A rapid and simple micromethod to measure picograms of Norepinephrine (NE) and Dopamine (DA) in small samples of brain tissue and pituitary gland

Zschaeck, L.L. de; Ramírez, Victor D.

doi:10.1007/bf01266305

Cited by 23 publications

(4 citation statements)

References 11 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To further validate the biological response of the CN to locally infused AMPH, DA concentration in perfusates from conscious, unreastrained proestrus rats perfused with either KRP or saline were measured by an established REA [5,52]. This psychostimulant was highly effective in stimulat ing a prompt increase in DA release followed by a return to basal output within 1 h. In 2 diestrous rats (data not shown) similar responses were observed.…”

Section: Discussionmentioning

confidence: 85%

See 1 more Smart Citation

In vivo Output of Dopamine and Metabolites from the Rat Caudate Nucleus as Estimated with Push-Pull Perfusion On-Line with HPLC-EC in Unrestrained, Conscious Rats

et al. 1984

View full text Add to dashboard Cite

In the present experiment we used push-pull perfusion (PPP) on-line with high performance liquid chromatography with electrochemical detection (HPLC-EC) to measure the concentration of neuroactive substances collected in perfusates from the caudate nucleus (CN) of conscious, freely moving rats. To validate the suitability of such an approach, both chromatographic and biological procedures were used. The chromatographic performances of four pure standards, dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindole acetic acid (5-HIAA) were examined under the conditions of the experiment and the release of these four chemicals by amphetamine (AMPH) locally infused into the CN or systemically administered to conscious rats used as an index of biological validation. Distinct dose-response curves were obtained for each standard injected into the HPLC-EC singly or mixed together in perfusion medium (modified Krebs-Ringer’s Phosphate, KRP, pH 7.4). Moreover, each standard in the chromatogram appeared as a well-defined elution band with a different retention time. The brain perfusate samples did not contain factors interfering with the normal operation of the HPLC-EC or measurement of the the concentration of added standards. The recovery of pure DA, DOPAC, 5-HIAA and HVA added to the perfusate samples was 97, 87, 98 and 114%, respectively. No decrements in peak heights were observed in the chromatograms when a 1-ng dose mixture of the four standards dissolved in medium and maintained at 4°C was injected into the HPLC-EC at regular intervals for a 60-min period after initial preparation. In addition, similar concentration levels of DA (8.03 ± 0.72 vs. 8.51 ± 0.62 ng/mg) were detected in male rat corpus striatum (CS) fragments extracted with either perfusion medium or with 0.1 VHCIO4; however, higher levels of DOPAC were found using perfusion medium than 0.1 N NClO₄ (1.85 ± 0.16 vs. 1.15 ± 0.15 ng/mg). To validate biologically the preparation, AMPH was infused either locally into the CN or administered systemically. Infusion of this psychostimulant directly into the CN of a diestrous rat evoked a clear dose-response in DA output (from a basal level of 10–15 pg/min to a maximum of 150 pg/min) without modifying HVA or 5-HIAA output. However, an apparent decrease in DOPAC output immediately following the 10^–6 M dose of AMPH was noticed. When two consecutive doses of AMPH (10^–3 M) were infused into the CN of a diestrous rat at 2-hour intervals, similar increases in DA output were recorded. The basal and AMPH-stimulated output of DA from the CN of proestrous and diestrous rats were also estimated by measuring the concentration of this amine in perfusate samples with an already validated radioenzymatic assay (REA). Comparable data was obtained (5–15 pg/min, DA basal output), corroborating the authenticity of DA determinations by HPLC-EC. In addition, when AMPH was administered intraperitoneally the expected changes in behavior and DA output were also obser...

show abstract

Section: Discussionmentioning

confidence: 85%

“…Acidified medium was used to prepare the DA standard curves. The REA has been described in detail elsewhere [5,52]. Several modifications of the assay were made in order to allow a larger sample size to be used and thus improve detection.…”

Section: Experimental Designmentioning

confidence: 99%

In vivo Output of Dopamine and Metabolites from the Rat Caudate Nucleus as Estimated with Push-Pull Perfusion On-Line with HPLC-EC in Unrestrained, Conscious Rats

et al. 1984

View full text Add to dashboard Cite

show abstract

“…The tissue homogenates and the effluent samples were then centrifuged [5]. On the same day as the perifusion 20-pl sam ples were assayed using a modified, radioenzymatic assay [20] with a sensitivity of 32 pg dopamine per tube when a criterion of twice the blank value is used. Recent modifications to these assay proce dures are described elsewhere [5],…”

Section: Experiments I: Development O F Amphetamine-stimulated Dopaminmentioning

confidence: 99%

Experimental Studies on the Development of Sex Differences in the Release of Dopamine from Striatal Tissue Fragments in vitro

