In situ hybridization histochemical localization of prodynorphin messenger RNA in the central nervous system of the rat

The distribution of preprodynorphin messenger RNA-containing perikarya in the central nervous system of the rat was determined with in situ hybridization histochemistry using a ³⁵S-labelled complementary RNA probe. All of the regions of the central nervous system reported by other investigators to contain perikarya that synthesize prodynorphin-derived peptides, except the pedunculopontine tegmental nucleus, the accessory trigeminal nucleus, and the ventral nucleus of the trapezoid body, also contained perikarya that synthesize preprodynorphin messenger RNA. However, the olfactory bulb, the anterior olfactory nucleus, the islands of Calleja, the CA1-CA3 fields of the hippocampus, the septohippocampal nucleus, the diagonal band of Broca, the basal and cortical amygdaloid nuclei, the entopeduncular nucleus, the subthalamic nucleus, the superior colliculus, the Edinger-Westphal nucleus, the dentate nucleus, the raphes linearis and pontis, the dorsal cochlear nucleus, the medial vestibular nucleus, the inferior olive, and the dorsal motor nucleus of the vagus nerve also contained preprodynorphin messenger RNA-synthesizing perikarya. These observations suggest that prodynorphin-derived peptides have a much more pervasive role in central nervous system function than previously suspected. However, before the physiological significance of these observations can be judged, it will be necessary to determine whether all of the novel sites of preprodynorphin messenger RNA synthesis are sites of prohormone synthesis and conventional processing. J. Comp. Neurol. 384:211-232, 1997. © 1997 Wiley-Liss, Inc.     

强啡肽B免疫反应阳性神经元在大鼠下丘脑的分布及与精氨酸加压素的共存

实验应用免疫细胞化学 PAP法显示了强啡肽 B免疫活性阳性神经元在大鼠下丘脑形态特征和分布特点。结果表明 :强啡肽 B正常条件下免疫活性阳性神经元仅在下丘脑的视上核、室旁核、环核、附属神经分泌核和室管膜。正中隆起处有强啡肽 B免疫活性阳性纤维分布。侧脑室注射秋水仙素后在下丘脑视前区、前区、室周核、前连合核、交叉上核、交叉后核、弓状核、背侧区、背内侧核、穹隆周核、下丘脑外侧区、腹内侧核、室周大细胞区等核区显示了强啡肽 B免疫活性阳性神经元。双标记免疫组织化学方法还显示强啡肽 B与精氨酸加压素主要共存于下丘脑室旁核 ,次为视上核 ,附属神经分泌核和下丘脑外侧区     

Kappa opioid receptor-like immunoreactivity in guinea pig brain: Ultrastructural localization in presynaptic terminals in hippocampal formation

Physiological and pharmacological studies have suggested that kappa opioid receptors (KORs) may be located presynaptically in the guinea pig hippocampal formation. In the present study, KOR-like immunoreactivity (-LI) was examined by using a rabbit antibody raised against a synthetic peptide from the carboxyl terminus of a cloned rat kappa receptor (KT). The specificity of affinity-purified KT antibody was confirmed by Western blotting, enzyme-linked immunosorbent assay, immunolabeling of KORs expressed in Xenopus oocytes, and immunocytochemical preadsorption controls. Specificity also was demonstrated by the light microscopic distribution of KT-LI in sections through the forebrain and the pons, which was largely consistent with the distribution of KORs previously reported, and resembled that of immunoreactivity for dynorphin B, an endogenous ligand for KORs. Detailed analysis of the hippocampal formation revealed that KT-LI was located predominantly in thin processes in the granule cell and inner molecular layers of the dentate gyrus. A few KT-labeled processes were also present in stratum lacunosum-moleculare of the CA1 region and all layers of the CA3 region of the hippocampus. By electron microscopy, KT-LI was restricted to unmyelinated axons and axon terminals, and was associated with plasma membranes, large dense-core vesicles, and cytoplasmic surfaces of small vesicles. In the dentate gyrus, immunolabeled terminals formed asymmetric synapses with granule cell perikarya and large unlabeled dendrites. In the CA3 region of hippocampus, KT-LI was present in small unmyelinated axons. The results of this study 1) demonstrate the specificity of the KT antibody, 2) show that the distribution of KT labeling corresponds well with previous KOR and dynorphin localization in many regions, and 3) provide ultrastructural evidence that KORs are located presynaptically in the guinea pig hippocampal formation. © 1996 Wiley-Liss, Inc.     

