Brain Nerve Terminals Research Articles

Vesicle exocytosis stimulated by alpha-latrotoxin is mediated by latrophilin and requires both external and stored Ca2+.

alpha-Latrotoxin (LTX) stimulates massive neurotransmitter release by two mechanisms: Ca2+-dependent and -independent. Our studies on norepinephrine secretion from nerve terminals now reveal the different molecular basis of these two actions. The Ca2+-dependent LTX-evoked vesicle exocytosis (abolished by botulinum neurotoxins) is 10-fold more sensitive to external Ca2+ than secretion triggered by depolarization or A23187; it does not, however, depend on the cation entry into terminals but requires intracellular Ca2+ and is blocked by drugs depleting Ca2+ stores and by inhibitors of phospholipase C (PLC). These data, together with binding studies, prove that latrophilin, which is linked to G proteins and inositol polyphosphate production, is the major functional LTX receptor. The Ca2+-independent LTX-stimulated release is not inhibited by botulinum neurotoxins or drugs interfering with Ca2+ metabolism and occurs via pores in the presynaptic membrane, large enough to allow efflux of neurotransmitters and other small molecules from the cytoplasm. Our results unite previously contradictory data about the toxin's effects and suggest that LTX-stimulated exocytosis depends upon the co-operative action of external and intracellular Ca2+ involving G proteins and PLC, whereas the Ca2+-independent release is largely non-vesicular.

Functional Evidence that BDNF Is an Anterograde Neuronal Trophic Factor in the CNS

In this report, we have tested the hypothesis that brain-derived neurotrophic factor (BDNF) is an anterograde neurotrophic factor in the CNS and have focused on central noradrenergic neurons that synthesize BDNF. Double-label immunocytochemistry for BDNF and dopamine-beta-hydroxylase (DBH), a marker for noradrenergic neurons, demonstrated that BDNF is partially localized to noradrenergic nerve fibers and terminals in the adult rat brain. To test the functional importance of this anterograde BDNF, we analyzed transgenic mice carrying a DBH-BDNF minigene. Increased synthesis of BDNF in noradrenergic neurons of DBH-BDNF mice caused elevated TrkB tyrosine kinase activation throughout postnatal life in the neocortex, a noradrenergic target region. This afferently regulated increase in TrkB receptor activity led to long-lasting alterations in cortical morphology. To determine whether noradrenergic neuron-expressed BDNF also anterogradely regulated neuronal survival, we examined a second noradrenergic target, neonatal facial motoneurons. One week after axotomy, 72% of facial motoneurons were lost in control animals, whereas only 30-35% were lost in DBH-BDNF transgenic mice. Altogether, these results indicate that BDNF is anterogradely transported to fibers and terminals of noradrenergic neurons, that anterogradely secreted BDNF causes activation of TrkB in target regions, and that this secretion has functional consequences for target neuron survival and differentiation. This presynaptic secretion of BDNF may provide a cellular mechanism for modulating neural circuitry, in either the developing or mature nervous system.

Immunofluorescent identification of endomorphin-2-containing nerve fibers and terminals in the rat brain and spinal cord.

Endomorphin-1 and -2 are potent and selective agonists for the mu-opioid receptor which have recently been isolated from bovine brain extracts. In the present study we used polyclonal antibodies specific for endomorphin-2 to determine its immunocytochemical distribution in the rat brain and spinal cord Endomorphin-2-like immunoreactivity was confined to varicose fibers with an overall discrete distribution within the central nervous system. The immunostaining was completely abolished by preincubation of the antiserum with endomorphin-2 but not with endomorphin-1. Endomorphin-2-like immunoreactivity was enriched in many but not all brain regions known to contain dense mu-opioid receptors, including nucleus accumbens, septum, midline thalamic nuclei, hypothalamic and amygdala nuclei, locus coeruleus, periaqueductal gray and spinal cord dorsal horn. In contrast, endomorphin-2 was absent from the cortex, striatum, hippocampus, nucleus of the solitary tract and dorsal root ganglia. Dual-labeling experiments revealed that endomorphin-2-immunoreactive nerve fibers did not co-contain any other opioid peptide. Thus, the present findings strongly suggest that endomorphin-2 may be a natural ligand for the mu-opioid receptor likely to be involved in the modulation of nociceptive transmission and reward-seeking behavior.

