Brain β-endorphin-like immunoreactivity in adult rats given β-endorphin neonatally

β-endorphin-like immunoreactivity was measured by radioimmunoassay in the brains of adult rats treated neonatally with β-endorphin, naloxone, or vehicle. After treatment with β-endorphin, the decreases observed in β-endorphin-like immunoreactivity in the hypothalamus, pineal, midbrain, pons-medulla, hippocampus, striatum, frontal cortex, occipital cortex, and posterior cortex were highly significant but the 23% decrease in the thalamus was not significantly different from that of control rats. Neonatal administration of naloxone only resulted in a significant decrease in β-endorphin-like immunoreactivity in the hypothalamus. In contrast, no differences were discernible in content of either β-endorphin-like immunoreactivity or ACTH-like immunoreactivity in the pituitary of rats treated with β-endorphin, naloxone, or vehicle in the neonatal period. These same rats had shown an increased threshold to painful thermal stimulation by the tail-flick test after administration of either β-endorphin or naloxone at birth. The results suggest that neonatally injected β-endorphin may alter the levels of β-endorphin-like immunoreactivity in rat brain as well as the response to pain.     

The cloned μ, δ and κ receptors and their endogenous ligands: Evidence for two opioid peptide recognition cores

The opioid peptides are derived from three prohormone precursors referred to as proopiomelanocortin (POMC), proenkephalin (ProEnk) and prodynorphin (ProDyn). Following specific cleavage, several biologically active peptides are generated that can bind the μ, δ and κ receptors. The present study examines the receptor binding affinities of the POMC, ProEnk and ProDyn peptides to the cloned μ, δ and κ receptors expressed transiently in transfected COS-1 cells. Consistent with previous findings using brain homogenates, competition studies demonstrate that no opioid peptide family can be exclusively associated with a specific opioid receptor type. Short ProEnk peptides, such as Leu- and Met-enkephalin are selective for δ, but C-terminally extended peptides such as Met-Enk-Arg-Gly-Leu and Met-Enk-Arg-Phe have a high affinity to μ, δ and κ. Similarly, Peptide E, the BAM peptides, and metorphamide have a high affinity for all three opioid receptors types. While dynorphin A peptides and - and β-neoendorphin have a preference for κ, they also bind the cloned δ and μ receptors. Our findings do not easily fit a simple ‘message-address’ model where the Try-Gly-Gly-Phe core is extended and this gradually alters selectivity. Rather, the pattern appears more discontinuous, and would fit better with the idea of two similar but distinct cores; a Tyr-Gly-Gly-Phe Met- or Leu core that is necessary and sufficient for μ and δ but not κ and a Tyr-Gly-Gly-Phe-Met or Leu core with an Arg-X extension that is equally necessary and sufficient for κ.     

Release into ventriculo-cisternal perfusate of β-endorphin- and met-enkephalin-immunoreactivity: effects of electrical stimulation in the arcuate nucleus and periaqueductal gray of the rat

To examine the resting and evoked release of the endogenous opioid peptides β-endorphin and Met-enkephalin from brain, we examined the levels of the respective immunoreactivities in the lateral ventricle-cisterna magna perfusate of the halothane-anesthetized rat. Ten Hz but not 100 Hz stimulation in the arcuate nucleus (ARC) of the hypothalamus released β-endorphin immunoreactivity (β-EPir) to the perfusate, whereas 100 Hz but not 10 Hz stimulation in the periaqueductal gray (PAG) of the mid brain released Met-enkephalin immunoreactivity (MEir). MEir was not released by stimulation in ARC and β-EPir was not released by stimulation in PAG. Characterization of the released β-EPir and MEir by high performance liquid chromatography showed that authentic β-endorphin and Met-enkephalin were the major constituents of β-EPir and MEir, respectively. Systemic administration of the dopaminergic antagonist haloperidol increased plasma, but not perfusate levels of β-EPir. Both the opioid antogonist naloxone and the NMDA antagonist MK-801 failed to affect β-EPir or MEir release. ARC and PAG stimulation inhibited a nociceptive reflex (tail-dip in 52.5°C water), and naloxone did not reliably reverse this inhibition. These data support the previously suggested possibility of opioid mediation of stimulation induced analgesia, although we were unable to confirm the theory by naloxone reversibility in this study. Furthermore, the data support the assumption that measurement of opioid peptides in cerebrospinal fluid is a relevant approach in research aimed at elucidating the physiological and pathophysiological roles of endogenous opioid peptides.     

