The opioid octapeptide Met-enkephalin-Arg-Gly-Leu: Characterization and distribution in rat spinal cord

The regional quantitation, immunohistochemical localization and molecular heterogeneity of Met⁵-enkephalin-Arg⁶-Gly⁷-Leu⁸ were examined in rat spinal cord with a specific radioimmunoassay. A rostrocaudal gradient in Met⁵-enkephalin-Arg⁶-Gly⁷-Leu⁸ content was observed; the highest levels occurred in sacral cord. Dorsal cord content was higher than that of ventral cord at all spinal segments. Immunohistochemical staining supported and refined the latter observation: a dense network of perikarya and fibers was found in Laminae I and II of the dorsal horn. Cell bodies were frequently observed in lamina IV. Additional terminals were seen around the central canal and in the ventral gray matter, often outlining perikarya of motor neurons. Total Met⁵-enkephalin-Arg⁶-Gly⁷-Leu⁸ immunoreactivity could be fractionated into two main components using gel filtration chromatography. Nearly half of the total immunoreactivity eluted as a high molecular weight peptide; the other half which co-eluted with Met⁵-enkephalin-Arg⁶-Gly⁷-Leu⁸ was further identified to be authentic Met⁵-enkephalin-Arg⁶-Gly⁷-Leu⁸ on reverse phase high pressure liquid chromatography. The present data, in conjunction with our previous study of Met⁵-enkephaline and Met⁵-enkephalin-Arg⁶-Phe⁷ indicates that all opioid peptides derived from preproenkephalin A are present in spinal cord and most likely are stored in the same neurons. Immunohistochemical localization of Met⁵-enkephalin-Arg⁶-Gly⁷-Leu⁸ in dorsal and ventral cord suggest a role for this peptide in both sensory and motor integration.     

Increased 2-deoxyglucose uptake in hippocampus, globus pallidus and substantia nigra after cerebral ischemia

Ten minutes of complete cerebral ischemia in the rat results in a short period of increased ¹⁴C-2-deoxyglucose uptake in hippocampus, globus pallidus and substantia nigra, regions which selectively develop ischemic nerve cell changes, possibly as a result of damage to a transmitter system.     

Met-enkephalin: potent contraversive rotation after microinjection in rat substantia nigra

This study reports a novel action of Met⁵-enkephalin in the rat substantia nigra, i.e. potent contraversive rotation that is dose-dependent, site-specific, mimicked by morphine and blocked by naloxone. These results suggest that this pentapeptide may play an important modulatory role in the control of movement in this region of the central nervous system. Alterations in endogenous Met-enkephalin levels in the substantia nigra may be a contributive factor in Parkinson's disease, a movement disorder related to neuropathology of the substantia nigra.     

Severe tactual memory deficits in monkeys after combined removal of the amygdala and hippocampus

Monkeys with bilateral removals of both the amygdaloid complex and hippocampal formation were far more severely impaired on a tactual memory task than were monkeys with removal of either structure alone. These data parallel earlier findings on visual memory in monkeys and suggest that: (i) the memory deficit following combined ablations of the amygdala and hippocampus is multimodal; and (ii) the global anterograde amnesia observed in patients with medial temporal-lobe damage is also due to combined damage to these two structures.     

Distribution of Met-enkephalin-Arg,Phe in rat spinal cord

The distribution of Met⁵-enkephalin-Arg⁶,Phe⁷ (YGGFMRF) in rat spinal cord was determined by a specific RIA and compared with that of Met⁵-enkephalin. The concentration of YGGFMRF on a per mg protein basis was highest in sacral cord and successively decreased in more rostral segments. A similar rostro-caudal distribution was observed for Met⁵-enkephalin. Regional microdissection revealed the dorsal grey matter to be highest in YGGFMRF content followed by ventral gray, ventral white and dorsal white matter; a similar pattern was observed for Met⁵-enkephalin. The ratio of Met⁵-enkephalin. The ratio of Met⁵-enkephalin: YGGFMRF concentration was5.4 ± 0.15 (S.E.M.) on average in all regions measured, indicating a very close quantitative relationshp between the two molecules. Our data suggest that YGGFMRF may act as a precursor of or cotransmitter with Met⁵-enkephalin in spinal cord tissue.     