Nitric oxide causes glutamate release from brain synaptosomes.

We determined the ability of pathological levels of nitric oxide (NO) to cause glutamate release from isolated rat brain nerve terminals using a fluorometric assay. It was found that NO (0.7 and 2 microM) produced (4 and 10 nmol/mg of synaptosomal protein) Ca2+-independent glutamate release from synaptosomes (after 1 min of exposure). Spermine/NO complex (spermine NONOate; a slow NO donor) and potassium cyanide (an inhibitor of cytochrome oxidase) also caused Ca2+-independent glutamate release. Preincubation of synaptosomes with 5 microM 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (an inhibitor of soluble guanylyl cyclase) had no effect on NO-induced Ca2+-independent glutamate release. Ca2+-independent glutamate release produced by NO was greater in a low-oxygen medium. NO, spermine NONOate, and potassium cyanide inhibited synaptosomal respiration with a similar order of potency with respect to their ability to cause glutamate release. Because NO has been shown previously to inhibit reversibly cytochrome oxidase in competition with oxygen, our findings in this study suggest that NO (and cyanide) causes glutamate release following inhibition of mitochondrial respiration at the level of cytochrome oxidase. Thus, elevated NO production leading to mitochondrial dysfunction, glutamate release, and excitotoxicty may contribute to neuronal death in neurological diseases.

High calcium permeability of serotonin 5-HT3 receptors on presynaptic nerve terminals from rat striatum.

The serotonin 5-HT3 receptor, a ligand-gated ion channel, has previously been shown to be present on a subpopulation of brain nerve terminals, where, on activation, the 5-HT3 receptors induce Ca2+ influx. Whereas postsynaptic 5-HT3 receptors induce depolarization, being permeant to Na+ and K+, the basis of presynaptic 5-HT3 receptor-induced calcium influx is unknown. Because the small size of isolated brain nerve terminals (synaptosomes) precludes electrophysiological measurements, confocal microscopic imaging has been used to detect calcium influx into them. Application of 100 nM 1-(m-chlorophenyl)biguanide (mCPBG), a highly specific 5-HT3 receptor agonist, induced increases in internal free Ca2+ concentration ([Ca2+]i) and exocytosis in a subset of corpus striatal synaptosomes. mCPBG-induced increases in [Ca2+]i ranged from 1.3 to 1.6 times over basal values and were inhibited by 10 nM tropisetron, a potent and highly specific 5-HT3 receptor antagonist, but were insensitive to the removal of external free Na+ (substituted with N-methyl-D-glucamine), to prior depolarization induced on addition of 20 mM K+, or to voltage-gated Ca2+ channel blockade by 10 microM Co2+/Cd2+ or by 1 microM omega-conotoxin MVIIC/1 microM oemga-conotoxin GVIA/200 nM agatoxin TK. In contrast, the Ca2+ influx induced by 5-HT3 receptor activation in NG108-15 cells by 1 microM mCPBG was substantially reduced by 10 microM Co2+/Cd2+ and was completely blocked by 1 microM nitrendipine, an L-type Ca2+ channel blocker. We conclude that in contrast to the perikaryal 5-HT3 receptors, presynaptic 5-HT3 receptors appear to be uniquely calcium-permeant.

18F]CFT [(18F)WIN 35,428], a radioligand to study the dopamine transporter with PET: characterization in human subjects.

