Studies on the specificity of uptake and release of radiolabelled histamine in rat brain slices

Previously it has been shown that radiolabelled histamine is taken up by brain slices and may subsequently be released by depolarizing stimuli in a calcium-dependent manner, indicating the involvement of neurons in uptake and release of histamine.The present study demonstrates that after incubation of brain slices with low (nM) concentrations of [³H]histamine the amine may be taken up by (and released from) dopaminergic and serotonergic neurons (nerve terminals). Thus 6-hydroxydopamine- and 5,7-dihydroxytryptamine-induced lesions not only reduced the uptake of [³H]dopamine (in striatal slices) and [³H]serotonin (in hippocampal slices), but also, though to a lesser extent, that of [³H]histamine. Immunocytochemical findings revealed that the neurotoxins did not visibly affect histaminergic neurons. Lesioning of noradrenergic neurons appeared not to alter significantly the uptake of [³H]histamine. Further, various drugs acting on either catecholamine-, serotonin- or opioid-receptors and known to cause presynaptic inhibition of the release of [³H]dopamine or [³H]wrotonin from striatal or hippocampal slices also inhibited [³H]histamine release.It is concluded that incubation of brain slices with low concentrations of [³H]histamine does not result in a selective labelling of histaminergic neurons. The possibility that, unlike other monoamines, histamine is not subject to high-affinity uptake by the nerve terminals from which it was released, is discussed.     

UPTAKE,METABOLISM, AND RELEASE OF CAMP IN SELENASTRUM CAPRICORNUTUM (CHLOROPHYCEAE)1

Uptake rate, intracellular metabolism, and extracellular release of cAMP were examined in batch cultures of the unicellular alga Selenastrum capricornutum Printz (Chlorophyceae). cAMP uptake was linearly dependent on external substrate concentration (0.5 nM to 1 mM) over a broad range of culture cell densities (1.7-5 × 10⁶cells. mL^?1) to mid-to-late log phase growth. Chromatographic analyses indicated that the majority of assimilated [³H]-cAMP was converted within 30 min to radiolabelled material which co-chromatographed with ADP/ATP and adenosine. About 20% of the (³H]-label originally added to cells as a [³H]-cAMP was released to the extracellular medium within 10 min, and chromatographic analyses demonstrated that most of the radiolabelled material was released as cAMP. Analyses with cell suspensions preincubated in KCN or DCMU suggested that cAMP release, but not uptake, was repressed by respiratory or photosynthetic electron flow inhibitors. However, the data were consistent with a passive CAMP uptake mechanism only if much of the assimilated CAMP was bound or compartmentalized.     

6-AMINODOPAMINE-INDUCED DEGENERATION OF CATECHOLAMINE NEURONS

the effects of 6-aminodopamine on central and peripheral catecholamine neurons using fluorescence histochemical and isotope techniques have been investigated. Systematic administration of 6-aminodopamine (20 mg/kg intraveneously) produced a rapid (within 1 h) and long-lasting depletion of endogenous noradrenaline in adrenergic nerves of mouse atrium and iris with a concomitant loss of [³H]noradrenaline uptake. The effects were dosedependent. Accumulations of noradrenaline in non-terminal axons were observed histochemically, indicating that 6-aminodopamine induces neuronal damage. Desipramine completely blocked the 6-aminodopamine induced noradrenaline depletion and reduction in [³H]noradrenaline uptake, indicating that 6-aminodopamine has to be taken up by the axonal ‘membrane pump’ to produce its effects. Themonoamine oxidase inhibitor, nialamide, potentiated the effect of 6-aminodopamine on [³H]noradrenaline uptake. 6-Aminodopamine did not affect the cell bodies of the adrenergic neurons and there was a reappearance of adrenergic nerves and recovery of [³H]noradrenaline uptake. 6-Aminodopamine does not seem to pass the blood-brain barrier after systemic injection. Intraventricular injection of 6-aminodopamine in rats led to a considerable reduction in endogenous whole brain noradrenaline and [³H]noradrenaline uptake in slices from cerebral cortex and hypothalamus. Similar, but less pronounced effects were observed on dopamine neurons in the caudate nucleus. Histochemically, pronounced accumulations of transmitter were observed in the axons of the catecholamine neurons. The results obtained favour the view that 6-aminodopamine is able to produce an acute and selective degeneration of catecholamine neurons similar to that seen after the neurotoxicagent, 6-hydroxydopamine. Both compounds seemed to be approximately equally potent in their neurotoxicity, although 6-aminodopamine seemed to be more generally toxic.     