Immunocytochemical analysis of [Met]enkephalin-Arg-Gly-Leu immunoreactive structures in the rat superior cervical ganglion

Indirect immunofluorescence and immunoelectron microscopy were employed to analyze the enkephalinergic systems in the rat superior cervical ganglion (SCG). These systems were identified using specific antiserum against [Met⁵]Enkephalin-Arg⁶-Gly⁷-Leu⁸ (ENK-8), a peptide which is derived only from proenkephalin A. Abundant ENK-8 like immunoreactive (ENK-8-LI) neurons and fibers were observed in the SCG, but their distribution patterns were heterogenous; ENK-8-LI neurons were localized preferentially in the caudal two-thirds of the SCG, while immunoreactive fibers were found to be distributed more densely in the rostral one-third than in the remaining part of the SCG. Most of the ENK-8-LI neurons were large and had ultrastructural features resembling those of principal cells, some were identified electron microscopically as small intensely fluorescent (SIF) cells. ENK-8-LI fibers were varicose in appearance and surrounded the perikarya of neurons. Since most of these fibers were not detected after experimental decentralization of the SCG and since ENK-8-LI terminals were seen to contain small lucent vesicles, most of the former were thought to be preganglionic fibers. Immunoreactive fibers mainly formed synaptic contacts with the dendrites of non-immunoreactive principal cells, but a small proportion of ENK-8-LI principal cells also received synaptic input from them. Occasionally, immunoreactive fibers formed synapses with the processes or the soma of both ENK-8-LI and non-immunoreactive SIF cells. On the basis of these findings, we conclude that: (1) preganglionic ENK-8-LI fibers terminate mainly on the principal cells, which are devoid of ENK-8-LI structures; (2) the majority of ENK-8-LI neurons are principal cells, while the remainder are SIF cells; (3) inputs to these cells mainly involve structures lacking ENK-8 immunoreactivity; and (4) there are, however, a small number of ENK-8-LI preganglionic fibers which terminate on ENK-8-LI principal cells and SIF cells.     

Distribution of immunoreactive metorphamide (adrenorphin) in discrete regions of the rat brain: Comparison with met-enkephalin-Arg-Gly-Leu

The distribution of immunoreactive (ir)-metorphamide (adrenorphin) in 101 microdissected rat brain and spinal cord regions was determined using a highly specific radioimmunoassay. The highest concentration of metorphamide in brain was found in globus pallidus (280.1 fmol/mg protein). High concentrations of ir-metorphamide (>120 fmol/mg protein) were found in 9 nuclei, including central amygdaloid nucleus, lateral preoptic area, anterior hypothalamic nucleus, hypothalamic paraventricular nucleus, interpeduncular nucleus, periaqueductal grey matter and nucleus of the solitary tract. Moderate concentrations of the peptide (between 60 and 120 fmol/mg protein) were found in 47 brain nuclei such as nucleus accumbens, bed nucleus of stria terminalis, several septal and amygdaloid nuclei, most of the hypothalamic nuclei, ventral tegmental area, red nucleus, raphe nuclei, lateral reticular nucleus, area postrema and others. Low concentrations or ir-metorphamide (<60 fmol/mg protein) were measured in 41 nuclei, e.g., cortical structures, hippocampus, caudate nucleus, thalamic nuclei, supraoptic nucleus, substantia nigra, vestibular nuclei, cerebellum (nuclei and cortex). The olfactory bulb has the lowest metorphamide concentration (5.8 fmol/mg protein). Spinal cord segments exhibit very low peptide concentrations.     

Met-enkephalin-Arg-Gly-Leu in the organ of Corti: high performance liquid chromatography and immunoelectron microscopy

The opioid octapeptide Met-enkephalin-Arg⁶-Gly⁷-Leu⁸ (MERGL) was identified and quantified in the guinea pig cochlea using high performance liquid chromatography and a specific radioimmunoassay. The presence of MERGL immunostaining in efferent endings (coming from the brainstem) within the inner spiral bundle and the tunnel spiral bundle was demonstrated using a pre-embedding immunoelectronmicroscopic technique. Axo-dendritic synapses were observed between the MERGL immunostained varicosities and auditory dendrites. It is hypothesized that MERGL could act, together with Met-enkephalin and other pro-enkephalin A-related peptides, as an efferent neuromodulator or neurotransmitter in the organ of Corti.     