Becker

Ramírez²

1981

Neuroendocrinology

View full text Add to dashboard Cite

Sex differences have been reported in the amphetamine-stimulated release of dopamine from rat striatal tissue fragments in vitro. The present experiments were conducted to further investigate these sex differences. Employing an in vitro perifusion system, we examined the amphetamine-stimulated release of dopamine from striatal tissue of male and female rats: (1) at 4, 19, and 31 days of age; (2) as adults, after receiving hormone treatments neonatally that are known to alter the pattern of gonadotropin release, and (3) as adults, following postpubertal ovariectomy (OVX) or castration (CAST). There were no sex differences in dopamine release at 4, 19 or 31 days of age. Amphetamine elicited significant dopamine release from striatal tissue at all of these ages, regardless of the sex of the donor. There was significant amphetamine-stimulated dopamine release from striatal tissue obtained from both feminized males (CAST at day 2, tested as adults) and control males (CAST at day 15, tested as adults). Striatal tissue obtained from both androgenized females (500 µg testosterone propionate on day 2, OVX day 15, tested as adults) and control females (OVX day 15, tested as adults) also showed significant amphetamine-stimulated dopamine release. These later results are in marked contrast to the results obtained when female rats were ovariectomized postpubertally. Striatal tissue obtained from female rats, OVX as adults, fails to show significant dopamine release when stimulated with amphetamine. In adult OVX females, this attenuation of amphetamine-stimulated dopamine release is seen as early as 11 days after OVX and persists for at least 45 days. However, the effect of CAST in adult males does not differ from its effects in immature males. Striatal tissue obtained from adult males 45 days after CAST, shows significant amphetamine-stimulated release of dopamine. The experiments reported here confirm our previous results of a sex difference in the responsiveness of striatal tissue to amphetamine stimulation. We conclude that this sex difference in amphetamine-stimulated dopamine release from the striatum is not a function of the organizational effects of neonatal androgen exposure, but develops later in life, perhaps in association with puberty.

show abstract

“…The tissue homogenate and effluent samples were then centrifuged (Beckman J-21B centrifuge. JCF-2 swinging bucket rotor) at 5,000 rpm for 45 min at 4 C. On the same day of the perifusion, 20-//1 samples were assayed using a modified, sensitive microradioenzymatic assay [24] with a sensitivity close to 32 pg/tube for both NE and DA, when a criterion o f 2 SD from blank values is used.…”

Section: Methodsmentioning

confidence: 99%

Dynamics of Endogenous Catecholamine Release from Brain Fragments of Male and Female Rats

Becker

Ramírez

1980

Neuroendocrinology

View full text Add to dashboard Cite

A continuous flow perifusion system was used to measure the in vitro release rate, and percent release of endogenous norepinephrine (NE) and dopamine (DA) from rat brain fragments. Catecholamines (CA) were measured simultaneously in effluent fractions and in tissue fragments following perifusion using a modified radioenzymatic assay. Employing this perifusion system allows not only quantification of endogenous release, but also permits study of the dynamics of the release process. To validate the system, NE and DA release were measured in effluent from medial basal hypothalamus (MBH) and caudate nucleus (CN) fragments under various temperature and ionic conditions. K⁺-stimulated release (60 mM KCl), but not the basal release rate, is temperature and calcium dependent. Marked regional differences are seen in the parameters considered. DA release rate from the CN in male rats was 30 times greater than from the MBH. The percent of DA release was 2.5 times greater and there was a 3 times greater final tissue concentration of DA in the CN than in the MBH. Although the release rate of NE from CN fragments was higher than that of the MBH, it did not reach statistical difference. However, the percent of NE release from the CN was close to 4 times greater than the percent release from the MBH, and final tissue concentrations of NE were 87.9 pg/ mg in the CN compared to 1,240 pg/mg in the MBH (postperifusion values). In a group of female rats, only the percent release of NE from the MBH was significantly different from that of similar tissue from male rats. However, the release rate and percent release of DA from the MBH of female rats were significantly lower than that of male rats. It appears that under this in vitro condition there are marked regional and sex differences in the metabolism of CA between the CN and the MBH fragments of male and female rats. Implications of these findings are briefly discussed.

show abstract

A rapid and simple micromethod to measure picograms of Norepinephrine (NE) and Dopamine (DA) in small samples of brain tissue and pituitary gland

Cited by 23 publications

References 11 publications

In vivo Output of Dopamine and Metabolites from the Rat Caudate Nucleus as Estimated with Push-Pull Perfusion On-Line with HPLC-EC in Unrestrained, Conscious Rats

In vivo Output of Dopamine and Metabolites from the Rat Caudate Nucleus as Estimated with Push-Pull Perfusion On-Line with HPLC-EC in Unrestrained, Conscious Rats

Experimental Studies on the Development of Sex Differences in the Release of Dopamine from Striatal Tissue Fragments in vitro

Dynamics of Endogenous Catecholamine Release from Brain Fragments of Male and Female Rats

Contact Info

Product

Resources

About