[1]
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The Met-enkephalin content in tissue micropunches of the substantia nigra zona compacta, ventral tegmental area and the caudate nucleus in BALB/c and CBA mouse strains was measured by radioimmunoassay. The CBA strain had significantly higher Met-enkephalin levels in all 3 regions than the BALB/c strain. These differences should be taken into account when relating the intensity of dopamine-induced behaviors to the number of dopamine neurons.
[2]
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Recent studies have focused on differences in anatomical and biochemical features of dopamine (DA) systems in various mouse strains. These include differences in DA levels, metabolites, enzymes involved in DA synthesis, D2-DA receptors and the number of DA neurons in the substantia nigra zona compacta (SNZC) and the ventral tegmental area (VTA)I, 3,8-10,16,19,20. Variations in the spontaneous locomotor and drugelicited activity reflect differences in DA cell number. For example, the BALB/c mice, which have 20-50% (ref. 19) more DA cells than the CBA strain, exhibit more spontaneous locomotion, amphetamine-induced stereotypy and haloperidol-induced catalepsy 6'7. It is suggested that mice of the BALB/c strain exhibit more of these DA-related behaviors than the CBA, because they contain more DA neurons in the SNZC and VTA, and therefore synthesize and release more DA in the striatum and nucleus accumbens septi (NAS) 2°. However, these strains are likely to be different in other morphological and biochemical phenotypes, in addition to DA cell number, which could also account for the differences in DA-related behavior. For example, the endogenous neuropeptide cholecystokinin (CCK), neurotensin, substance P and enkephalins have been found in SNZC and VTA and shown to be involved in DA-related behaviors 5'14,18. Specifically, microinjections of morphine or analogues of the endogenous opioid peptides into the midbrain DA regions have been shown to increase locomotion and stereoty-py2A2A3,15. These effects resemble those seen after the pharmacological activation of DA neurons and can be antagonized either by D A antagonists or with the opiate antagonist naloxone.
[3]
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In this study we sought to determine if there are differences in the levels of Met-enkephalin in specific midbrain DA areas in the BALB/c and CBA strains.
[4]
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Seven-to 8-week-old male mice (Cumberland View Farms. Clinton, TN) were housed in groups of 6, under controlled conditions of temperature (23-25 °C) and illumination (lights on 05.00-19.00 h) and provided with ad lib food and water. After 2-3 days under these conditions, the mice were sacrificed by cervical dislocation, the brains removed rapidly and frozen on dry ice. The brains were mounted on specimen chucks, equilibrated and coronal thin (30 /~m for histology) and thick (200/~m for neurochemistry) sections cut in a cryostat (-14 °C). Using the stained sections and standard atlases for guidance.
[5]
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Correspondence: M.K. Sanghera, Department of Psychiatry, University of Texas Health Science Center, Dallas, TX 75235, U.S.A. bilateral tissue micropunches (0.5 mm inner diameter) of the SNZC and VTA were pooled from 5 animals. Single, bilateral determinations were also made from the caudate nucleus (1.0 mm i.d. of micropunch).
[6]
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Brain samples were pooled in Eppendorf type tubes containing 250/~1 of a solution of 1.6 g glycine dissolved in 100 ml 0.1 N HCI. The glycine-HCl solution lowered the pH sufficiently to inhibit proteolytic enzyme activity and minimizes non-specific binding to carrier proteins. The brain samples were sonicated and flash-frozen on dry ice and stored at -70 °C until assayed. Protein was determined on the remaining pellets 17, using bovine serum albumin standards.
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Samples were thawed at room temperature and spun down. Normal saline (200/~1) was added to 200 /~1 supernatant. Met-enkephalin was then extracted by passing the samples through ODS Sep-pak cartridges. The extracted peptide was eluted with 2 ml of 1% trifluoroacetic acid in 80% methanol. Samples were oxidized by adding 4 ~1 of a 0.06% (w/v) H202 solution. The supernatant was evaporated at 60 °C under nitrogen and the extracted peptide was reconstituted in 500 ~1 assay buffer (0.05 M sodium phosphate, pH 7.4, containing 2.5 mg of human serum albumin per milliliter, 0.02% (w/v) sodium azide, 0.1% mercaptoethanol and a few drops of Phenol red). The pH of reconstituted samples was adjusted back to neutrality by sequential additions of 5 /~1 of 1 N sodium hydroxide.