Immunocytochemical survey of haloperidolinduced immunoreactive changes of [Met]enkephalin-Arg-Gly-Leu in the rat forebrain

It has already been demonstrated that chronic treatment with the dopamine receptor blocker, haloperidol, results in an increase of proenkephalin-A-derived peptides in the caudate-putamen (CP). To examine this phenomenon at the cellular level, we used immunocytochemistry to investigate the effects of haloperidol on [Met]enkephalin-Arg⁶-Gly⁷-Leu⁸ (MEAGL) immunoreactivity in the rat forebrain. After daily haloperidol (5 mg/kg, IP, for 6 days) or haloperidol decanoate (70 mg/kg, IM, given once or twice) treatment, immunoreactive neurons appeared diffusely in the whole CP and in the core part of the nucleus accumbens (Acb) and less frequently in the outer shell part of the Acb and the cell-dense layer of the tuberculum olfactorium (TuO). Increase of MEAGL-immunoreactive fibers in the CP, Acb and TuO was also detected after these treatments, a particularly prominent increase being found in the striopallidal terminals in the globus pallidus and ventral pallidum. Haloperidol or haloperidol decanoate had no effect on MEAGL immunoreactivity in the cerebral cortex, amygdala, or hypothalamus. Reserpine treatment (5 mg/kg, IP, for 6 days) caused similar effects on the dorsal and ventral striopallidal system and the direct injection of 6-hydroxydopamine (10 μ/5 μl) into the CP led to the appearance of MEAGL-immunoreactive neurons in accordance with the depleted dopaminergic terminal area. These findings suggest that haloperidol influences enkephalinergic neurons region specifically and that in the dorsal and ventral striopallidal enkephalinergic system haloperidol increases MEAGL immunoreactivity in cell bodies, fibers and terminals by blocking intrastriatal dopaminergic neurotransmission.     

经颅重复性低频磁刺激后大鼠苍白球及黑质HMW MAP-2表达的变化

观察经颅重复性低频磁刺激 (rTMS)对大鼠基底节输出结构高分子量微管相关蛋白 (HMWMAP 2 )阳性标记变化的影响。采用磁刺激器给予大鼠头部 1Hz,10 0mT的重复性刺激 ,然后用免疫组织化学ABC法检测苍白球外侧部 (GPe)、腹侧苍白球 (VGP)及黑质 (SN)等部位HMWMAP 2的免疫阳性产物。较之对照组动物 ,rTMS后苍白球外侧部和腹侧苍白球HMWMAP 2免疫反应物未见减少 ,且染色明显增强 ,阳性标记的纤维增粗 ;黑质特别是黑质网状部 (SNR)亦可见到染色明显增强的HMWMAP 2免疫标记出现。rTMS能够使基底节输出结构中HMWMAP 2的表达发生变化 ,推测可能通过树突重塑而致突触传递的变化等而发挥其临床治疗作用。     

In vivo release of [H]GABA in cat caudate nucleus and substantia nigra. I. Bilateral changes induced by a unilateral nigral application of muscimol

Push-pull cannulae were implanted in both caudate nuclei and both substantiae nigrae (SN) of halothane-anesthetized cats and the release of [³H]GABA, continuously synthesized from [³H]glutamine, was measured in these structures during 4 h of superfusion. In some experiments, multi-unit neuronal activity was recorded at the tip of the nigral push-pull cannulae, using a bipolar electrode. Two hours after the onset of superfusion with [³H]glutamine, 10⁻⁶M of muscimol was added (for 1 h) in the superfusion medium delivered to one SN. This treatment increased locally the release of [³H]GABA and enhanced the neuronal activity of the nigral cells in the zona reticulata. An increased release of [³H]GABA was also observed in the contralateral SN, in association with an inhibition of the activity of the zona reticulata cells. The unilateral nigral application of muscimol also induced asymmetric changes in the release of [³H]GABA in both caudate nuclei, since [³H]GABA release was increased ipsilaterally and reduced on the contralateral side. The present findings are considered in relation to possible GABAergic neuronal populations affected by this local pharmacological treatment.     

