It seems appropriate in this Special Issue of Journal of Neuroendocrinology, which marks the new ... more It seems appropriate in this Special Issue of Journal of Neuroendocrinology, which marks the new Pan American Neuroendocrine Society (PANS), to recognize the first group of international scientists who were linked by their common purpose to promote neuroendocrinology throughout the world. This article is protected by copyright. All rights reserved.
In this paper, by means of a superfusion technique, we have examined the effect of ovine prolacti... more In this paper, by means of a superfusion technique, we have examined the effect of ovine prolactin on dopaminergic neurons innervating the dorsal striatum, ventral striatum and the mediobasal hypothalamus of male rats. Fragments from dorsal striatum were superfused with ovine prolactin dissolved in normal medium (Krebs-Ringer phosphate) or medium containing tetrodotoxin (TTX, 10 6 M). Ovine prolactin stimulated the in vitro release of dopamine from dorsal and ventral striatal fragments. In dorsal striatal fragments a linear dose-dependent dopamine release was observed only when fragments were superfused with Krebs-Ringer phosphate-TTX medium. In addition, [LeuS]enkephalin (10-6 and 10-5 M) decreased the prolactin-induced in vitro dopamine release from dorsal striatal fragments superfused with Krebs-Ringer phosphate-TTX medium. Ovine prolactin (10 9-105 M) did not elicit changes in dopamine, 3,4-dihydroxyphenylacetic acid or 5-hydroxyindoleacetic acid outputs from mediobasal hypothalamic fragments superfused with Krebs-Ringer phosphate medium containing TTX. The possible regulatory mechanisms of ovine prolactin on dopaminergic neurons are discussed.
In the present experiment we examined the spontaneous in vitro dopamine (DA) efflux from superfus... more In the present experiment we examined the spontaneous in vitro dopamine (DA) efflux from superfused corpus striatum (CS) of male rats at 3-hr intervals throughout a 24-hr photoperiod (lights on 0500-1900 hr). Maximal mean efflux, and post-superfusion DA concentrations were obtained at 0600 hr. With the exception of 0600 hr, mean efflux was lower during the light compared to the dark phase. Interestingly, the direction of the efflux profiles also varied as a function of time demonstrating increasing, decreasing and relatively stable profiles over the superfusion period. These changes in overall mean efflux, post-superfusion tissue concentration and efflux rate profile direction indicate that circadian processes play a complex role upon the synthesis/release process of DA from the nigrostriatal dopaminergic system that is revealed under the dynamic conditions of in vitro superfusion of isolated CS fragments.
Journal of Psychiatry Neuroscience Jpn, May 1, 1996
Our laboratory has pioneered the use of bovine serum albumin (BSA) linked to different positions ... more Our laboratory has pioneered the use of bovine serum albumin (BSA) linked to different positions of the ring structure of progesterone to investigate steroid-membrane interactions. The complex can be radioiodinated to demonstrate the existence of specific membrane progesterone binding sites in the rat brain. Not only are these progesterone complexes specific ligands, but they also elicit functional responses in the central nervous system (CNS), particularly in the corpus striatum (CS) where progesterone-BSA conjugates linked at C-3 and C-11 positions (P-3-BSA and P-1 1-BSA) alter amphetamine-evoked dopamine release. In this communication we will report our current studies that use radioiodinated progesterone-BSA conjugates (P-3-125I-BSA, P-6-1251-BSA, and P-11-125I-BSA) and estradiol-BSA conjugates linked at C-6 position (178-E-6-125I-BSA and 17-E-6-BSA) to demonstrate the existence of specific membrane binding sites for progesterone and estrogen in several regions of the rat brain. In addition, initial studies to isolate and purify these membrane binding sites from digitonin-solubilized P2-membrane fractions by affinity chromatography are reported. The data indicate that these sites are part of a complex membrane receptor for either estrogen or progesterone, the so-called membrane estrogen receptor (mER) and the membrane progesterone receptor (mPR), respectively.