We have characterized the usage of [18F]CFT (also known as [18F]WIN 35,428) as a radioligand for in vivo studies of human dopamine transporter by PET. CFT was labeled with 18F to a high specific activity, and dynamic PET scans were conducted in healthy volunteers at various time points up to 5 h from [18F]CFT injection. The regional distribution of [18F]CFT uptake correlated well with the known distribution of dopaminergic nerve terminals in the human brain and also with that of other dopamine transporter radioligands. Striatal binding peaked at 225 min after injection and declined thereafter, demonstrating the reversible nature of the binding to the dopamine transporter. Therefore, due to the relatively long half-life of 18F (109.8 min), PET scans with [18F]CFT could easily be conducted during the binding equilibrium, allowing estimation of Bmax/Kd values (i.e., binding potential). Binding potentials for putamen and caudate measured at equilibrium were 4.79+/-0.11 and 4.50+/-0.23, respectively. We were able to also visualize midbrain dopaminergic neurons (substantia nigra) with [18F]CFT in some subjects. In conclusion, the labeling of CFT with 18F allows PET scans to be conducted at binding equilibrium, and therefore a high signal-to-noise ratio and reliable quantification of binding potential can be achieved. With a high resolution 3D PET scanner, the quantification of extrastriatal dopamine transporters should become possible.

Interactions between presynaptic calcium channels and proteins implicated in synaptic vesicle trafficking and exocytosis.

Monoclonal antibodies were generated by immunizing mice with chick brain synaptic membranes and screening for immunoprecipitation of solubilized omega conotoxin GVIA receptors (N-type calcium channels). Antibodies against two synaptic proteins (p35--syntaxin 1 and p58--synaptotagmin) were produced and used to purify and characterize a ternary complex containing N-type channels associated with these two proteins. These results provided the first evidence for a specific interaction between presynaptic calcium channels and SNARE proteins involved in synaptic vesicle docking and calcium-dependent exocytosis. Immunoprecipitation experiments supported the conclusion that syntaxin 1/SNAP-25/VAMP/synaptotagmin I or II complexes associate with N-type, P/Q-type, but not L-type calcium channels from rat brain nerve terminals. Immunofluorescent confocal microscopy at the frog neuromuscular junction was consistent with the co-localization of syntaxin 1, SNAP-25, and calcium channels, all of which are predominantly expressed at active zones of the presynaptic plasma membrane facing post-synaptic folds rich in acetylcholine receptors. The interaction of proteins implicated in calcium-dependent exocytosis with presynaptic calcium channels may locate the sensor(s) that trigger vesicle fusion within a microdomain of calcium entry.

In vivo radiotracers for vesicular neurotransmitter transporters

The vesicular monoamine transporter is a specific presynaptic protein involved in the transport of monoamines from the cytosol to storage vesicles of monoaminergic nerve terminals. Recently, radioligands for this transporter have been developed and utilized for in vivo positron emission tomographic (PET) imaging of monoaminergic nerve terminals in the human brain. In this review, the characteristics of vesicular transport and storage that provided the impetus for development of these radioligands are presented and discussed.

Brain myosin V is a synaptic vesicle-associated motor protein: evidence for a Ca2+-dependent interaction with the synaptobrevin-synaptophysin complex.

Brain myosin V is a member of a widely distributed class of unconventional myosins that may be of central importance to organelle trafficking in all eukaryotic cells. Molecular constituents that target this molecular motor to organelles have not been previously identified. Using a combination of immunopurification, extraction, cross-linking, and coprecipitation assays, we demonstrate that the tail domain of brain myosin V forms a stable complex with the synaptic vesicle membrane proteins, synaptobrevin II and synaptophysin. While myosin V was principally bound to synaptic vesicles during rest, this putative transport complex was promptly disassembled upon the depolarization-induced entry of Ca2+ into intact nerve endings. Coimmunoprecipitation assays further indicate that Ca2+ disrupts the in vitro binding of synaptobrevin II to synaptophysin in the presence but not in the absence of Mg2+. We conclude that hydrophilic forces reversibly couple the myosin V tail to a biochemically defined class of organelles in brain nerve terminals.

A study of the neurotoxic effect of MDMA ('ecstasy') on 5-HT neurones in the brains of mothers and neonates following administration of the drug during pregnancy.