Distribution and characterization of opioid peptides derived from proenkephalin A in human and rat central nervous system

In various areas of rat and human brain and spinal cord the distributions of opioid peptides derived from the proenkephalin A precursor, the heptapeptide [Met]enkephalin-Arg⁶-Phe⁷ (MERF), the octapeptide [Met]enkephalin-Arg⁶-Gly⁷-Leu⁸ (MERGL), and bovine adrenal medulla dodecapeptide (BAM-12P), were determined by a combination of radioimmunoassay, gel filtration, and high-performance liquid chromatography.In the human central nervous system the highest concentrations were seen in the striatum (pallidum > caudate nucleus > putamen) and in substantia nigra, hypothalamus, and periaqueductal gray. Similarly, in rat brain high levels were found in striatum and hypothalamus.Bovine adrenal medulladocosa peptide (BAM-22P) only occurred in the rat brain, but could not be detected in human brain. No MERF, MERGL, BAM-12P, or BAM-22P could be found in either rat or human pituitary.In contrast to MERF, MERGL and BAM-12P, peptides derived from the proenkephalin B precursor, dynorphin₁₋₈ and dynorphin B, showed high concentrations only in substantia nigra and pallidum, but quite low levels in the other regions of human brain and spinal cord.The present study provides evidence that the proenkephalin A precursor known from adrenal medulla also exists in the rat and human central nervous system. Moreover, the identification of BAM-12P in these tissues indicates that cleavage of the precursor molecule must also involve sites different from those with paired basic amino acids.     

Application ofl-glutamic acid and substance P to the substantia nigra modulates in vivo [H]serotonin release in the basal ganglia of the cat

L-Glutamic acid or substance P were applied to the caudate nucleus (CN) or substantia nigra (SN) and their effects on local, spontaneous, in vivo [3H]serotonin ([3H]5-HT) release as well as [3H]5-HT release in the contralateral CN and SN were studied using cats implanted with push-pull cannulae. L-Glutamic acid (5 x 10(-5) M), when applied to the CN or SN inhibited the local release of [3H]5-HT but did not affect release in the contralateral CN and SN. In the SN, the L-glutamic acid effect was blocked by L-glutamic acid diethylester. Substance P (10(-7) M) applied to the SN induced an increase in [3H]5-HT release that was delayed in onset. Furthermore, [3H]5-HT release was elevated in the contralateral CN immediately upon the application of substance P to the SN. These results suggest that L-glutamic acid and substance P may control 5-HT transmission in the basal ganglia.     

Vulnerability to stressor-induced disturbances in self-stimulation from the dorsal and ventral A10 area: Differential effects of intraventricular D-Ala-Met-enkephalinamide, D-Ala, N-Me-Phe, Gly-Ol-enkephalin, and D-Pen, D-Pen- enkephalin administration

D-Ala²-Met⁵-enkephalinamide (DALA) (1.0 μg/μl) was administered intraventricularly to mice responding for electrical stimulation from the dorsal or ventral aspects of the VTA immediately prior to footshock (Experiment 1). Predictably, footshock reduced self-stimulation from the dorsal but not the ventral VTA immediately, 24, and 168 h following the stressor. Intraventricular DALA administration effected a partial attenuation of stressor-induced self-stimulation reductions from the dorsal VTA immediately and 24 h poststressor. Deficits appeared among DALA-Shocked mice responding for brain stimulation from the ventral VTA during comparable test intervals. The long-term depressant influence of footshock on self-stimulation from the dorsal VTA was abolished among DALA-treated mice and DALA-associated reductions in self-stimulation from the ventral A10 region among stressed mice were not evident 1 week later. Administration of D-Ala², N-Me-Phe⁴, Gly-Ol⁵-enkephalin (DAGO) (0.01 μg/μl) or D-Pen², D-Pen⁵-enkephalin (DPDPE) (1.0 μg/μl) intraventricularly prior to footshock effected an immediate and a delayed antagonism, respectively, of the stressor on self-stimulation from the dorsal VTA, which persisted for 1 week. Prophylactic administration of 0.001 μg/μl DAGO or 0.01 μg/μl DPDPE prior to the stressor failed to influence self-stimulation from the ventral VTA (Experiment 2). Administration of 0.01 μg/μl DAGO or 1.0 μg/μl DPDPE among mice responding for brain stimulation from the dorsal VTA following footshock produced a weak therapeutic effect immediately poststressor, but effected protracted amelioration of footshock-induced reductions of self-stimulation from the dorsal VTA (Experiment 3). Taken together, μ, δ, and μ-δ activation influenced self-stimulation differentially from the dorsal and ventral VTA according to the temporal order of opioid peptide challenge relative to stressor imposition. These data are discussed with respect to stressors, motivational alterations, and the putative modulating influence of endogenous enkephalin activity in subareas of the VTA.     