Dopamine-releasing protein (DARP) is a potent regulator of dopamine (DA) release known to be invo... more Dopamine-releasing protein (DARP) is a potent regulator of dopamine (DA) release known to be involved in the development of rat catecholaminergic systems. In the present study, we examined the internalization and transport of DARP-36aa, a synthetic peptide derived from the N-terminal sequence of DARP, in rat C6 glioma cells. A colloidal gold DARP-36aa conjugate (DARP-36aa: AU) and biotinylated DARP-36aa were employed to visualize internalization and intracellular transport of DARP-36aa. Electron microscopy demonstrated that DARP-36aa: AU was rapidly incorporated into C6 glioma cells. Internalization via clathrin-coated pits and vesicles was clearly observed followed by transport and sorting of DARP-36aa: AU into multivesicular bodies, tubulo-vesicular endosomes, and lysosomes. Internalization of DARP-36aa: AU was also examined in primary mesencephalic cell cultures where a similar pattern of internalization and transport via clathrin-coated pits and vesicles was observed. Fluorescence microscopy using a biotinylated DARP-36aa/avidin-rhodamine conjugate revealed that DARP-36aa is diffusely distributed on the plasmalemma prior to internalization at 4 degrees C. Following a 30-min incubation at 37 degrees C DARP-36aa was concentrated in the cytosol, particularly in areas surrounding cellular projections and the perikaryon. Immunocytochemical studies employing biotinylated DARP-36aa and an anti-clathrin heavy chain antibody demonstrated that DARP-36aa and clathrin colocalize during DARP-36aa internalization. We also observed a marked increase in tyrosine phosphorylation of a 45-kD protein in response to DARP-33a stimulation in C6 glioma cells. Genistein, a specific tyrosine kinase inhibitor, significantly inhibited DARP-36aa: AU internalization and transport in C6 glioma cells. These findings suggest that tyrosine kinase activity may result in DARP-36aa receptor-mediated endocytosis.
In vivo release of dopamine (DA) and its metabolites were determined following a direct infusion ... more In vivo release of dopamine (DA) and its metabolites were determined following a direct infusion of 3,4-dihydroxyphenylalanine (L-DOPA) through a push pull cannula in the caudate nucleus of unanesthetized, freely behaving rats. L-DOPA infusions increased the release of DA and dihydroxyphenylacetic acid (DOPAC) beginning with 10 s M L-DOPA, while homovanillic acid (HVA) was released consistently only following 10 ~ M L-DOPA. Maximal release of DA preceded that of DOPAC which preceded that of HVA. No salient changes in 5-hydroxyindoleacetic acid or behavior were observed folowing any L-DOPA dose.
Eight female and eight male New Zealand White rabbits were outfitted with push-pull cannulae aime... more Eight female and eight male New Zealand White rabbits were outfitted with push-pull cannulae aimed at the tuberal region of the hypothalamus and were used in 19 perfusion experiments. Animals were treated under 2 conditions: a control condition in which female (n = 4) and male (n = 4) rabbits were perfused only with artificial medium for 5-9 h, and an experimental condition in which female (n = 4) and male (n = 5) rabbits were subjected to perfusion with artificial medium, followed by 6 pulses of progesterone (P4) (10 min on, 30 min off; 10 ng/ml) during a 240-min period. Two female rabbits were also subjected to perfusion with artificial medium, followed by 6 pulses of cholesterol (10 min on, 30 min off; 10 ng/ml) during a 240-min period. The LHRH concentration in perfusates collected every 10 min was measured by RIA. In the 4 females undergoing 6- to 7-h control push-pull perfusions, spontaneous pulses were observed, with about 1 pulse every 60 min, and a variable amplitude of the LHRH signal, with a mean release of about 0.91 pg/10 min. In the 4 does treated with pulsatile P4 at 10 ng/ml, the mean LHRH release rate increased significantly from 0.91 +/- 0.13 to 1.66 +/- 0.20 pg/10 min (P less than 0.035), primarily due to an increase in the amplitude of the LHRH pulses, which were significantly greater than controls. This response occurred with an apparent mean latency of about 50 min. Cholesterol pulses did not affect the spontaneous activity of the LHRH neural apparatus. In the 4 males undergoing 5- to 9-h control push-pull perfusions, spontaneous LHRH pulses were observed with about 1 pulse every 60 min, and the mean release increased from 1.25 +/- 0.56 to 1.54 +/- 0.55 (P less than 0.035, by Wilcoxon's matched pairs signed rank test) in the late afternoon primarily due to an increase in the amplitude of the pulses. P4 pulses did not affect the spontaneous activity of the LHRH neural apparatus compared to that in the control animals. Overall, these results clearly demonstrate that although spontaneous pulsatile LHRH release from the hypothalamus of awake unrestrained female and male rabbits is similar, pulses of P4 can activate only the female LHRH neural apparatus, with an apparent latency to peak LHRH release of approximately 50 min. In addition, there appears to be an increase in LHRH mean release levels in the late afternoon to early evening in male rabbits.