1. It is well established that 3,4-methylenedioxymethamphetamine (MDMA or 'ecstasy') is neurotoxic and produces long term degeneration of cerebral 5-hydroxytryptamine (5-HT) nerve terminals in many species. Since MDMA is used extensively as a recreational drug by young people, it is being ingested by many women of child bearing age. We have therefore examined the effect of administering high doses of MDMA to rats during pregnancy on the cerebral content of both the dams and the neonates. 2. MDMA (20 mg kg-1, s.c.) was injected twice daily on days 14-17 of the gestation period. The initial dose produced a marked hyperthermic response in the dam which was progressively attenuated in both peak height and area under the curve following further doses of the drug. The body weight of the dams decreased during the period of treatment. 3. There was a modest decrease in litter size (-20%) of the MDMA-treated dams. 4. The concentration of 5-HT and its metabolite 5-HIAA was decreased by over 65% in the hippocampus and striatum and 40% in the cortex of the dams 1 week after parturition. In contrast, the content of 5-HT and 5-HIAA in the dorsal telencephalon of the pups of the MDMA-treated dams was the same as that seen in tissue from pups born to control animals. 5. Administration of MDMA (40 mg kg-1, s.c.) to adult rats increased thiobarbituric acid reacting substances (TBARS) in cortical tissue 3 h and 6 h later, indicating increased lipid peroxidation. No increase in TBARS was seen in the cortical tissue of 7-10 day neonates injected with this dose of MDMA 3 h or 6 h earlier. 6. The data suggest that exposure to MDMA in utero during the maturation phase does not produce damage to 5-HT nerve terminals in the foetal rat brain, in contrast to the damage seen in the brains of the mothers. This may be due to MDMA being metabolized to free radical producing entities in the adult brain but not in the immature brain or, alternatively, to more effective or more active free radical scavenging mechanisms being present in the immature brain.

Direct observation of serotonin 5-HT3 receptor-induced increases in calcium levels in individual brain nerve terminals.

Confocal microscopy was used to assess internal calcium level changes in response to presynaptic receptor activation in individual, isolated nerve terminals (synaptosomes) from rat corpus striatum, focusing, in particular, on the serotonin 5-HT3 receptor, a ligand-gated ion channel. The 5-HT3 receptor agonist-induced calcium level changes in individual synaptosomes were compared with responses evoked by K+ depolarization. Using the fluorescent dye fluo-3 to measure relative changes in internal free Ca2+ concentration ([Ca2+]i), K+-induced depolarization resulted in variable but rapid increases in apparent [Ca2+]i among the individual terminals, with some synaptosomes displaying large transient [Ca2+]i peaks of varying size (two- to 12-fold over basal levels) followed by an apparent plateau phase, whereas others displayed only a rise to a sustained plateau level of [Ca2+]i (two- to 2.5-fold over basal levels). Agonist activation of 5-HT3 receptors induced slow increases in [Ca2+]i (rise time, 15-20 s) in a subset (approximately 5%) of corpus striatal synaptosomes, with the increases (averaging 2.2-fold over basal) being dependent on Ca2+ entry and inhibited by millimolar external Mg2+. We conclude that significant increases in brain nerve terminal Ca2+, rivaling that found in response to excitation by depolarization but having distinct kinetic properties, can therefore result from the activation of presynaptic ligand-gated ion channels.

Rapid chelation of calcium entering isolated rat brain nerve terminals during stimulation inhibits neurotransmitter release

The intracellular actions of calcium chelators on the release of the neurotransmitter glutamate from isolated rat brain nerve terminals (synaptosomes) were examined. Preloading synaptosomes with the rapid calcium-binding chelator 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′- tetraacetic acid (BAPTA) resulted in a decrease in K +-stimulated glutamate release to nearly half that of controls, whereas preloading with the calcium chelator EGTA, whose action is less rapid than that of BAPTA, was without effect. Inhibition of glutamate release was also observed on preloading synaptosomes with dibromo-BAPTA, but not with dinitro-BAPTA. K +-stimulated, Ca 2+-dependent synaptosomal protein phosphorylation was not affected after preloading with BAPTA. The results suggest that the calcium-dependent intracellular component essential for triggering the secretory response in mammalian brain nerve terminals resides near the calcium channels, binding; calcium rapidly on its entry during stimulation.