Distribution of met-enkephalin-arg-gly-leu-immunoreactive peptides in rat brain: Presence of multiple molecular forms

An antiserum to the opioid octapeptide met⁵-enk-arg⁶-gly⁷-leu⁸ was used to measure the distribution and molecular weight heterogeneity of met⁵-enk-arg⁶-gly⁷-leu⁸-immunoreactive (IR) peptides in rat brain. High concentrations of met⁵-enk-arg⁶-gly⁷-leu⁸-IR peptides were found in the striatum and hypothalamus; low concentrations were observed in the cortex and cerebellum. Intermediate levels of immunoreactivity were found in other brain regions. Thus the distribution of met⁵-enk-arg⁶-gly⁷-leu⁸-IR peptides closely parallels the distribution of met⁵-enkephalin. The immunoreactivity present in brain regions was characterized by gel filtration and reverse-phase high pressure liquid chromatography. In the striatum, more than 85% of the met⁵-enk-arg⁶-gly⁷-leu⁸-IR peptides eluted from a Biogel P-30 column at the position of the authentic octapeptide; the identity of this material was further confirmed by HPLC. Gel filtration of extracts prepared from the medulla-pons resulted in two peaks of immunoreactive material of equal size; one eluted at approximately 8000 daltons and one eluted in the position of the octapeptide. Enzymatic digestion (trypsin + car☐ypeptidase B) of the 8000 dalton-IR peptide resulted in the generation of met⁵-enkephalin-IR. Extracts prepared from other brain regions contained varying amounts of this high molecular weight form of met⁵-enk-arg⁶-gly⁷-leu⁸.     

Dopamine-mediated actions of ephedrine in the rat substantia nigra

Although ephedrine is a centrally active stimulant, its effect on midbrain dopamine neurons is not known. To study the effect of ephedrine on dopamine-containing cells, current-clamp microelectrode recordings were made from substantia nigra pars compacta (SNC) neurons in horizontal brain slice preparations. Ephedrine (100-1000 microM) slowed spontaneous firing and produced a modest concentration-dependent hyperpolarization of membrane potential (EC50 279 microM), with a concomitant net decrease in membrane resistance. These effects were blocked by the D2-like dopamine antagonist sulpiride (1 microM). Electrically evoked inhibitory synaptic potentials mediated by GABAB receptors were reduced 28% by ephedrine. However, ephedrine did not reduce fast synaptic potentials mediated by GABAA or ionotropic glutamate receptors. Inhibition of the GABAB response appeared to be mediated by a postsynaptic mechanism because ephedrine also reduced baclofen-induced hyperpolarization by 28%. Both ephedrine-induced hyperpolarization and inhibition of baclofen-induced hyperpolarization were abolished when slices were superfused with the tyrosine hydroxylase inhibitor alpha-methyl-para-tyrosine (AMPT). Despite perfusion with AMPT, the ability of ephedrine to cause hyperpolarization was restored after perfusing the slice with dopamine (30 microM). Taken together, these results suggest that ephedrine causes hyperpolarization and suppresses GABAB receptor-mediated effects by releasing endogenous dopamine. However, the high concentrations required to observe these effects in vitro suggest that biologically relevant central effects of ephedrine are more likely to be mediated either by non-dopamine systems, such as those involving noradrenaline, or by dopamine systems outside the SNC.     