In the present experiment we used push-pull perfusion (PPP) on-line with high performance liquid ... more 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 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 degrees C was injected into the HPLC-EC at regular intervals for a 60-min period after initial preparation.(ABSTRACT TRUNCATED AT 250 WORDS)
The mechanism by which adrenal medulla transplants to the brain of Parkinson's patients a... more The mechanism by which adrenal medulla transplants to the brain of Parkinson's patients ameliorate the symptoms of this neurodegenerative disease is unknown. It has been postulated but not proven that these transplants secrete a neurotrophic factor(s) responsible for the activation of the dopamine (DA) neurons. DA-releasing protein (DARP) was identified by immunocytochemistry in cells of the adrenal gland but not in tissues such as the liver or spleen. In the adrenal gland the immunopositive DARP cells were distributed both in the cortex as well as in the medulla. In the former, the distribution was rather uniform covering a broad area of the zona glomerulosa and fasciculata without positive cells in the zona reticularis. In the adrenal medulla the DARP signal was found in segregated cells forming clusters similar to the homotypic distribution of norepinephrine and epinephrine cells. The immunopositive cells for DARP were oval or spindle in shape and the staining was localized in the cytoplasm surrounding the nucleus. In addition, in this paper we present evidence confirming that the adrenal gland is the source of a protein that after immunopurification in nanogram amounts releases DA from corpus striatum fragments superfused in vitro. These data clearly demonstrate the existence of DARP in the adrenal gland and support the hypothesis that the effectiveness of the adrenal medulla transplants to the brain of Parkinson's patients is in part due to the secretion of neurotrophic factors. We submit that DARP is one of such factors.
In the present study, we have investigated the effects of intermittent dibutyryl cyclic AMP (dbcA... more In the present study, we have investigated the effects of intermittent dibutyryl cyclic AMP (dbcAMP, 5 X 10(-8) M), butyrate (5 X 10(-8) M) and forskolin (10(-4) M) on immunoreactive luteinizing hormone-releasing hormone (LHRH) release from superfused hypothalamic fragments from intact male rats of age 25, 30, 45, or 60-75 day (adult). The results indicate that at 25 days of age, male rat hypothalami were most responsive to cyclic AMP (162% of preinfusion basal LHRH release); by 30 days of age, dbcAMP also elicited increased LHRH release (120% of basal). By 45 days of age, the dbcAMP effect on in vitro LHRH release was slightly inhibitory (78% of basal); however, by adulthood, the effect of this cyclic nucleotide on LHRH release was minimal (92% of basal). Butyrate also induced age-dependent modifications in in vitro LHRH release from male rat hypothalami, with slight increases following butyrate delivery at 25 days of age (115%), slight decreases at 30 (82%) and 45 days of age (68%), and little change in adulthood (94%). This latter finding emphasizes the importance of using butyrate as a control for butyryl derivatives of cyclic AMP, which are known to liberate butyric acid as a product of hydrolysis of parent compounds. To address the possibility that the lack of effectiveness of dbcAMP in the older animal preparations was solely due to an increasing sensitivity of male rat medio-basal hypothalamus fragments to butyrate, we examined the effect of forskolin (10(-4) M), an adenylate cyclase stimulator, on LHRH release.(ABSTRACT TRUNCATED AT 250 WORDS)
The effects of varying doses (1.25, 2.5, and 5.0 mg/kg, ip) of D-amphetamine sulfate (AMPH) on ei... more The effects of varying doses (1.25, 2.5, and 5.0 mg/kg, ip) of D-amphetamine sulfate (AMPH) on eight individual behaviors (Rearing, Grooming, Sniffing, Stationary, Gnawing, Head Bobbing, "Sleeping," and Licking) of Castrate + Oil-treated, Castrate + Testosterone Propionate (TP)-treated, and Intact male rats were examined. For Stationary, Sniffing, and "Sleeping" at 1.25 mg/kg AMPH and Rearing and Sniffing at the 2.5 mg/kg dose a significantly greater duration in the behavioral score was obtained for Castrate + Oil versus Castrate + TP and Intact males. These results indicate the complexity of the AMPH dose-response effects upon measurable behaviors, the alteration in the duration of these effects as a function of the hormonal condition of the male rat, and the importance of examining discrete components of behavior when hormone-amphetamine interactions are examined.