Association of Drosophila cysteine string proteins with membranes

Cysteine string proteins are putative synaptic vesicle proteins that lack a transmembrane domain. Our analysis shows that Drosophila cysteine string proteins are extensively modified by hydroxylamine-sensitive fatty acylation. This modification could be responsible for association of csp's with membranes. Extensive deacylation of Dcsp's by a 20 h incubation in 1 M hydroxylamine, pH 7.0, or methanolic KOH produces a protein of 6–7 kDa lower mass than untreated Dcsp's. Surprisingly, the hydroxylamine treatment does not cause release of Dcsp's from membranes. On the other hand, alkaline stripping of membranes isolated from Drosophila brain by 0.1 M sodium carbonate, pH 11.5, causes a significant release of Dcsp's from membranes into the cytosol. These results indicate that fatty acylation may not form the main anchor of Dcsp's in membranes. Taking advantage of the endocytotic block in the Drosophila mutant shibire ts1, we analyzed the acylation states of Dcsp's in two stages during synaptic vesicle recycling and found no evidence for an acylation/ deacylation cycle of Dcsp's in the brain nerve terminals.

Inhibition of nicotine‐evoked [3H] dopamine release by pyridino N‐substituted nicotine analogues: A new class of nicotinic antagonist

AbstractA series of N‐substituted nicotine analogues were synthesized and evaluated for their ability to inhibit nicotine‐evoked [3H]dopamine ([3H]DA) release from rat striatal slices and for displacement of [3H]nicotine binding from rat striatal membranes. Of the eleven compounds examined, the most efficacious analogues in the [3H]DA release assay were those that contained a pyridino N‐alkyl substituent of three carbons or more in length. Structure‐activity relationships indicate that potency in the [3H]DA release assay increases with increase in alkyl chain length. Introduction of an aromatic or unsaturated residue into the pyridino‐N substituent also afforded compounds with significant antagonist activity. The most efficacious and potent compound in the series was S‐N‐octylnicotinium iodide (NONI). NONI had a potency approximately two‐fold that of the classical nicotinic antagonists, mecamylamine (MEC) and dihydro‐β‐erythroidine (DHBE). In addition, over the concentration range examined, NONI completely blocked the effect of nicotine to evoke [3H]DA release, whereas the classical antagonists inhibited but did not completely block nicotine's effect. Furthermore, NONI did not possess agonist activity at concentrations which completely blocked nicotine‐evoked [3H]DA release. Results from competition assays for [3H]nicotine binding revealed that all the analogues were able to displace [3H]nicotine binding with a lower affinity as compared to the reference compound DHBE. A significant correlation between alkyl chain length and affinity for the [3H]nicotine binding site was observed. Moreover, there was a lack of correlation between displacement of [3H]nicotine binding and inhibition of nicotine‐evoked [3H]DA release, suggesting that different nicotinic receptor subtypes are responsible for modulation of DA release and [3H]nicotine binding in striatum. The pKa values determined for S(—)nicotine and one of the active antagonists, S‐N‐allyl‐nicotinium iodide (NANI), indicates that the quaternary ammonium analogues exist predominantly in their unprotonated forms at physiological pH. Molecular modeling studies suggest that these antagonists may interact with the nicotinic receptor in a novel binding mode which is different from the mode of interaction of nicotine with this receptor. This structure‐activity data may provide useful information on the antagonist pharmacophore of the nicotinic receptor subtypes responsible for modulation of DA release and for nicotine binding in brain. Thus, it is proposed that these antagonists bind to the receptor in their unprotonated forms and that the binding mode involves interaction of the quaternary pyridinium‐N atom with the anionic site of the receptor. The unprotonated pyrrolidine‐N atom serves as the hydrogen bond acceptor, which reverses the roles the nitrogens normally play in the binding of nicotine to the receptor. The N‐alkyl substituent most likely binds to a site that extends beyond the normal agonist pharmacophore volume, which may prevent the receptor protein from achieving its open‐channel quaternary form. In conclusion, this study describes a new class of efficacious nicotinic antagonists which inhibit nicotine‐evoked [3H]DA release, from DA nerve terminals in brain and displace [3H]nicotine binding from brain membranes, providing new tools for unraveling the role of neuronal nicotinic receptors. © 1995 Wiley‐Liss, Inc.