[8]
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Met-enkephalin levels were measured by the highly specific assay developed by Clement-Jones et al. 4. The assay achieves high specificity by taking advantage of the tendency of the carboxy-terminal methionine residue in Met-enkephalin to undergo spontaneous oxidation to methionine sulfoxide. In the assay, sets of standards and all samples were therefore exposed to oxidizing conditions prior to assay, thus converting the carboxy-terminal methionine residues to methionine sulfoxide. The use of an antiserum, which recognizes the free carboxy-terminal methionine sulfoxide residues of Met-enkephalin sulfoxide then allows highly specific Met-enkephalin measurements. The Met-enkephalin sulfoxide antiserum was raised in a rabbit immunized with Met-enkephalin sulfoxide coupled via its amino-terminal to bovine thyroglobulin by the glutaraldehyde method. The antiserum has no significant cross-reactivity with Leu-enkephalin, fl-endorphin, fl-lipoprotein, Met-enkephalin Arg-Phe, Met-enkephalin Arg-Gly-Leu, or a large number of other hypothalamic, pituitary or gastrointestinal peptides 4.
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Statistical analysis of data consisted of 2-way unbalanced analysis of variance using the method of weighted squares of means to calculate F-ratios and F-probabilities. Multiple comparisons were made by Duncan's new multiple range test for unequal sample size, ct = 0.05.
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Analysis of variance revealed significant difference (P < 0.001) for strains (F-ratio = 27.55), brain areas (F-ratio = 102.64) and no significant interaction for strain x brain area. Multiple comparisons with the Duncan's test revealed significant differences at P < 0.05 level.
[11]
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The results of this study indicate that there are significant differences in Met-enkephalin levels in the SNZC, VTA and caudate nucleus of BALB/c and CBA mouse strains, in addition to the differences in DA cell number. These differences in Met-enkephalin levels could either directly, or indirectly via an opioid peptide-DA interaction, account for the observed differences in DA-related behavior between these two strains. Duncan's test revealed significant differences at P ~< 0,05 level.
[12]
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Evidence favoring an opiate-DA interaction in the midbrain DA systems is substantial in the rat. Anatomical studies have shown that there is a distinct overlap between the distribution of enkephalin immunofluorescence, opiate receptors and DA cell bodies and terminals in the SN, VTA, striatum and the NAS. However, it is unclear whether this opiate-DA interaction is one of excitation or inhibition of the DA system 2'12'13't5.
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Data from this study, in combination with behavioral and biochemical data, would suggest that the opioid peptide Met-enkephalin is inhibitory to midbrain DA systems in influencing DA-related behaviors in the BALB/c and CBA strains. Thus in the latter strain the presence of high levels of Met-enkephatins, as compared to the BALB/c strain, may produce a greater inhibition of DA neuronal firing. Tlais could occur either directly in the SNZC/VTA, or indirectly via excitation of an inhibitory input. The striatonigral GABAergic input to the DA neuron in SNZC is a likely candidate for such an action. Stimulation of such an inhibitory input would result in decreased levels of DA being released from terminals resulting in lower spontaneous and drug-induced activities. If, however, the overall action of the opiates is one of direct excitation of DA neurons, our data would suggest that the opioid peptide has only a minor role in determining the intensity of DA-related behaviors.
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In addition to the opiate peptides, other substances, for example CCK, substance P, and neurotensin, have been shown to be present in midbrain regions and, to be involved in DA-related behaviors 5a8. It is possible that levels of these peptides are also different in the BALB/c and CBA mouse strains. These possibilities therefore must also be determined before any cause-and-effect relationship between DA-related behaviors and the number of midbrain DA neurons can be made.
[15]
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Met-enkephalin antiserum was kindly donated by Dr. P.J. Lowry, St. Bartholomew's Hospital, London, U.K. The technical assistance of Ms. Blanca Gonzalez and the secretarial assistance of Ms. Laura Boynton is gratefully acknowledged. Supported by Grant MH-39699-01.