Characterization of dynorphin-containing neurons on dissociated dentate gyrus cell cultures

In the dentate gyrus, the synthesis of the opioid peptide, dynorphin, is modulated by a variety of stimuli. In order to elucidate the cellular and molecular mechanisms regulating the synthesis of dynorphin in the hippocampus, we have established a routine primary cell culture of dentate granule neurons and identified granule-like neurons by a characteristic marker, dynorphin, in these cultures. Cultures were prepared from 7-day-old rat pups and maintained in medium with 2% fetal bovine serum. These cultures contained approximately 20% neurons and survived for over 4 weeks. After 2 weeks in culture, neurons expressing dynorphin-A and its messenger RNA were detected using immunocytochemistry and in situ hybridization, respectively. In dentate cultures, enkephalin-, cholecystokinin-, neuropeptide Y- and substance P-positive cells were observed in addition to dynorphin-positive cells with immunocytochemistry. The results suggest that dentate gyrus cell cultures provide a valid in vitro model for studying molecular mechanisms regulating prodynorphin gene expression.     

海上氦氧150 m饱和-182 m巡回潜水对潜水员血浆AVP、DynA、ACTH和CRH含量的影响

目的　研究海上大深度饱和潜水对潜水员血浆精氨酸加压素 (AVP)、强啡肽 A(Dyn A)、促肾上腺皮质激素 (ACTH)和促皮质素释放激素 (CRH)含量的影响。方法　用放射免疫法检测 8名潜水员在饱和潜水前后血浆 AVP、Dyn A、ACTH和 CRH的含量。结果　饱和潜水后潜水员血浆 AVP(14 4 8.5± 16 33.6 ) ng/L、Dyn A(4 9.4± 36 .8) ng/L、ACTH(392 .1± 2 4 5 .6 ) ng/L 和 CRH(378.1±4 4 .6 ) ng/L 含量显著或非常显著地高于饱和潜水前 (P<0 .0 5或 <0 .0 1)。结论　大深度饱和潜水能引起潜水员血浆 AVP、Dyn A、ACTH和 CRH含量的改变 ,所观察的 4种神经肽可能参与了大深度饱和潜水引起的机体应激反应     

Dynorphin A-(1–13) attenuates basal forebrain-lesion-induced amnesia in rats

The effects of dynorphin A-(1–13), an endogenous κ opioid agonist, on basal forebrain (BF)-lesion-induced amnesia in rats were investigated using step-through-type passive avoidance task. The BF was lesioned by injecting the cholinergic neurotoxin ibotenic acid (6 μg/side). The number of rats achieving the cut-off time (600 s) of step-through latency (STL) in BF-lesioned group significantly decreased as compared with that in sham-operated group. Dynorphin A-(1–13) (0.3 μg) significantly increased the number of rats achieving the cut-off time of STL in BF-lesioned rats. These results suggest that dynorphins play an improving role in the impairment of memory processes in BF-lesioned rats.     

Spinal release of immunoreactive dynorphin A(1−8) with the development of peripheral inflammation in the rat

Microprobes bearing immobilised antibodies to dynorphin A₍₁₋₈₎ were used to study the basal and evoked release of this prodynorphin derived peptide in the spinal cord of urethane anaesthetised normal rats and those with a peripheral inflammation. In the absence of any active peripheral stimulus the antibody microprobes detected immunoreactive (ir)-dynorphin A₍₁₋₈₎ in two areas (lamina I and laminae IV–V) in the dorsal horn of the spinal cord of normal rats. With the development of unilateral ankle inflammation over 3 to 5 days following subcutaneous injections of Freund's complete adjuvant, a basal presence of ir-dynorphin A₍₁₋₈₎ was found in both the dorsal and ventral horn regions of both sides of the spinal cord. Lateral compression of the ankles of the normal animals did not release ir-dynorphin A₍₁₋₈₎ during the period of stimulation, but this neuropeptide was detected in increased amounts in the ventral horn following the stimulus. By contrast, compression of inflamed ankles produced elevated levels of ir-dynorphin A₍₁₋₈₎ during the period of stimulus application at three major sites in the ipsilateral spinal grey matter. The largest peak was in the deep dorsal horn/upper ventral horn (laminae VI–VII), with further sites of significant release in the mid dorsal horn (laminae II–V) and the lower ventral horn. The observation that ir-dynorphin A₍₁₋₈₎ is physiologically released in the ventral and deep dorsal in addition to the superficial dorsal horn of the rat suggests an involvement of dynorphins in several aspects of spinal function.     