The Interactions between Nitric Oxide and Brain Nerve Terminals as Studied by Electron Paramagnetic Resonance

It has been proposed that nitric oxide (NO) toxicity is due to damage to mitochondrial iron-sulfur centers, resulting in inhibition of mitochondrial respiration and the appearance of an EPR-detectable (g = 2.04) iron-sulfur dinitrosyl complex - Fe(RS)2(NO)2. We show that the addition of nitroprusside (an NO and NO+ donor) to rat brain synaptosomes generates large (> 30 μM) concentrations of EPR-detectable iron-sulfur-dinitrosyl complexes. However, there was no correlation between the size of the g = 2.04 EPR signal and the inhibition of synaptosomal respiration. No significant loss of intensity was seen from the mitochondrial iron-sulfur protein EPR signals. The results are consistent with previous data demonstrating that cytochrome oxidase, not iron-sulfur enzymes, is the primary target for NO inhibition of brain cell respiration (Brown, G.C. and Cooper, C.E. (1994) FEBS Lett. 356, 295-298).

Kinetic properties of the sodium‐calcium exchanger in rat brain synaptosomes.

1. The kinetic properties of the internal Na+ (Na+i)- dependent 45Ca2+ influx and external Na+ (Na+o)-dependent 45Ca2+ efflux were determined in isolated rat brain nerve terminals (synaptosomes) under conditions which the concentrations of internal Na+ ([Na+]i), external Na+ ([Na+]o), external Ca2+ (Ca2+]o), and external K+ ([K+]o) were varied. Both fluxes are manifestations of Na(+)-Ca2+ exchange. 2. Ca2+ uptake was augmented by raising [Na+]i and / or lowering [Na+]o. The increase in Ca2+ uptake induced by removing external Na+ was, in most instances, quantitatively equal to the Na+i-dependent Ca2+ uptake. 3. The Na+i-dependent Ca2+ uptake (measured at 1 s) was activated with an apparent half-maximal [Ca2+]o (KCa(o)) of about 0.23 mM. External Na+ inhibited the uptake in a non- competitive manner: increasing [Na+]o from 4.7 to 96 mM reduced the maximal Na+(i)-dependent Ca2+ uptake but did not affect KCa(o). 4. The inhibition of Ca2+ uptake by Na+o was proportional to ([Na+]o)2, and had a Hill coefficient (nH) of approximately 2.0. The mean apparent half-maximal [Na+]o for inhibition (KI(Na)) was about 60mM, and was independent of [Ca2+]o between 0.1 and 1.2mM; this, too, is indicative of non-competitive inhibition. 5. Low concentrations of alkali metal ions (M+) in the medium, including Na+, stimulated the Na+i-dependent uptake. The external Na+ and K+ concentrations required for apparent half-maximal activation (KM(Na) and KM(K), respectively) were 0.12 and 0.10mM. Thus, the relationship between Ca2+ uptake and [Na+]o was biphasic: uptake was stimulated by [Na+]o < or = 10 mM, and inhibited by higher [Na+]o. 6. The calculated maximal Na+i-dependent Ca2+ uptake (Jmax) was about 1530 pmol (mg protein) -1s-1 at 30 degrees C saturating [Ca2+]o and external M+ concentration ([M+]o), and with negligible inhibition by external Na+. 7. Internal Na+ activated the Ca2+ uptake with an apparent half-maximal concentration (KNa(i)) of about 20 mM and a Hill coefficient, nH, of approximately 3.0. 8. The Jmax for the Na+o-dependent efflux of Ca2+ from 45Ca(2+)-loaded synaptosomes treated with carbonyl cyanide p-trifluormethoxy-phenylhydrazone (FCCP) and caffeine (to release stored Ca2+ and raise the internal Ca2+ concentration ([Ca2+]i) was about 1800-2000 pmol (mg protein -1s-1 at 37 degrees C. 9. When the membrane potential (Vm) was reduced (depolarized) by increasing [K+]o, the Na+i-dependent Ca2+ influx increased, and the Na+o-dependent Ca2+ efflux declined. Both fluxes changed about 2-fold per 60 mV change in Vm. This voltage sensitivity corresponds to the movement of one elementary charge through about 60% of the membrane electric field. The symmetry suggests that the voltage-sensitive step is reversible. 10. The Jmax values for both Ca2P influx and efflux correspond to a Na+-Ca2+ exchange-mediated flux of about 425-575 jumol Ca2P (1 cell water)-' s-' or a turnover of about one quarter of the total synaptosome Ca2P in 1 s. We conclude that the Na+-Ca2P exchanger may contribute to Ca2P entry during nerve terminal depolarization; it is likely to be a major mechanism mediating Ca2P extrusion during subsequent repolarization and recovery.