Audiogenic seizure susceptibility of C57BL/6 ↔ DBA/2 allophenic mice

Allophenic mice composed of cells from a strain (DBA/2) susceptible to sound-induced seizures and cells from a strain (C57BL/6) resistant to them were produced by embryo aggregation techniques. Twenty-eight allophenic mice were tested for audiogenic seizure susceptibility. The results were compared with the genotypic composition of the coat melanocytes. For those animals with a predominance of one genotype or the other in the coat, their seizure phenotype was the same as that of the strain most represented in the coat. In contrast, those animals with major contributions ofboth genotypes in the coat demonstrated the entire spectrum of susceptibility phenotypes. Such results are likely to be a manifestation of a relatively small target tissue for the genes influencing the development of audiogenic seizure susceptibility.     

Activation of periaqueductal grey pools of β-endorphin by analgetic electrical stimulation in freely moving rats

Electrical stimulation of the ventral midbrain periaqueductal grey (PAG) elicited an antinociception (analgesia) in freely moving rats. Stimulated animals displayed a pronounced decrease in levels of immunoreactive (ir)-β-endorphin (β-EP) in the midbrain PAG. This depletion was selective in that: (1) animals placed in the chamber and not stimulated revealed neither an analgesia nor an alteration in levels of ir-β-EP. (2) No change in levels of ir-β-EP was detectable in other brain regions. (3) Both stimulated rats and rats placed in the chamber and not stimulated revealed a rise in circulating ir-β-RP: the magnitude of this rise did not, however, differ between these groups. (4) Levels of ir-Met-enkephalin, ir-Leu-enkephalin and ir-dynorphin A were modified neither in the PAG nor in other CNS tissues. The data demonstrate that electrical stimulation of the midbrain PAG selectively influences (presumably activates) pools of β-EP therein. Together with our finding that destruction of PAG-localized β-EP neurones to block stimulation-analgesia¹⁴. the data suggest that an activation of intrinsic pools of β-EP underlies stimulation-produced analgesia elicited from the PAG in the rat.     

β-endorphin and methionine-enkephalin levels in discrete brain regions, spinal cord, pituitary gland and plasma of morphine tolerant-dependent and abstinent rats

The effect of morphine tolerance-dependence, protracted and naloxone-precipitated abstinence on the levels of β-endorphin and methionine-enkephalin in discrete brain regions, spinal cord, pituitary gland and plasma was determined in the male Sprague-Dawley rats. Among the brain regions examined, the levels of β-endorphin in descending order were: hypothalamus, amygdala, midbrain, hippocampus, corpus striatum, pons and medulla and cortex. The levels of β-endorphin in midbrain, hypothalamus, and pituitary of morphine tolerant-dependent rats were decreased significantly. During protracted withdrawal β-endorphin levels were decreased in amygdala, spinal cord and pituitary. During naloxone-precipitated abstinence β-endorphin levels were increased in corpus striatum, midbrain and cortex. In addition, in naloxone-precipitated abstinence β-endorphin levels were decreased in pituitary gland and hippocampus but increased in plasma. The levels of methionine-enkephalin in brain regions in decreasing order were: corpus striatum, pons and medulla, amygdala, hypothalamus, midbrain, hippocampus and cortex. The levels of methionine-enkephalin in pons and medulla, amygdala, hippocampus and pituitary gland were decreased in morphine tolerant-dependent rats. During protracted abstinence from morphine, methionine-enkephalin levels in spinal cord, amygdala, pons and medulla, midbrain, cortex, corpus striatum and pituitary gland were decreased. The levels of methionine-enkephalin in hypothalamus and corpus striatum of naloxone-precipitated abstinent rats were increased but were decreased in amygdala and pituitary gland. These results suggest that during morphine tolerance-dependence and during protracted abstinence β-endorphin and methionine-enkephalin levels in discrete brain regions and pituitary gland are decreased. During precipitated abstinence β-endorphin levels are increased in brain regions (except hippocampus) and plasma but decreased in pituitary, whereas methionine-enkephalin levels in amygdala and pituitary gland are decreased except in corpus striatum and hypothalamus where they are increased. The pituitary levels of β-endorphin where reduced in all three conditions. However, the levels after withdrawal were not significantly different from those in tolerant—dependent animals.     