Metabolism of glycoconjugates in hypothalamic neurons and glial cells: Comparison of incorporation of [3H]fucose and [3H]N-acetylmannosamine by electron microscopic autoradiography

Summary Incorporation of two glycoconjugate precursors, [³H]fucose and [³H]N-acetylmannosamine, in the arcuate nucleus of the rat hypothalamus is compared 30 min after intraventricular administration. Electron microscopic autoradiographs were analyzed by a sampling technique which provides information about (i) the distribution of radioactivity in the tissue, (ii) the relative volumes, and (iii) the amount of radioactivity per unit volume of the various tissue compartments. With both precursors, the highest incorporation was found in glial cell bodies, being about five to six times that of neurons. This holds true for all three types of glia. The concentration of radioactivity in the neuropil consisting of neuronal and glial processes was exceedingly low, although, due to its large volume, it contained the highest fraction of total tissue radioactivity. The limitations imposed on the interpretation of the data in terms of synthesis of fucosyland sialoglycoconjugates are discussed. It is proposed that the observations support the concept that high rates of synthesis for glycoproteins (and possibly gangliosides) are a characteristic metabolic property of glial cells.Dedicated to Prof. Emil Tonutti on the occasion of his 70th birthday     

Adult development of the hippocampal-serotonin system of C57BL/6N mice; analysis of high-affinity uptake of 3H-5HT in slices and synaptosomes

The saturable and specific high-affinity uptake of [³H]serotonin ([³H]5HT) (5 × 10^?8 M) was studied in slices from the hippocampus, parietal cortex, septum-preoptic area, and hypothalamus of male 2, 6, 12 and 24–32 month old C57BL/6N mice. Hippocampal [³H]5-HT uptake showed a significant biphasic relationship to age, with lower values in the 2 and 24–32 month old mice compared to 6 month old mice. No significant age effects were seen in the other regions, or in [³H]norepinephrine high-affinity uptake in the hippocampus.Studies of the high-affinity uptake mechanism in synaptosomal preparations were made in a subgroup of 12 and 24 month old mice. A micro-assay using a tissue-harvester measured high-affinity uptake on 8–30 μl of the P₂ suspension (crude-synaptosomal preparation). The high-affinity uptake was linear for 4 min at 37°C and inhibited in both the adult and aged tissue by 10^?5 M cold 5-HT (83 and 78% respectively), 10^?5 M fluoxetine (85 and 82% respectively) and 10^?3 M NaCN (57 and 57% respectively). Kinetic analysis of the [³H]5HT high-affinity uptake in the hippocampus (3 min, 37°C) revealed the same apparent K_m for serotonin at both ages (6.7 x 10^?8 M), but a 44% decrease in V_max in the aged hippocampal synaptosomal high-affinity uptake compared to adults (120 vs 215 pmol of 5-HT/g-tissue/3 min).These results are discussed in relationship to the reported age effects on the intrinsic neurons of the hippocampus.     

Pharmacokinetics and biodistribution of a new anti-episialin monoclonal antibody 139H2 in ovarian-cancer-bearing nude mice