In this report, using a push-pull perfusion technique, we examined in vivo the effects of exogeno... more In this report, using a push-pull perfusion technique, we examined in vivo the effects of exogenous dopamine (DA) on the output of neurochemical substances from the caudate nucleus (CN) of freely behaving rats. Exogenous DA, at concentrations of 10(-5) M, 5 x 10(-5) M and 10(-4) M, in modified Krebs-Ringer phosphate medium (KRP) was infused directly into the CN for 15 min each. Exogenous DA at the doses tested elicited increases in 3,4-dihydroxyphenylacetic acid (DOPAC) output in a dose-dependent manner. In addition, the higher two doses of exogenous DA also induced increases in homovanillic acid (HVA) output from the rat CN. The increases in DOPAC output by 5 x 10(-5) M DA was partially blocked by the inclusion of 10(-3) M nomifensine in KRP. Interestingly, exogenous DA-induced increases in HVA output were little affected by the nomifensine treatment. However, the catabolism of exogenous DA was almost completely eliminated by a prior 6-hydroxydopamine lesion in the ipsilateral substantia nigra. Furthermore, infusions of exogenous DA did not change 5-hydroxyindoleacetic acid output from the CN. In conclusion, our results confirm in vivo that (a) DA catabolic pathway via DOPAC intermediate predominates over the alternative pathway via 3-methoxytyramine, (b) increases in extracellular DA will lead to increases in DOPAC and HVA levels in extracellular space and (c) the majority of the DA is oxidized by intradopaminergic monoamine oxidase.
It seems appropriate in this Special Issue of Journal of Neuroendocrinology, which marks the new ... more It seems appropriate in this Special Issue of Journal of Neuroendocrinology, which marks the new Pan American Neuroendocrine Society (PANS), to recognize the first group of international scientists who were linked by their common purpose to promote neuroendocrinology throughout the world. This article is protected by copyright. All rights reserved.
In this paper, by means of a superfusion technique, we have examined the effect of ovine prolacti... more In this paper, by means of a superfusion technique, we have examined the effect of ovine prolactin on dopaminergic neurons innervating the dorsal striatum, ventral striatum and the mediobasal hypothalamus of male rats. Fragments from dorsal striatum were superfused with ovine prolactin dissolved in normal medium (Krebs-Ringer phosphate) or medium containing tetrodotoxin (TTX, 10 6 M). Ovine prolactin stimulated the in vitro release of dopamine from dorsal and ventral striatal fragments. In dorsal striatal fragments a linear dose-dependent dopamine release was observed only when fragments were superfused with Krebs-Ringer phosphate-TTX medium. In addition, [LeuS]enkephalin (10-6 and 10-5 M) decreased the prolactin-induced in vitro dopamine release from dorsal striatal fragments superfused with Krebs-Ringer phosphate-TTX medium. Ovine prolactin (10 9-105 M) did not elicit changes in dopamine, 3,4-dihydroxyphenylacetic acid or 5-hydroxyindoleacetic acid outputs from mediobasal hypothalamic fragments superfused with Krebs-Ringer phosphate medium containing TTX. The possible regulatory mechanisms of ovine prolactin on dopaminergic neurons are discussed.
In the present experiment we examined the spontaneous in vitro dopamine (DA) efflux from superfus... more In the present experiment we examined the spontaneous in vitro dopamine (DA) efflux from superfused corpus striatum (CS) of male rats at 3-hr intervals throughout a 24-hr photoperiod (lights on 0500-1900 hr). Maximal mean efflux, and post-superfusion DA concentrations were obtained at 0600 hr. With the exception of 0600 hr, mean efflux was lower during the light compared to the dark phase. Interestingly, the direction of the efflux profiles also varied as a function of time demonstrating increasing, decreasing and relatively stable profiles over the superfusion period. These changes in overall mean efflux, post-superfusion tissue concentration and efflux rate profile direction indicate that circadian processes play a complex role upon the synthesis/release process of DA from the nigrostriatal dopaminergic system that is revealed under the dynamic conditions of in vitro superfusion of isolated CS fragments.