Method for isolation of kappa-opioid binding sites by dynorphin affinity chromatography

A kappa-opioid receptor subtype was purified from a digitonin extract of frog brain membranes, using affinity chromatography. The affinity resin was prepared by coupling dynorphin (1-10) to AH Sepharose 4B. The purified receptor binds 4,750 pmol [3H]ethylketocyclazocine (EKC) per mg protein (5,600-fold purification over the membrane-bound receptor) with a Kd of 9.1 nM. The addition of cholesterol-phosphatidylethanolamine (2:1) enhanced 3.6-fold the binding activity of the purified material, which gives a purification very close to the theoretical. The purified receptor protein exhibits high affinity for kappa-selective ligands. The purified fraction shows one major band (65,000 Mr) in sodium dodecyl sulfate (SDS) gel electrophoresis.     

Enkephalin-containing polypeptides in human cerebrospinal fluid

The chemical nature of peptides in human CSF with the enkephalin core sequence from proenkephalin A and proenkephalin B, was investigated. Direct measurements with radioimmunoassay (RIA) were run on enkephalin, enkephalin hexapeptides, dynorphin A, dynorphin A1-8 and dynorphin B. The hexapeptides occurred in about 3 times higher concentration than the corresponding enkephalins. The only dynorphin RIA which gave positive results was the one for dynorphin A. However, most dynorphin A immunoactive material showed higher apparent molecular weight (MW; 3 and 5 kdalton) than the standard (2 kdalton). To identify and quantitate every possible proenkephalin derived peptide with the enkephalin sequence, chromatographic fractions were treated with trypsin. The products, Leu-enkephalin-Arg6 (from proenkephalin B) and Met-enkephalin-Lys6/Arg6 (from proenkephalin A) were measured by specific RIAs and identified by HPLC. In the higher (greater than 5 kdalton) MW interval, there was about 10-fold higher yield of Met-enkephalyl than Leu-enkephalyl hexapeptides. In the intermediate 1-3 kdalton MW interval, most activity derived from proenkephalin B. Finally, from the low MW region, there was about 5 times more proenkephalin A peptides. The main dynorphin A peak at 5 kdalton was transferred to a major Leu-enkephalin-Arg6 peak by trypsin degradation. The data indicate the presence of a whole family of peptides from the two proenkephalin genes in human CSF. Precursors to the peptides supposed to be the active members in the proenkephalin families occur in relatively high concentrations and may provide good markers for activity in these peptide systems.     

Preprodynorphin mRNA‐expressing neurones in the rat parabrachial nucleus: subnuclear localization, hypothalamic projections and colocalization with noxious‐evoked fos‐like immunoreactivity

The dorsal lateral subnucleus of the rat pontine parabrachial nucleus is a major target for ascending nociceptive information from the spinal cord. With in situ hybridization histochemistry, using a radiolabelled cRNA probe, we demonstrate that neurones in and near the dorsal lateral subnucleus express preprodynorphin mRNA. The cRNA probe was constructed from a PCR product amplified from rat genomic DNA. Sequencing of the PCR product revealed that it corresponded to the sequence 466–1101 of the rat preprodynorphin gene exon 4. Tract tracing experiments, using injection of cholera toxin subunit B into the hypothalamic median preoptic nucleus, showed a retrograde labelling pattern of neurones in the parabrachial nucleus that was almost identical to that of the preprodynorphin mRNA expressing neurones. Double-labelling, combining immunohistochemical detection of tracer and in situ hybridization, revealed that the retrogradely labelled neurones expressed preprodynorphin mRNA. A similar double-labelling, combining in situ hybridization with immunohistochemical detection of noxious-evoked fos following formalin injection into one hindpaw of awake animals, showed that almost all fos-immunoreactive neurones in the dorsal lateral parabrachial subnucleus also expressed preprodynorphin mRNA. Quantitative analysis suggested that the evoked fos immunoreactivity was accompanied by an increased preprodynorphin mRNA expression. The findings provide evidence that neurones in the dorsal lateral subnucleus produce dynorphin and project to the median preoptic nucleus, and that noxious stimulation in awake animals synaptically activates the dynorphinergic neurones in this subnucleus. These observations are consistent with the idea of a functional and chemical heterogeneity among different parabrachial subnuclei that serves to produce specific homeostatic responses to stimuli that changes the physiological status of the organism, including tissue damage.     