Summary The new murine anti-episialin monoclonal antibody (mAb) 139H2 has been selected for its strong reactivity with a series of human ovarian cancer xenografts. In the present report we describe the characteristics of mAb 139H2 investigated in vitro as well as in vivo. Scatchard plot analysis using the human ovarian cancer cell line NIH:OVCAR-3 showed an affinity constant of 1 × 10⁸ M^–1 and the expression of 7 × 10⁶ antigenic sites/cell. Reactivity with OVCAR-3 xenograft tissue was intense, localized at the cell membrane, heterogeneously distributed, and mainly detectable at the apical site of the cell. Administration of radiolabelled mAb 139H2 to nude mice bearing s.c. OVCAR-3 xenografts showed specific uptake in the tumour up to 9% of the injected dose/g. The maximum uptake in the tumour was retained for 3.5 days and mAb 139H2 cleared from the tumour with a half-life of 5.5 days. The half-life in blood was 50 h and no antibody-antigen complex formation could be detected. Poor uptake and no retention in episialin-negative WiDr colon cancer xenografts demonstrated specificity. Administration of an excess of an unlabelled irrelevant mAb did not influence the uptake in the OVCAR-3 xenografts or in other tissues. In contrast, tumour uptake decreased after addition of 300 µg or more unlabelled mAb 139H2 to a tracer dose of radiolabelled mAb 139H2. The uptake of mAb 139H2 in OVCAR-3 xenografts appeared inversely related to the tumour size.Supported by the Dutch Cancer Society     

Studies on the mechanisms of L-dopa-induced depletion of 5-hydroxytryptamine in the mouse brain

The administration of L-dopa to mice causes an increase in the brain concentrations of dopa and dopamine which is related temporally to a reduction in the brain concentration of 5HT. These effects occur concurrently with a reduction in the conversion of intravenously administered ³H-tryptophan to ³H-5HT without an alteration in the accumulation of ³H-tryptophan in the brain. The L-dopa-induced changes in the brain concentrations of dopa, dopamine and 5HT are not altered by pretreatment with drugs (imipramine, chlorimipramine, benztropine, cocaine) which inhibit the neuronal uptake of amines. Current evidence suggests that L-dopa is decarboxylated in 5HT neurons to dopamine, which displaces 5HT from intraneuronal storage sites.     

Uptake of GABA by neuronal and nonneuronal cells in dispersed cell cultures of postnatal rat cerebellum

A study was made of the time course and kinetics of [³H]GABA uptake by dispersed cell cultures of postnatal rat cerebellum with and without neuronal cells. The properties of GABA neurons were calculated from the biochemical difference between the two types of cultures. It was found that for any given concentration of [³H]GABA, or any time up to 20 min, GABA neurons in cultures 21 days in vitro had an average velocity of uptake several orders of magnitude greater than that of nonneuronal cells. In addition, the apparent K_m values for GABA neurons for high and low affinity uptake were 0.33 × 10⁻⁶ M and 41.8 × 10⁻⁴ M, respectively. For nonneuronal cells, the apparent K_m for high affinity uptake was 0.29 × 10⁻⁶ M. The apparent V_max values for GABA neurons for high and low affinity uptake were 28.7 × 10⁻⁶ mol/g DNA/min and 151.5 mmol/g DNA/min, respectively. For nonneuronal cells, the apparent V_max for high affinity uptake was 0.06 × 10⁻⁶ mol/g DNA/min. No low affinity uptake system for nonneuronal cells could be detected after correcting the data for binding and diffusion. By substituting the apparent kinetic constants in the Michaelis-Menten equation, it was determined that for GABA concentrations of 5 × 10⁻⁹ M to 1 mM or higher over 99% of the GABA should be accumulated by GABA neurons, given equal access of all cells to the label. In addition, high affinity uptake of [³H]GABA by GABA neurons was completely blocked by treatment with 0.2 mM ouabain, whereas that by nonneuronal cells was only slightly decreased. Most (75–85%) of the [³H]GABA (4.4 × 10⁻⁶ M) uptake by both GABA neurons and nonneuronal cells was sodium and temperature dependent.     