Journal of Psychiatry Neuroscience Jpn, May 1, 1996
Our laboratory has pioneered the use of bovine serum albumin (BSA) linked to different positions ... more Our laboratory has pioneered the use of bovine serum albumin (BSA) linked to different positions of the ring structure of progesterone to investigate steroid-membrane interactions. The complex can be radioiodinated to demonstrate the existence of specific membrane progesterone binding sites in the rat brain. Not only are these progesterone complexes specific ligands, but they also elicit functional responses in the central nervous system (CNS), particularly in the corpus striatum (CS) where progesterone-BSA conjugates linked at C-3 and C-11 positions (P-3-BSA and P-1 1-BSA) alter amphetamine-evoked dopamine release. In this communication we will report our current studies that use radioiodinated progesterone-BSA conjugates (P-3-125I-BSA, P-6-1251-BSA, and P-11-125I-BSA) and estradiol-BSA conjugates linked at C-6 position (178-E-6-125I-BSA and 17-E-6-BSA) to demonstrate the existence of specific membrane binding sites for progesterone and estrogen in several regions of the rat brain. In addition, initial studies to isolate and purify these membrane binding sites from digitonin-solubilized P2-membrane fractions by affinity chromatography are reported. The data indicate that these sites are part of a complex membrane receptor for either estrogen or progesterone, the so-called membrane estrogen receptor (mER) and the membrane progesterone receptor (mPR), respectively.
Dopamine-releasing protein (DARP) is a potent regulator of dopamine (DA) release known to be invo... more Dopamine-releasing protein (DARP) is a potent regulator of dopamine (DA) release known to be involved in the development of rat catecholaminergic systems. In the present study, we examined the internalization and transport of DARP-36aa, a synthetic peptide derived from the N-terminal sequence of DARP, in rat C6 glioma cells. A colloidal gold DARP-36aa conjugate (DARP-36aa: AU) and biotinylated DARP-36aa were employed to visualize internalization and intracellular transport of DARP-36aa. Electron microscopy demonstrated that DARP-36aa: AU was rapidly incorporated into C6 glioma cells. Internalization via clathrin-coated pits and vesicles was clearly observed followed by transport and sorting of DARP-36aa: AU into multivesicular bodies, tubulo-vesicular endosomes, and lysosomes. Internalization of DARP-36aa: AU was also examined in primary mesencephalic cell cultures where a similar pattern of internalization and transport via clathrin-coated pits and vesicles was observed. Fluorescence microscopy using a biotinylated DARP-36aa/avidin-rhodamine conjugate revealed that DARP-36aa is diffusely distributed on the plasmalemma prior to internalization at 4 degrees C. Following a 30-min incubation at 37 degrees C DARP-36aa was concentrated in the cytosol, particularly in areas surrounding cellular projections and the perikaryon. Immunocytochemical studies employing biotinylated DARP-36aa and an anti-clathrin heavy chain antibody demonstrated that DARP-36aa and clathrin colocalize during DARP-36aa internalization. We also observed a marked increase in tyrosine phosphorylation of a 45-kD protein in response to DARP-33a stimulation in C6 glioma cells. Genistein, a specific tyrosine kinase inhibitor, significantly inhibited DARP-36aa: AU internalization and transport in C6 glioma cells. These findings suggest that tyrosine kinase activity may result in DARP-36aa receptor-mediated endocytosis.
In vivo release of dopamine (DA) and its metabolites were determined following a direct infusion ... more In vivo release of dopamine (DA) and its metabolites were determined following a direct infusion of 3,4-dihydroxyphenylalanine (L-DOPA) through a push pull cannula in the caudate nucleus of unanesthetized, freely behaving rats. L-DOPA infusions increased the release of DA and dihydroxyphenylacetic acid (DOPAC) beginning with 10 s M L-DOPA, while homovanillic acid (HVA) was released consistently only following 10 ~ M L-DOPA. Maximal release of DA preceded that of DOPAC which preceded that of HVA. No salient changes in 5-hydroxyindoleacetic acid or behavior were observed folowing any L-DOPA dose.