GABA-gated chloride ion influx and receptor binding studies in C57BL6J and DBA2J mice

GABA-gated chloride ion influx was measured in brain 'microsac' preparations of young (20-22-day-old) and older (40-42-day-old) C57BL6J and DBA2J mice. The young DBA2J mice are susceptible to audiogenic seizures. GABA sensitivity was reduced in young DBA2J mice as compared to age-matched C57BL6J mice or older mice of either strain. Age and strain differences in ligand binding to GABA/benzodiazepine receptor complex and glutamate receptor could not account for this finding. These results provide evidence for a defect in GABA-gated chloride ion influx in audiogenic seizure-susceptible DBA2J mice.     

In vivo microdialysis study on changes in septal dynorphin and β-endorphin activities in active and hibernating Columbian ground squirrels

State-dependent changes in extracellular concentration of endogenous opioids in the septum of Columbian ground squirrels were examined in the hibernating and euthermic states using in vivo microdialysis. The order of estimated extracellular concentration was found to be: hibernating > interbout euthermia > non-hibernating euthermia for dynorphin A and interbout euthermia > hibernating > non-hibernating euthermia for β-endorphin. The apparent turnover rates of dynorphin A during hibernation was 15 times greater than that during euthermic non-hibernation phase and that of β-endorphin was 8-fold greater. These results demonstrate that subfamilies of endogenous opioids may vary differentially in their activities at different stages of an annual hibernation cycle and may reflect their different roles in the regulation of hibernation.     

Characterization of cortical spindles in DBA/2 and C57BL/6 inbred mice

Continuous twenty-four hour EEG recordings were conducted on freely-moving DBA/2 and C57BL/6 inbred mice. No brief spindle episodes (BSEs: 6-7 cps, 1-5 sec duration, high amplitude spindle bursts) were seen in the waking EEG of C57BL/6 mice. BSEs were a conspicuous element of the EEG during active waking (AW) and quiet waking (QW) in DBA/2 mice. BSEs occurred at a 10X faster rate in QW than in AW and had a longer duration. Sleep spindle bursts resembling BSEs were seen in both C57BL/6 and DBA/2 mice, and occasionally were observed to follow a K-complex. Rostropontine, but not midpontine, brainstem transection released spindles in both strains. Pentobarbital produced spindles in both strains. The waveforms of the waves comprising BSEs, sleep spindles, transection-induced spindles and barbiturate spindles were quite similar, though differing in frequencies and amplitude. Genetic factors may be critical for the lack of BSEs during AW and QW in C57BL/6 mice and for the occurrence of BSEs during AW in DBA/2 mice. In contrast, most other rodents whow a third pattern: BSEs only during QW. Since C57BL/6 mice can generate spindles under some circumstances, the absence of spindles during waking reflects some alteration in the mechanisms that control the initiation of BSEs rather than a lack of the circuits required to generate a BSE. These mechanisms are distinct from those processes of arousal that produce the background EEG desynchronization of waking. Following both rostropontine and midpontine transection, the background EEG is desynchronized, yet after rostropontine, but not midpontine transection, BSEs occur freely, at a rate over 200 per hour.     

A multivariate morphometric analysis of hippocampal anatomical variation in C57BL/6 ↔ BALB/c chimeric mice

We investigated hippocampal anatomy in artificially-produced chimeras derived by the aggregation of embryos from two widely-studied inbred mouse strains, C57BL/6J and BALB/cJ. Contrary to expectations, the chimeras were not always intermediate between the parental strains. For a number of characters, the chimeras exceeded qualitatively as well as quantitatively the phenotypical range displayed by both inbred parental strains. These findings imply that if only one parent is available for comparison, for instance, in studies involving a normally inviable genotype, separating effects of this genotype from idiosyncratic effects inherent to the chimeric model will be very difficult, if not impossible.     

Strain differences in pituitary β-endorphin and ACTH content in inbred mice

The whole pituitary contents of β-endorphin and ACTH were found to vary widely among 5 inbred strains of mice. β-endorphin values were 2.5-fold different and ACTH values 1.5-fold. Strains low in β-endorphin were also low in ACTH. The existence of genetic differences raises the possibility that there exist, or can be developed, strains with extremely low or high levels of these peptides that would aid research directed at elucidating the physiology of opioid peptides.     