Ciliary ganglion neurons in cell culture: high affinity choline uptake and autoradiographic choline labeling

Chick ciliary ganglion neurons grown in dissociated cell culture have a high affinity uptake mechanism for choline that has the properties expected for cholinergic neurons. The uptake has an apparent K_m of ca. 0.3 μM and is blocked by addition of 10 μM hemicholinium-3 or replacement of Na⁺ by Li⁺ in the uptake medium. When the choline uptake mechanism is used to label ciliary ganglion neuron-myotube cultures autoradiographically, over 99% of the neurons are labeled. A few cells with neuronal morphologies in such cultures (<1%) are labeled by γ-[³H]aminobutyric acid uptake. The number of [³H]choline-labeled neurons and the amount of Na⁺-dependent choline uptake is the same for ciliary ganglion neurons grown with and without skeletal myotubes. Rat superior cervical ganglion neurons, grown in cell culture under conditions that induce them to synthesize acetylcholine and form cholinergic synapses, are labeled by [³H]choline uptake, though not as heavily as ciliary ganglion neurons. In contrast, chick dorsal root ganglion neurons, a presumed population of noncholinergic neurons, are not labeled by [³H]choline uptake. Thus high affinity choline uptake can be used to label autoradiographically the cholinergic neurons tested, while at least one population of noncholinergic neurons remains unlabeled.     

EFFECT OF STRESS ON THE DISPOSITION OF CATECHOLAMINES LOCALIZED IN VARIOUS INTRANEURONAL STORAGE FORMS IN THE BRAIN STEM OF THE RAT

Abstract— The utilization of [³H]norepinephrine newly taken up or newly synthesized from [³H]tyrosine was studied in the brain stem of normal and stressed rats up to 5 h after the intracistemal injection of [³H]norepinephrine or [³H]tyrosine. The biphasic disappearance of the exogenous as well as of the endogenously synthesized [³HJnorepinephrine revealed that the amine is localized in at least two main compartments (A and B). The half-life of the amine newly taken up or newly synthesized, mainly localized in compartment A, is of short duration (between 15 and 30 min); the amine stored for a longer period of time and mainly distributed in compartment B is utilized more slowly (half-life, 180 to 260 min). A stress of short duration (15 min) induced by electric shocks applied to the feet increased the utilization of [³HJnorepinephrine newly taken up or newly synthesized from [³H]dopamine or [³H]tyrosine, but has no effect on the [³H]norepinephrine stored for a longer time period, indicating that the amine in compartment A is released in preference to that stored in compartment B. A stress of longer duration (180 min) increased the utilization of [³H] norepinephrine in both compartments and induced a sustained increased in norepinephrine synthesis as shown by the enhanced formation of [³H]norepinephrine from [³H]tyrosine in brain stem slices in vitro. The electrical stress was without effect on [³H]norepinephrine uptake. As for [³H]norepinephrine, the 15 min of stress enhanced the utilization of [³H] dopamine newly taken up or newly synthesized from [³H]tyrosine and had no effect on [³H]dopamine stored for a longer time period. These results suggest an increased release of both [³H]dopamine and [³H]norepinephrine from noradrenergic terminals of the rat brain stem. Finally, the 15 min of stress appeared to have no effect on the utilization of [³H] serotonin newly synthesized from [³H]tryptophan in serotonergic neurons of the brain stem, whereas the 180 min of stress increased the utilization of 5-HT in this structure.     

Catecholaminergic innervation of GRF-containing neurons in the rat hypothalamus revealed by electron-microscopic cytochemistry

Summary The Catecholaminergic innervation of neurons containing growth hormone-releasing factor (GRF) was examined by use of a method which combined either 5-hydroxydopamine (5-OHDA) uptake or autoradiography after intraventricular injection of ³H-noradrenaline with immunocytochemistry for GRF in the same tissue sections at the electron-microscopic level. In the ventrolateral part of the arcuate nucleus of the rat hypothalamus a large number of immunonegative axon terminals were found to make synaptic contact with GRF-like immunoreactive (GRF-LI) cell bodies and processes. ³H-noradrenaline autoradiography or 5-OHDA-labeling combined with GRF immunocytochemistry revealed that axon terminals labeled with ³H-noradrenaline or 5-OHDA make synaptic contact with the GRF-LI nerve cell bodies and processes. These findings indicate that catecholamine-containing neurons innervate GRF neurons to regulate GRF secretion via synapses in the rat arcuate nucleus.     

