin and NO equally regulate the serotoninergic system's function in the snail brain. The effects of serotonin and NO are interconnected and interdependent. A possible role of the NO as a second messenger during serotonin activation and as a serotonin co-transmitter in presynaptic neurones, is discussed.     

Interrelations between monoaminergic afferents and corticotropin-releasing factor-immunoreactive neurons in the rat central amygdaloid nucleus: ultrastructural evidence for dopaminergic control of amygdaloid stress systems

Ample evidence implicates corticotropin-releasing factor (CRF)-producing neurons of the central amygdaloid nucleus (CeA) in vegetative, endocrine, and behavioral responses to stress and anxiety in laboratory rats. Monoaminergic systems are involved in modulating these responses. In the present paper, interrelations between CRF-immunoreactive (ir) neurons, and noradrenergic, serotonergic, and dopaminergic afferents were studied using single and double immunolabeling for light and electron microscopy in the rat CeA. Dopaminergic axons formed dense plexus in the CeA overlapping with the localization of CRF-ir neurons, and their terminals formed frequent associations with CRF-ir somata. Contacts of serotonergic axons on CRF-ir neurons were few, and contacts of noradrenergic axons were the exception. Ultrastructurally, symmetric synapses of dopaminergic terminals on CRF-ir somata and dendrites were found. More than 83% of CRF-ir somata were contacted in single ultrathin sections. About half of these possessed two or more contacts. Of non-ir somata, 37% were contacted by dopaminergic terminals, and only 13% of these had two or more contacts. Correlative in situ hybridization indicated that CeA CRF-ir neurons may express receptor subtype dopamine receptor subtype 2. In conclusion, dopaminergic afferents appear to specifically target CeA CRF neurons. They are thus in a position to exert significant influence on the rat amygdaloid CRF stress system.     

Adenosine A2B receptor activation stimulates glucose uptake in the mouse forebrain

ATP consumption during intense neuronal activity leads to peaks of both extracellular adenosine levels and increased glucose uptake in the brain. Here, we investigated the hypothesis that the activation of the low-affinity adenosine receptor, the A_2B receptor (A_2BR), promotes glucose uptake in neurons and astrocytes, thereby linking brain activity with energy metabolism. To this end, we mapped the spatiotemporal accumulation of the fluorescent-labelled deoxyglucose, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), in superfused acute hippocampal slices of C57Bl/6j mice. Bath application of the A_2BR agonist BAY606583 (300 nM) triggered an immediate and stable (>10 min) increase of the velocity of 2-NBDG accumulation throughout hippocampal slices. This was abolished with the pretreatment with the selective A_2BR antagonist, MRS1754 (200 nM), and was also absent in A_2BR null-mutant mice. In mouse primary astrocytic or neuronal cultures, BAY606583 similarly increased ³H-deoxyglucose uptake in the following 20 min incubation period, which was again abolished by a pretreatment with MRS1754. Finally, incubation of hippocampal, frontocortical, or striatal slices of C57Bl/6j mice at 37 °C, with either MRS1754 (200 nM) or adenosine deaminase (3 U/mL) significantly reduced glucose uptake. Furthermore, A_2BR blockade diminished newly synthesized glycogen content and at least in the striatum, increased lactate release. In conclusion, we report here that A_2BR activation is associated with an instant and tonic increase of glucose transport into neurons and astrocytes in the mouse brain. These prompt further investigations to evaluate the clinical potential of this novel glucoregulator mechanism.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-015-9474-3) contains supplementary material, which is available to authorized users.     

Interaction between aflatoxin B1 and oxytetracycline in isolated rat hepatocytes

Isolated rat hepatocytes were used as an in vitro model to investigate A possible interaction between oxytetracycline (OXT) and aflatoxin B₁ (AFB₁). LDH leakage, RNA and protein synthesis and glycogen accumulation were measured in the presence of both drugs, either separately or in combination. The evolution of LDH leakage during the incubation was identical in untreated and treated cells. AFB₁ inhibited RNA and protein synthesis at a concentration of 10^–7 M and 10^–6 M, respectively, and higher, whereas OXT did not influence RNA synthesis but inhibited protein synthesis at the highest tested concentration, 10^–3 M. As far as glycogen is concerned, rats were injected with glucagon before sacrifice in order to obtain a constant synthesis rate in isolated hepatocytes. AFB₁ inhibited the accumulation of glycogen from 10^–6 M upward. This effect was never observed before 90 min of incubation. OXT had no effect on glycogen synthesis. In the presence of both drugs, no interaction was demonstrated as far as RNA and protein synthesis were concerned, but OXT opposed the inhibition induced by AFB₁ on glycogen accumulation. If the in vivo protection, provided by OXT against AFBI-induced toxicity, is due to a direct interference in the toxic mechanisms of the mycotoxin, these results show that OXT does not influence the AFB₁-inhibition of RNA and protein synthesis. The latter are early and sensitive parameters inhibited by AFB₁. On the contrary, taking into consideration the results on glycogen accumulation, it seems more interesting to investigate further this metabolism.Abbreviations AFB₁ Aflatoxin B₁ - OXT Oxytetracycline - DMEM Dulbecco's Modified Eagle's Medium - LDH Lactate Dehydrogenase - DMSO Dimethyl Sulfoxide - BSA Bovine Serum Albumin