Melatonin exerts its analgesic actions not by binding to opioid receptor subtypes but by increasing the release of β-endorphin an endogenous opioid

The occurrence of systematic diurnal variations in pain thresholds has been demonstrated in human. Salivary melatonin levels change following acute pain when other factors that could explain the change have been removed or controlled. Melatonin-induced analgesia is blocked by naloxone or pinealectomy. By using selective radioligands [3H]-DAMGO, [3H]-DPDPE, [3-U69593, and 3H]-nociceptin, we have shown that the bovine pinealocytes contain delta and mu, but not kappa or ORL1 opioid receptor subtypes. In the present study, by using melatonin receptor agonists (6-chloromelatonin or 2-iodo-N-butanoyl-5-methoxytryptamine) or melatonin receptor antagonist (2-phenylmelatonin), we have shown that these agents do not compete with opioid receptor subtypes. However, we observed a time-dependent release of beta-endorphin an endogenous opioid peptide, by melatonin from mouse pituitary cells in culture. Hence, it is suggested that melatonin exerts its analgesic actions not by binding to opioid receptor subtypes but by binding to its own receptors and increasing the release of beta-endorphin.     

Regional distribution of β-lipotropin converting enzymes in rat pituitary and brain

Among the brain areas studied only pars distalis and pars intermedia are found to contain β-lipotropin activating enzyme indicating that these may be the exclusive organs for a physiologically significant conversion of β-lipotropin into β-endorphin. β-Endorphin inactivating enzyme is found to be rather uniformly distributed in all the pituitary and brain regions, α- and γ-endorphins are presumably formed by the action of this enzyme on β-endorphin.     

Age-related changes in cortico-releasing factor, somatostatin, neuropeptide Y, methionine enkephalin and β-endorphin in specific rat brain areas

We investigated the age-related changes in the tissular protein, cortico-releasing factor (CRF), somatostatin (SOM), neuropeptide Y(NPY), methionine enkephalin (M-ENK) and beta-endorphin (beta-END) levels in frontal cortex, hippocampus, striatum and hypothalamus of young (4-month-old), mature (18-month-old) and senescent (26-month-old) Wistar male rats, bred in a specific pathogen free environment. Between the age of 4 and 18 months, the tissular protein levels increased in all 4 structures studied. The CRF and SOM levels increased in the hippocampus, while the NPY levels decreased. During this time, the NPY content increased in the striatum, whereas the SOM and M-Enk striatal levels decreased. Concomitantly, the NPY and beta-End levels decreased in the hypothalamus. Interestingly, no significant variations were found to occur in the frontal cortex whatever the neuropeptide studied. Between the age of 18 and 26 months, no significant changes in the tissular protein levels were detected, except in the hippocampus. The changes in the neuropeptide concentrations observed during this period depended on the neuropeptide and the brain structure studied. The CRF and beta-End levels decreased in the frontal cortex and the hypothalamus, respectively. The NPY peptidergic systems seem to be preferentially affected by aging processes since 3 out of the 4 structures studied--the frontal cortex, the striatum and the hypothalamus--showed a decrease in their tissular NPY content. During the same period, none of the 5 neuropeptides studied were affected in the hippocampus.     

Pharmacology of β-phenylethylamine-induced seizures in mice

1. 1. The endogenous trace amine β-phenylethylamine (PE) produced tonic-clonic seizures in male Swiss mice when administered in doses of 125–200 mg/kg. The number of mice exhibiting PE-induced seizures, the latency to onset of first seizure and the latency to loss of the righting reflex were dose dependent.

2. 2. Pretreatment with the benzodlazepines diazepam, chlordiazepoxide, midazolam and clonazepam significantly reduced the incidence of PE-induced seizures. Similarly, increasing brain gamma-amlnobutyric acid (GABA) levels by injection of the GABA-transamlnase inhibitors aminooxyacetic acid or gabaculine afforded significant protection against pea's convulsant effects.

3. 3. The data suggest that PE when administered at high doses may interfere either directly or Indirectly with GABAergic neurotransmission in the central nervous system. In addition, since PE induces an epileptiform spiking pattern and produces seizures in rodents It appears possible that the amine may play a role in some forms of human epilepsy.