Eight female and eight male New Zealand White rabbits were outfitted with push-pull cannulae aime... more Eight female and eight male New Zealand White rabbits were outfitted with push-pull cannulae aimed at the tuberal region of the hypothalamus and were used in 19 perfusion experiments. Animals were treated under 2 conditions: a control condition in which female (n = 4) and male (n = 4) rabbits were perfused only with artificial medium for 5-9 h, and an experimental condition in which female (n = 4) and male (n = 5) rabbits were subjected to perfusion with artificial medium, followed by 6 pulses of progesterone (P4) (10 min on, 30 min off; 10 ng/ml) during a 240-min period. Two female rabbits were also subjected to perfusion with artificial medium, followed by 6 pulses of cholesterol (10 min on, 30 min off; 10 ng/ml) during a 240-min period. The LHRH concentration in perfusates collected every 10 min was measured by RIA. In the 4 females undergoing 6- to 7-h control push-pull perfusions, spontaneous pulses were observed, with about 1 pulse every 60 min, and a variable amplitude of the LHRH signal, with a mean release of about 0.91 pg/10 min. In the 4 does treated with pulsatile P4 at 10 ng/ml, the mean LHRH release rate increased significantly from 0.91 +/- 0.13 to 1.66 +/- 0.20 pg/10 min (P less than 0.035), primarily due to an increase in the amplitude of the LHRH pulses, which were significantly greater than controls. This response occurred with an apparent mean latency of about 50 min. Cholesterol pulses did not affect the spontaneous activity of the LHRH neural apparatus. In the 4 males undergoing 5- to 9-h control push-pull perfusions, spontaneous LHRH pulses were observed with about 1 pulse every 60 min, and the mean release increased from 1.25 +/- 0.56 to 1.54 +/- 0.55 (P less than 0.035, by Wilcoxon's matched pairs signed rank test) in the late afternoon primarily due to an increase in the amplitude of the pulses. P4 pulses did not affect the spontaneous activity of the LHRH neural apparatus compared to that in the control animals. Overall, these results clearly demonstrate that although spontaneous pulsatile LHRH release from the hypothalamus of awake unrestrained female and male rabbits is similar, pulses of P4 can activate only the female LHRH neural apparatus, with an apparent latency to peak LHRH release of approximately 50 min. In addition, there appears to be an increase in LHRH mean release levels in the late afternoon to early evening in male rabbits.
In the present experiment we used push-pull perfusion (PPP) on-line with high performance liquid ... more 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 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 degrees C was injected into the HPLC-EC at regular intervals for a 60-min period after initial preparation.(ABSTRACT TRUNCATED AT 250 WORDS)
The mechanism by which adrenal medulla transplants to the brain of Parkinson's patients a... more The mechanism by which adrenal medulla transplants to the brain of Parkinson's patients ameliorate the symptoms of this neurodegenerative disease is unknown. It has been postulated but not proven that these transplants secrete a neurotrophic factor(s) responsible for the activation of the dopamine (DA) neurons. DA-releasing protein (DARP) was identified by immunocytochemistry in cells of the adrenal gland but not in tissues such as the liver or spleen. In the adrenal gland the immunopositive DARP cells were distributed both in the cortex as well as in the medulla. In the former, the distribution was rather uniform covering a broad area of the zona glomerulosa and fasciculata without positive cells in the zona reticularis. In the adrenal medulla the DARP signal was found in segregated cells forming clusters similar to the homotypic distribution of norepinephrine and epinephrine cells. The immunopositive cells for DARP were oval or spindle in shape and the staining was localized in the cytoplasm surrounding the nucleus. In addition, in this paper we present evidence confirming that the adrenal gland is the source of a protein that after immunopurification in nanogram amounts releases DA from corpus striatum fragments superfused in vitro. These data clearly demonstrate the existence of DARP in the adrenal gland and support the hypothesis that the effectiveness of the adrenal medulla transplants to the brain of Parkinson's patients is in part due to the secretion of neurotrophic factors. We submit that DARP is one of such factors.