Differential sensitivity of opioid-induced feeding to naloxone and naloxonazine

The high-affinity mu-1 opioid binding site has been implicated in some opioid responses (e.g., supraspinal analgesia) but not others (e.g., respiratory depression) by comparing the actions of naloxone, a short-acting, non-selective antagonist, and naloxonazine, an irreversible and selective mu-1 antagonist. The mu-1 site has been implicated in the opioid component modulating free feeding and deprivation-induced feeding, but not glucoprivic feeding. The present study compared naloxone and naloxonazine antagonism of hyperphagia induced by morphine, ethylketocyclazocine (EKC), dynorphin and d-ala²,d-leu⁵-enkephalin (DADL) in rats. Morphine produced a dose-dependent (0.01–5 mg/kg) hyperphagia in midly food-deprived rats that was blocked by naloxone (0.01–10 mg/kg). Naloxonazine (10 mg/kg) shifted the morphine hyperphagia dose-response curve to the right. These effects could not be fully accounted for by the intrinsic hypophagic properties of these antagonists. EKC produced a dose-dependent (0.5–5 mg/kg) hyperphagia which was blocked by naloxone (10 mg/kg) only at low effective EKC doses. Naloxonazine (10 mg/kg) failed to affect EKC hyperphagia. Naloxone, but not naloxonazine also blocked dynorphin and DADL hyperphagia. These results indicate that feeding induced by opiate and opioid agonists are differentially mediated by the mu-1 and other opioid binding sites; these data contrast with the modulation by the mu-1 site of the supraspinal analgesia induced by each of these agonists.     

Neurotransmitter regulation of dopamine neurons in the ventral tegmental area

Over the last 10 years there has been important progress towards understanding how neurotransmitters regulate dopaminergic output. Reasonable estimates can be made of the synaptic arrangement of afferents to dopamine and non-dopamine cells in the ventral tegmental area (VTA). These models are derived from correlative findings using a variety of techniques. In addition to improved lesioning and pathway-tracing techniques, the capacity to measure mRNA in situ allows the localization of transmitters and receptors to neurons and/or axon terminals in the VTA. The application of intracellular electrophysiology to VTA tissue slices has permitted great strides towards understanding the influence of transmitters on dopamine cell function, as well as towards elucidating relative synaptic organization. Finally, the advent of in vivo dialysis has verified the effects of transmitters on dopamine and γ-aminobutyric acid transmission in the VTA. Although reasonable estimates can be made of a single transmitter's actions under largely pharmacological conditions, our knowledge of how transmitters work in concert in the VTA to regulate the functional state of dopamine cells is only just emerging. The fact that individual transmitters can have seemingly opposite effects on dopaminergic function demonstrates that the actions of neurotransmitters in the VTA are, to some extent, state-dependent. Thus, different transmitters perform similar functions or the same transmitter may perform opposing functions when environmental circumstances are altered. Understanding the dynamic range of a transmitter's action and how this couples in concert with other transmitters to modulate dopamine neurons in the VTA is essential to defining the role of dopamine cells in the etiology and maintenance of neuropsychiatrie disorders. Further, it will permit a more rational exploration of drugs possessing utility in treating disorders involving dopamine transmission.     

An ultrastructural study of dynorphin-immunoreactive nerve fibers and terminals in the celiac-superior mesenteric ganglion of the guinea pig

Dynorphin-immunoreactive nerve fibers and terminals were identified in the celiac-superior mesenteric ganglion of the guinea pig at the ultrastructural level with the peroxidase-antiperoxidase technique. The immunostained material was localized in the large dense core vesicles of the terminals but was also present diffusely in the axoplasm. The terminals formed numerous axodendritic and a few axosomatic contacts, interpreted as synapses, with the principal ganglion cells. These findings suggest that dynorphin plays a role as a neurotransmitter or neuromodulator in the ganglion and, taken together with earlier findings, indicate an involvement of dynorphin neurons in the intestino-intestinal inhibitory reflex.     