Glial responses associated with dopaminergic striatal reinnervation following lesions of the rat substantia nigra

Lesioning of dopaminergic substantia nigra pars compacta (SNpc) neurons leads to depletion of dopamine (DA) and dopaminergic axons in the dorsal striatum, followed by subsequent compensatory sprouting of dopaminergic fibers and striatal reinnervation. In this study, the response of striatal glia (microglia and astroglia) was compared with the degeneration and regeneration of dopaminergic axons following SNpc lesions. Following partial SNpc lesions, density of dopamine transporter (DAT) immunoreactive (-ir) terminals in the dorsal striatum returned to normal within 16 weeks of injury, suggesting that dopaminergic reinnervation of the striatum was complete. In conjunction, the glial responses in the dorsal striatum consisted of two peaks. The first peak in glial density occurred immediately after lesioning, peaking at 7 days, implying that it was likely to be associated with removal of debris from degenerating terminals. The second glial response commenced 8 weeks after lesioning and peaked some time after 16 weeks. The time of onset of the second peak suggests that it may be associated with the establishment of synapses rather than with axonal guidance.     

A comparative study on characterization and distribution of cholecystokinin binding sites among the rat, mouse and guinea pig brain

Using the in vitro quantitative receptor autoradiographical technique, [propionyl-3H]propionylated cholecystokinin octapeptide ([3H]pCCK-8) binding sites were investigated in tissue sections of rat, mouse and guinea pig brains. In all the tested animals, [3H]pCCK-8 bound very slowly to the tissue sections. Dissociation was also slow, and had a biphasic profile suggesting CCK-8 binding sites are heterogeneous. Dissociation rate constants were, however, unequal among these species. In the saturation binding studies, both Bmax and (Kd)app values varied among the animal species. The autoradiograms revealed marked species differences in [3H]pCCK-8 binding sites in the brain among 3 closely related species of rodents. [3H]pCCK-8 binding sites were undetectable in the nucleus accumbens/caudate-putamen and the amygdaloid complex of the mouse brain, and scarcely found in the ventromedialis of the hypothalamus of the mouse and guinea pig brain. Furthermore, moderate-to-high densities of [3H]pCCK-8 binding sites were observed in the cerebella of the mouse and guinea pig, whereas in the rat cerebellum the binding sites were undetected. The above-mentioned observations suggest the existence of species differences in the binding pattern of CCK-like peptides among closely related animal species. Furthermore, it would appear that CCK-like peptides in the brain may play different physiological roles among animal species.     

Comparative hydrolysis of extracellular adenine nucleotides and adenosine in synaptic membranes from porcine brain cortex, hippocampus, cerebellum and medulla oblongata

We have investigated the metabolism of extracellular adenine nucleotides and adenosine in porcine brain. The cortex synaptic plasma membranes hydrolyzed ATP to ADP, AMP and adenosine. We also observed a slow hydrolysis of adenosine with the concomitant accumulation of inosine. These results indicate that NTPDase1, NTPDase2, ecto-5'-nucleotidase, and adenosine deaminase are present in cortex synaptic membranes from porcine brain. We further showed that all these enzymes are also abundant in synaptic membranes from hippocampus, cerebellum, and medulla oblongata and compared their specific activities. Brain cortex and hippocampus exhibited higher activities of NTPDase1 and NTPDase2 than cerebellum and medulla oblongata. It was consistent with the high level of the expression of NTPDases in the two first structures. Adenosine deaminase activity was found in all brain structures analyzed; however, it was lower than the activity of ecto-nucleotidases. Taken together, our data suggest that investigated enzymes have a ubiquitous abundance in porcine brain, and observed differences in their activities in cortex, hippocampus, cerebellum, and medulla oblongata may correlate with the pattern of P2 receptor expression in these brain areas. In addition, low activity of adenosine deaminase may indicate that nonenzymatic mechanism(s) are responsible for the termination of P1 receptor signaling in porcine brain.