Ultrastructural radioautography of synthesis and migration of proteins and catecholamines in the rat adrenal medulla

Summary The synthetic pathways of proteins and catecholamines in the rat adrenal medullary cells were compared systematically at the ultrastructural level, within a 24 h period, with 2 tracers, L-tyrosine 3,5-³H and L-3,4-dihydroxy [ring 2,5,6-³H] phenylalanine (L-dopa³H). Young rats were injected with either of these tracers and sacrificed in pairs at close time intervals. With L-tyrosine ³H, the label was about equal over rough endoplasmic reticulum (RER) and secretory granules at 2 min after injection and remained almost constant in intensity over the secretory granules throughout the period of observation. A peak of radioactivity was also observed in the Golgi complex between 5 and 20 min after injection. This indicates that L-tyrosine ³H participates in the synthesis of both granule proteins and catecholamines as confirmed by the results obtained after injection of L-dopa ³H. With this tracer, radioactivity over RER, Golgi complex, cytosol and cell surface remained very low at all times and was undetectable at several time intervals. In contrast, radioactivity over secretory granules was very high at all time intervals. The present results thus confirm that in both adrenaline- and noradrenaline-storing cells, the protein moiety of chromaffin granules is synthetized in the RER, packaged in the Golgi complex and rapidly found in newly formed secretory granules. Following either L-tyrosine ³H or L-dopa ³H injection, catecholamine synthesis occurs only in or in close vicinity to chromaffin granules in both cell types at all time intervals. Acknowledgements. This work was supported by a grant from the Medical Research Council of Canada to the Multidisciplinary Research Group of Hypertension of the Clinical Research Institute of Montreal and by the Canadian Heart Foundation     

DEVELOPMENT OF THE BLOOD-BRAIN BARRIER FOR 6-HYDROXYDOPAMINE

The postnatal development of the blood-brain barrier for the neurotoxic action of 6-hydroxydopamine on central noradrenaline neurons has been investigated by recording the in vitro uptake of [³H]noradrenaline in slices from cerebral cortex, hypothalamus and spinal cord in rats treated with large doses of 6-hydroxydopamine at different ages. The [³H]noradranaline uptake was permanently and markedly reduced in all regions when the animals were treated at birth, certainly related to degeneration of noradrenaline neurons, caused by 6-OH-DA. In the cerebral cortex and hypothalamus an efficient protection against the effects of 6-OH-DA on [³H]noradrenaline uptake developed postnatally, while in the spinal cord this protection was never seen to become complete. The results obtained indicate a rapid formation of a blood-brain barrier for 6-OH-DA in the cerebral cortex between the 7th and 9th day after birth. In the hypothalamus the development of this barrier seemed to have a more gradual time-course, but appeared to be fully developed already at day 5 postnatally. Also in the spinal cord the barrier developed more gradually from birth to the adult age. It was observed, however, that both in the cerebral cortex and in the spinal cord, the blood-brain barrier developed, could not completely protect the central noradrenaline neurons from the neurotoxic actions of large doses of 6-OH-DA administered systemically to adult rats. Furthermore, the results obtained support the view that 6-OH-DA does not seem to apparently affect the outgrowth of remaining NA neurons which have not been destroyed by the 6-OH-DA treatment.     

Autoradiographic labeling of spinal cord neurons with high affinity choline uptake in cell culture

Embryonic chick spinal cord neurons grown in dissociated cell culture have a high affinity uptake mechanism for choline. We find that, in addition to acetylcholine synthesis, the accumulated choline is used for the synthesis of metabolites such as lipids that are retained in part by conventional fixation techniques. As a result autoradiographic methods can be used to identify the cells that have the uptake mechanism in spinal cord cultures. About 60% of the neurons are labeled by [³H]choline uptake in cultures prepared with spinal cord cells from 4-day-old embryos, and about 40% are labeled in cultures prepared with cord cells from 7-day-old embryos. Neurons that innervate skeletal myotubes in spinal cord-myotube cultures are consistently labeled by [³H]choline uptake. Neurons unlabeled by the procedure are viable: they exclude the dye trypan blue and accumulate ¹⁴C-amino acids for protein synthesis. Most of the neurons unlabeled by [³H]choline uptake can instead be labeled by uptake of γ-[³H]aminobutyric acid, and vice versa. These results suggest that high affinity choline uptake can be used to label cholinergic neurons in cell culture, and that at least some populations of noncholinergic neurons are not labeled by the procedure. It cannot yet be concluded, however, that all labeled neurons are cholinergic since more labeled neurons are obtained per cord than would be expected from the number of neurons making up identified cholinergic populations in vivo. A three- to fourfold increase in the amount of high affinity choline uptake is observed between Days 3 and 15 in culture for spinal cord cells obtained from 4-day-old embryos. The number of [³H]choline-labeled neurons in such cultures decreases slightly during the same period, suggesting that the increase in uptake reflects neuronal growth or development rather than an increase in population size. Both the magnitude of the uptake and the number of [³H]choline-labeled neurons are the same for spinal cord cells grown with and without skeletal myotubes.     