Rhythmogenesis in vasopressin cells

Many neurones in the central nervous system possess intrinsic pattern-generating properties, including vasopressin magnocellular neurosecretory cells. Synaptic input to vasopressin cells is not rhythmically patterned and yet these neurones fire action potentials in a 'phasic' activity pattern comprised of alternating periods of activity and silence that each last tens of seconds. This review describes the intrinsic and extrinsic mechanisms that generate phasic activity in vasopressin cells, highlighting recent work that has shown phasic activity to result from feedback modulation of synaptic inputs, and of intrinsic membrane properties, by peptides released from the dendrites of vasopressin cells.     

Topography of cocaine-induced gene regulation in the rat striatum: relationship to cortical inputs and role of behavioural context

Psychostimulants alter gene expression in projection neurons of the striatum, and such neuroplasticity is implicated in drug addiction and dependence. Evidence indicates that excitatory inputs from the cortex and thalamus are critical for these molecular changes. In the present study, we determined the topography of cocaine-induced changes in gene expression in the rat striatum and investigated whether these molecular alterations are associated with particular cortical inputs. Acute induction of c-fos (by 25 mg/kg of cocaine), and the c-fos response and dynorphin expression after repeated cocaine treatment (25 mg/kg, 4 days) were assessed as examples for short-term and longer-term molecular changes, respectively. In addition, we examined whether these molecular effects were influenced by the behaviour performed during cocaine action (running-wheel training vs. open field). Our results demonstrate that the overall topography of cocaine-induced gene regulation in the striatum is remarkably stable. Both acute and longer-term molecular changes were maximal in caudal dorsal striatal sectors that receive convergent inputs from the medial agranular and the sensorimotor cortex. In contrast, relatively minor or no effects were found in rostral and ventral striatal sectors. However, running-wheel training under the influence of cocaine enhanced the c-fos response to a subsequent cocaine challenge selectively in parts of the caudal sensorimotor striatum. These results indicate that cocaine produces molecular adaptations preferentially in cortico-basal ganglia circuits through the sensorimotor striatum, and that some of these neuronal changes are influenced by the behaviour performed during drug exposure.     

Immunoreactive dynorphin in human brain and pituitary

The distribution of immunoreactive dynorphin (ir-dyn) has been determined in various regions of human brain and pituitary by use of a highly specific radioimmunoassay. The concentrations of ir-dyn in the substantia nigra (24.5 pmol/g) and hypothalamus were among the highest in the 26 brain areas examined. Substantial amounts were also measurable in other extrapyramidal structures such as the caudate nucleus, pallidus and putamen. Lower concentrations of ir-dyn were detected in the amygdala, hippocampus, periaqueductal gray matter, colliculi, pons, medulla and area postrema, but only low amounts were found in the posterior lobe of the pituitary, while no ir-dyn was detectable in the anterior lobe. By gel permeation chromatography the brain immunoreactivity was shown to consist of 3-4 peaks of apparent molecular weights of about 12,000, 6000, 1800 and less than 1000. It was possible to demonstrate the high opioid potency of 2 of these peaks in the guinea-pig ileum longitudinal muscle bioassay after purification by immunoprecipitation. A comparison of the distribution pattern of ir-dyn revealed some parallels with enkephalin, whereas the distribution of ir-beta-endorphin is quite different.