Subcellular localization and kinetic properties of aromatase activity in rat brain

The conversion of testosterone to estradiol is catalyzed by cytochrome P450 aromatase. In situ aromatization is required for the full expression of the effects of testosterone in the brain. This study examined the subcellular distribution and reaction kinetics of aromatase in the adult rat brain. Preoptic area, hypothalamus and amygdala were homogenized in isotonic sucrose buffered with potassium phosphate. Tissue homogenates were fractionated by ultracentrifugation. Aromatase activity was measured using a previously validated 3H2O assay. Marker enzymes were measured to identify organelles in the different subcellular fractions. Aromatase activity in all 3 tissues was enriched 10-fold in microsomes, but not in other subcellular fractions. The addition of either a NADPH-generating system or 1 mM NADPH to the reaction mixture stimulated aromatase activity in all subcellular fractions, whereas NADH was only minimally effective. In general, substrate affinity constants were equivalent in all brain areas and subcellular fractions (approximately 10 nM) suggesting that one predominant catalytic form of the enzyme is present in the rat brain. One week after castration, aromatase activity was significantly reduced in all subcellular fractions of preoptic area and in the whole homogenate and microsomal fraction of the hypothalamus. Castration did not significantly alter aromatase activity in any subcellular compartment of amygdala. To more critically evaluate its subcellular localization, aromatase activity was measured in purified synaptosomes. Aromatase activity was not enriched in these preparations suggesting that it is not substantially associated with nerve terminals in rat brain.

Calcineurin-mediated protein dephosphorylation in brain nerve terminals regulates the release of glutamate.

In response to Ca2+ entry, several prominent brain nerve terminal phosphoproteins undergo dephosphorylation, but the relation between dephosphorylation and neurotransmitter release is unknown. Using the immunosuppressants cyclosporin A (CsA) and L-683,590 (FK-520) to inhibit specifically the Ca2+/calmodulin-dependent protein phosphatase calcineurin, we demonstrate here that Ca(2+)-dependent dephosphorylation in isolated rat brain nerve terminals (synaptosomes) is mediated by calcineurin. Pretreatment with micromolar CsA resulted in a 76-95% inhibition of stimulation-induced decreases in 32P-labeled dynamin (previously referred to as dephosphin), a phosphoprotein of M(r) = 145,000 (145-kDa protein), and a phosphoprotein of M(r) = 170,000 (170-kDa protein). Pretreatment with FK-520 also inhibited Ca(2+)-dependent dephosphorylation. Using hypotonic lysates of 32P-labeled synaptosomes, the addition of Ca2+ plus calmodulin, but not either agent alone, induced dynamin dephosphorylation. CsA and FK-520 had little to no effect on the release of glutamate induced by either K(+)-depolarization or the Ca2+ ionophore ionomycin. In contrast, calcineurin inhibition led to a substantial enhancement of glutamate release evoked by the K(+)-channel blocker 4-aminopyridine, an agent whose action most closely mimics physiological stimulation. Calcineurin inhibition had no effect on stimulation-induced changes in synaptosomal Ca2+ levels. Based on our findings, we hypothesize that Ca(2+)-dependent protein dephosphorylation resulting from calcineurin activation during physiological stimulation limits neurotransmitter release from brain nerve terminals, perhaps being dependent upon cyclic repolarization of the membrane during stimulation.