Inhibition of the uptake of 5-hydroxytryptamine, noradrenaline and dopamine into rat brain homogenates by various hydroxylated tryptamines

Recent work has shown that intracerebral injections of 5,6-dihydroxytryptamine (5,6-DHT) lead to a fairly selective and long lasting depletion of 5-HT in the rat CNS (BAUMGARTEN, BJORKLUND, LACHENMAYER, NOBIN and STENEVI, 1971; DALY, FUXE and JONSSON, 1973). This effect appears to result from a degeneration of the serotonin-containing neurons (BAUMGARTEN and LACHENMAYER, 1972a). 5,6-DHT does, however, to a lesser extent affect both NA and dopamine (DA) containing nerve terminals (BAUMGARTEN et al., 1971). In an attempt, therefore, to find compounds having a more specific toxic action we have investigated several other hydroxylated tryptamines. In order to obtain information about the differential affinities of these analogues for neuronal uptake sites we have examined their effects on the uptake of [³H]5-HT and (±)-[³H]NA into synaptosomes in homogenates of rat hypothalamus and of [³H]DA uptake into a similar preparation from the rat corpus striatum. It is known that the uptake of these putative transmitters in rat brain homogenates is predominantly into the synaptosome fraction (KANNENGIESSER, HUNT and RAYNAUD, 1973; COYLE and SNYDER, 1969).     

Electron-microscopic cytochemistry of the catecholaminergic innervation of TRH neurons in the rat hypothalamus

Summary The catecholaminergic innervation of thyrotropin-releasing hormone (TRH) neurons was examined by use of a combined method of 5-hydroxydopamine (5-OHDA) uptake or autoradiography after intraventricular injection of ³H-noradrenaline (³H-NA) and immunocytochemistry for TRH in the same tissue sections at the electron-microscopic level.TRH-like immunoreactive nerve cell bodies were distributed abundantly in the parvocellular part of the paraventricular nucleus (PVN), in the suprachiasmatic preoptic nucleus and in the dorsomedial nucleus of the rat hypothalamus. In the PVN, a large number of immunonegative axon terminals were found to make synaptic contact with TRH-like immunoreactive cell bodies and fibers. In the combined autoradiography or 5-OHDA labeling with immunocytochemistry, axon terminals labeled with ³H-NA or 5-OHDA were found to form synaptic contacts with the TRH immunoreactive nerve cell bodies and fibers. These findings suggest that catecholamine-containing neurons, probably noradrenergic, may innervate TRH neurons to regulate TRH secretion via synapses with other unknown neurons in the rat PVN.This study was supported by grants from the Ministry of Education, Science and Culture, Japan     

Comparison of synaptosomal and glial uptake of pipecolic acid and GABA in rat brain

The active uptake of [³H]pipecolic acid increased with incubation time and its uptake at 3 min was half of that at 20 min. [¹⁴C]GABA uptake rose earlier, and its uptake at 3 min was almost 80% of that at 20 min. On the other hand, a ratio (pellet/medium) of [³H]pipecolic acid uptake into glial cell-enriched fractions, was much less (0.4–0.6) than that of [¹⁴C]GABA (25.8–74.1). GABA, 10^–4 M, and pipecolic acid, 10^–4 M, produced a significant inhibition of [³H]pipecolic acid uptake into P₂ fractions. Pipecolic acid, 10^–4 M, significantly reduced the synaptosomal and glial uptake of [¹⁴C]GABA. GABA, 10^–4 M, affected neither spontaneous nor high K⁺-induced release of [³H]pipecolic acid from brain slices. It is suggested that pipecolic acid is involved in either synaptic transmission or in its modulation at GABA synapses in the central nervous system.     