Intranigral injection of dynorphin in combination with substance P on striatal dopamine metabolism in the rat

The effects of intranigral injection of dynorphin(1-13), substance P and their combination on striatal dopamine (DA) metabolism as well as circling behavior were studied in rats. Dynorphin(1-13) produced a significant dose-dependent increase of dihydroxyphenyl acetic acid (DOPAC) concentration as well as the number of contralateral circling. No change in dopamine (DA) concentration was observed. Substance P caused a dose-dependent increase of DOPAC concentration. At large doses it also produced an increase in DA concentration. Combined intranigral dynorphin(1-13) and substance P brought about an additive effect for the increase of DOPAC and the number of contralateral circling. No additive effect was found when dynorphin(1-13) and neurokinin A were injected together intranigrally.     

Effect of Acute Ethanol Administration on the Release of Opioid Peptides From the Midbrain Including the Ventral Tegmental Area

Background:  Experimental evidence suggests that ethanol alters the activity of the endogenous opioid peptide systems in a dose and brain-region dependent manner. These alterations may influence the processes of ethanol reward and reinforcement. Thus, it was the objective of this study to investigate the response of the 3 major opioid peptide systems (endorphins, enkephalins, and dynorphins) to acute ethanol administration, at the level of the midbrain including the ventral tegmental area (midbrain/VTA), a region important for drug, including ethanol reinforcement.
Methods:  Using the in vivo microdialysis technique coupled with specific solid-phase radioimmunoassay for β-endorphin, met-enkephalin, and dynorphin A_1–8, changes in the extracellular concentration of theses peptides at the level of midbrain/VTA were determined at distinct time points following the administration of 0.0 (saline), 0.8, 1.2, 1.6, 2.0, and 2.4 g ethanol/kg B.Wt.
Results:  A biphasic effect of ethanol on β-endorphin release was found, with low to medium (1.2, 1.6, and 2.0 g) but not high (2.4 g) doses of ethanol, inducing a significant increase in the dialysate content of β-endorphin. A late increase in the dialysate content of dynorphin A_1–8 was observed in response to the 1.2 g ethanol dose. However, none of the ethanol doses tested significantly altered the content of met-enkephalin in the dialysate.
Conclusions:  The present findings suggest that the ethanol-induced increase of β-endorphin release at the level of midbrain/VTA may influence alcohol reinforcement.     

Dynorphin B and spinal analgesia: induction of antinociception by the cannabinoids CP55,940, Δ-THC and anandamide

The endogenous opioid dynorphin B was evaluated for its role in cannabinoid-induced antinociception. Previous work in our laboratory has shown that the synthetic, bicyclic cannabinoid, CP55,940, induces the release of dynorphin B whilst the naturally occurring cannabinoid, Δ⁹-tetrahydrocannabinol (Δ⁹-THC), releases dynorphin A. The dynorphins contribute in part to the antinociceptive effects of both cannabinoids at the level of the spinal cord. The present study compares dynorphin B released from perfused rat spinal cord in response to acute administration of anandamide (AEA), Δ⁹-THC and CP55,940 at two time points, 10 min and 30 min post administration, and attempts to correlate such release with antinociceptive effects of the drugs. Dynorphin B was collected from spinal perfusates of rats pretreated with Δ⁹-THC, CP55,940 or AEA. The supernatant was lyophilized and the concentrations of dynorphin B were measured via radioimmunoassay. At a peak time of antinociception (10 min), CP55,940 and Δ⁹-THC induced significant two-fold increases in the release of dynorphin B. AEA did not significantly release dynorphin B. Upon a 30-min pretreatment with the drugs, no significant dynorphin B release was observed, although antinociceptive effects persisted for CP55,940 and Δ⁹-THC. Previous work indicates that Δ⁹-THC releases dynorphin A while AEA releases no dynorphin A. This study confirms that although all three test drugs produced significant antinociception at 10 min, the endocannabinoid, AEA, does not induce antinociception via dynorphin release. Thus, our data indicate a distinct mechanism which underlies AEA-induced antinociception.