The effects of selective blockade of delta and mu opiate receptors on ethanol consumption by C57BL/6 mice in a restricted access paradigm

The effects of naltrexone, naltrindole (a selective delta opiate receptor antagonist) and β-funaltrexamine (β-FNA; a selective mu opiate receptor antagonist) on alcohol intake by C57BL/6 mice in a restricted access paradigm were examined. During the pretreatment baseline phase, mice consumed an average of 1.3 g/kg during 1 h access sessions to a 12% alcohol solution. Treatment with naltrexone reduced alcohol consumption to about 50% of that the saline controls. Treatment with β-FNA had no effect on alcohol consumption whereas natrindole reduced consumption to the same extent as that observed with naltrexone. The pattern of findings indicate that naltrexone's ability to reduce alcohol consumption can be attributed to blockade of delta opiate receptors. Implications for treatment in human clinical trials are indicated.     

Genetic analysis of cerebellar folial pattern in crosses of C57BL/6J and DBA/2J inbred mice

Variation in the cerebellar folial pattern of mice is influenced by genetic elements [Inouye, M. and Oda, S.,J. Comp. Neurol., 190 (1980) 357–362]. In crosses of C57BL/6J and DBA/2J inbred mice, the presence or absence of a specific fissure, the intraculminate fissure, is largely determined by a single genetic locus (Cfp-1), which is located on distal Chromosome 4 [Neumann et al.,Brain Res., 524 (1990) 85–89]. In the present study, the mid-sagittal cerebellar folial pattern has been examined in crosses of C57BL/6J and DBA/2J mice and in BXD recombinant inbred strains. At least three loci, includingCfp-1, are involved in variation in vermian pattern formation. Genetic variation in thyroid hormone function may be involved in the inheritance of folial pattern. A locus (Cfp-2) that appears to be partially responsible for this negative genetic correlation in mice may be linked toAfp on Chromosome 5. This hypothesis was suggested by the negative correlation between neonatal serum T₄ level and the number of folia in rats given neonatal injections of thyroxine or propylthiouracil [Lauder, J.M. et al.,Brain Res., 76 (1974) 33–40].     

A comparative study of aggression related changes in brain serotonin in CBA, C57BL, and DBA mice

Spontaneous aggression was induced in 3 strains of male mice (CBA, C57BL, and DBA) by a new technique of socially intermixing them for 14 days and daily measuring the amount of scarring each received. In all strains, serotonin (5-HT) was lower in the ventromedial (VMH) and supraoptic (SO) hypothalamic areas and amygdala in the more aggressive as compared to the controls or less aggressive animals. The correlation coefficients between the amount of physical scarring and brain 5-HT in these areas ranged from 0.50 to 0.91 (P less than 0.05 to P less than 0.01). Hippocampal (HPC) 5-HT was also lower in the more aggressive animals in CBA mice. Also the most aggressive strain (CBA) tended to have the lowest SO, VMH, and amygdala 5-HT concentration. In all 3 strains the concentration of brain 5-HT from lowest to highest was: frontal cortex less than HPC less than amygdala less than VMH less than SO. The data suggest that there is an inverse relationship between hypothalamic and limbic 5-HT and aggressiveness. Also the data suggest that the technique of intermixing 3 strains of mice successfully induced spontaneous aggression which peaked around 14 days and was reliable and could be quantified.     

β-Endorphin Injected into the Nucleus of the Raphe Magnus Facilitates Metastatic Tumor Growth

Electrical stimulation of the periaqueductal gray of the rat's midbrain inducing analgesia leads to an increase in the number of artificial pulmonary metastases from the Walker 256 tumor. In an effort to investigate the influence of the pain suppression system and its associated peptides on this phenomenon, we activated the pain suppression system directly from the Nucleus of the Raphe Magnus, a non-opioid subsystem. After inducing analgesia by direct injection of β-endorphin on the Nucleus of the Raphe Magnus, we noted an increase in the number of artificial pulmonary metastases. This result could be blocked by pretreatment with naloxone. If the Nucleus of the Raphe Magnus was activated by electrical stimulation sufficient to induce analgesia, the metastatic effect was still present but markedly attenuated.