Release of 3H-gamma-aminobutyric acid (GABA) by inhibitory neurons of the crayfish

Inhibitory neurons innervating the muscle receptor organ (MRO) of crayfish were used to study the uptake and release of tritiated GABA. MROs that have been directly exposed to ³H GABA for 60–75 min release radioactivity during low-frequency electrical stimulation. When ganglia containing the inhibitory cell bodies are exposed to ³H GABA, the isotope travels along a pathway unique to the inhibitory axon, at rates that range between 160 and 240 mm per day. Electrical stimulation of inhibitory axons whose cell bodies have been exposed to ³H GABA for 4–5 hr produces release of isotope from isolated MROs. Low calcium, high magnesium exposure prevents the stimulus-dependent release of radioactivity. Thin layer chromatographic analyses indicate that GABA comprises at least a major fraction of the radioactivity collected from stimulated preparations. A number of unidentified radioactive compounds are usually present with GABA, and it is suggested that most of these are catabolites of GABA.     

Uptake and metabolism of female sex steroids by isolated small neurons and other cell fractions from the rat medial basal hypothalamus

Rat medial basal hypothalami (MBH) and sections of cerebral cortex (CC) were dissociated with trypsin to prepare single cells and subcellular fractions. They were then separated into four fractions on a discontinuous sucrose gradient. The small neurons in Fraction D were highly purified. Fraction A had synaptosomes, myelin and other cell particulates. Fraction B had glial cells, neurons and a few synaptosomes. Fraction C had large neurons and red blood cells. All four fractions contained LHRH, but most (62.5%) of this hormone was present in Fraction A. Dissociated cell suspensions were incubated with [³H]-steroids, with and without a 100-fold excess of unlabeled steroids, then separated on sucrose gradients. In most fractions the total uptake and specific uptake of [³H]-progesterone, [³H]-5α-pregnane-3,20-dione (5α-dihydroprogesterone) and [³H]-l7β-estradiol were greater for the dissociated cells from the MBH than the CC. The dissociated cells and cell particulates in all four fractions from the MBH and CC metabolized progesterone, 5α-dihydroprogesterone and l7β-estradiol.These results indicate that hypothalamic neurons contain small amounts of LHRH and retain the ability to take up and metabolize progesterone, 5α-dihydroprogesterone and 17β-estradiol.     

Estrogen Regulation of Dopamine Release in the Nucleus Accumbens: Genomic-and Nongenomic-Mediated Effects

Abstract: The ability of estrogen to modulate mesolimbic dopamine (DA) was examined using in vivo voltammetry. Estrogen priming (5 μg, 48 h) of ovariectomized (ovx) female rats resulted in a slight decrease in K⁺-stimulated DA release measured in the nucleus accumbens: this decrease was accompanied by a significant increase in both DA reuptake and DA clearance times. Following estrogen priming nomifensine, a potent inhibitor of the DA uptake carrier, was still able to potentiate K⁺-stimulated DA release and alter the time course of DA availability, but the response was attenuated compared with ovx controls. Direct infusion of 17β-estradiol hemisuccinate (17β-E, 20–50 pg) into the nucleus accumbens resulted in a biphasic potentiation of K⁺-stimulated release. An initial increase in release was observed 2 min after 17β-E infusion; this increase, although reduced by 15 min, was still significantly higher than control values. A subsequent potentiation was observed 60 min after the initial 17β-E infusion; this response remained for at least an additional 60 min. Nomifensine did not significantly alter K⁺-stimulated DA release following 17β-E infusion, but was still able to potentiate the total time DA was available extracellularly. These data suggest that the mesolimbic A10 DA neurons that terminate in the nucleus accumbens can be modulated in vivo by estrogen and that this modulation may be mediated by both genomic (long term) and nongenomic (short term) mechanisms.