In the present study, we have investigated the effects of intermittent dibutyryl cyclic AMP (dbcA... more In the present study, we have investigated the effects of intermittent dibutyryl cyclic AMP (dbcAMP, 5 X 10(-8) M), butyrate (5 X 10(-8) M) and forskolin (10(-4) M) on immunoreactive luteinizing hormone-releasing hormone (LHRH) release from superfused hypothalamic fragments from intact male rats of age 25, 30, 45, or 60-75 day (adult). The results indicate that at 25 days of age, male rat hypothalami were most responsive to cyclic AMP (162% of preinfusion basal LHRH release); by 30 days of age, dbcAMP also elicited increased LHRH release (120% of basal). By 45 days of age, the dbcAMP effect on in vitro LHRH release was slightly inhibitory (78% of basal); however, by adulthood, the effect of this cyclic nucleotide on LHRH release was minimal (92% of basal). Butyrate also induced age-dependent modifications in in vitro LHRH release from male rat hypothalami, with slight increases following butyrate delivery at 25 days of age (115%), slight decreases at 30 (82%) and 45 days of age (68%), and little change in adulthood (94%). This latter finding emphasizes the importance of using butyrate as a control for butyryl derivatives of cyclic AMP, which are known to liberate butyric acid as a product of hydrolysis of parent compounds. To address the possibility that the lack of effectiveness of dbcAMP in the older animal preparations was solely due to an increasing sensitivity of male rat medio-basal hypothalamus fragments to butyrate, we examined the effect of forskolin (10(-4) M), an adenylate cyclase stimulator, on LHRH release.(ABSTRACT TRUNCATED AT 250 WORDS)
The effects of varying doses (1.25, 2.5, and 5.0 mg/kg, ip) of D-amphetamine sulfate (AMPH) on ei... more The effects of varying doses (1.25, 2.5, and 5.0 mg/kg, ip) of D-amphetamine sulfate (AMPH) on eight individual behaviors (Rearing, Grooming, Sniffing, Stationary, Gnawing, Head Bobbing, "Sleeping," and Licking) of Castrate + Oil-treated, Castrate + Testosterone Propionate (TP)-treated, and Intact male rats were examined. For Stationary, Sniffing, and "Sleeping" at 1.25 mg/kg AMPH and Rearing and Sniffing at the 2.5 mg/kg dose a significantly greater duration in the behavioral score was obtained for Castrate + Oil versus Castrate + TP and Intact males. These results indicate the complexity of the AMPH dose-response effects upon measurable behaviors, the alteration in the duration of these effects as a function of the hormonal condition of the male rat, and the importance of examining discrete components of behavior when hormone-amphetamine interactions are examined.
In this report, using a push-pull perfusion technique, we examined in vivo the effects of exogeno... more In this report, using a push-pull perfusion technique, we examined in vivo the effects of exogenous dopamine (DA) on the output of neurochemical substances from the caudate nucleus (CN) of freely behaving rats. Exogenous DA, at concentrations of 10(-5) M, 5 x 10(-5) M and 10(-4) M, in modified Krebs-Ringer phosphate medium (KRP) was infused directly into the CN for 15 min each. Exogenous DA at the doses tested elicited increases in 3,4-dihydroxyphenylacetic acid (DOPAC) output in a dose-dependent manner. In addition, the higher two doses of exogenous DA also induced increases in homovanillic acid (HVA) output from the rat CN. The increases in DOPAC output by 5 x 10(-5) M DA was partially blocked by the inclusion of 10(-3) M nomifensine in KRP. Interestingly, exogenous DA-induced increases in HVA output were little affected by the nomifensine treatment. However, the catabolism of exogenous DA was almost completely eliminated by a prior 6-hydroxydopamine lesion in the ipsilateral substantia nigra. Furthermore, infusions of exogenous DA did not change 5-hydroxyindoleacetic acid output from the CN. In conclusion, our results confirm in vivo that (a) DA catabolic pathway via DOPAC intermediate predominates over the alternative pathway via 3-methoxytyramine, (b) increases in extracellular DA will lead to increases in DOPAC and HVA levels in extracellular space and (c) the majority of the DA is oxidized by intradopaminergic monoamine oxidase.
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Papers by Victor Ramirez