Barium evokes glutamate release from rat brain synaptosomes by membrane depolarization: involvement of K+, Na+, and Ca2+ channels.

During K(+)-induced depolarization of isolated rat brain nerve terminals (synaptosomes), 1 mM Ba2+ could substitute for 1 mM Ca2+ in evoking the release of endogenous glutamate. In addition, Ba2+ was found to evoke glutamate release in the absence of K(+)-induced depolarization. Ba2+ (1-10 mM) depolarized synaptosomes, as measured by voltage-sensitive dye fluorescence and [3H]-tetraphenylphosphonium cation distribution. Ba2+ partially inhibited the increase in synaptosomal K+ efflux produced by depolarization, as reflected by the redistribution of radiolabeled 86Rb+. The release evoked by Ba2+ was inhibited by tetrodotoxin (TTX). Using the divalent cation indicator fura-2, cytosolic [Ca2+] increased during stimulation by approximately 200 nM, but cytosolic [Ba2+] increased by more than 1 microM. Taken together, our results indicate that Ba2+ initially depolarizes synaptosomes most likely by blocking a K+ channel, which then activates TTX-sensitive Na+ channels, causing further depolarization, and finally enters synaptosomes through voltage-sensitive Ca2+ channels to evoke neurotransmitter release directly. Though Ba(2+)-evoked glutamate release was comparable in level to that obtained with K(+)-induced depolarization in the presence of Ca2+, the apparent intrasynaptosomal level of Ba2+ required for a given amount of glutamate release was found to be several-fold higher than that required of Ca2+.

Effect of Chronic Estradiol and Progesterone Treatments of Ovariectomized Rats on Brain Dopamine Uptake Sites

Dopamine released from brain nerve terminals is mainly removed from the synaptic cleft by an uptake mechanism. Despite their functional importance, modulation of the dopamine uptake sites is still not well known. Steroid hormones were shown to modulate brain dopamine transmission. The aim of this study was thus to investigate in ovariectomized rats the effects of 17 beta-estradiol and progesterone treatments on brain dopamine uptake sites. Treatments consisted of 17 beta-estradiol (10 micrograms/0.2 ml), progesterone (0.72 mg/0.2 ml), 17 beta-estradiol + progesterone, or the vehicle (0.3% gelatin in saline solution) twice daily for 2 weeks. The steroid treatments left the affinity of [3H]GBR 12935 binding to striatal homogenates unchanged (ovariectomized rats, 0.823 +/- 0.028 nM), whereas the density was increased by these steroids alone or in combination to a similar extent of 16-23%. Chronic treatment of ovariectomized rats with 17 beta-estradiol, progesterone, or their combination increased to the same extent and uniformly [3H]-GBR 12935 binding in the striatum as measured by autoradiography; the increase was similar in the substantia nigra pars compacta, whereas no steroid effect was observed in the nucleus accumbens and in the substantia nigra pars reticulata. In summary, chronic exposure to 17 beta-estradiol and/or progesterone increased dopamine uptake site density in the nigrostriatal dopaminergic system, whereas the nucleus accumbens and the substantia nigra pars reticulata were unaffected.