Modulatory Effect of Dopamine on High-Affinity Glutamate Uptake in the Rat Striatum

In vivo electrical stimulation of the frontal cortical areas was found to enhance sodium-dependent high-affinity glutamate uptake (HAGU) measured in rat striatal homogenates. This activating effect was counteracted by in vivo administration of apomorphine and by in vitro addition of dopamine (DA; 10(-8) M) in the incubation medium, and potentiated by in vivo haloperidol administration. At the doses used, the dopaminergic compounds had no effect on basal HAGU. alpha-Methylparatyrosine pretreatment was found to enhance slightly basal HAGU as well as the activating effects of cortical stimulation. Interestingly enough, lesion of dopaminergic neurons by substantia nigra injection of 6-hydroxydopamine (6-OHDA) did not cause any significant change either in basal HAGU or in the effect of cortical stimulation. Measurement of DA effects in vitro in experiments combined with in vivo manipulations of the dopaminergic nigrostriatal and corticostriatal systems showed that the capacity of DA to inhibit striatal HAGU depends directly on the level of the uptake activation reached over basal value. These results suggest that under physiological conditions, the dopaminergic nigrostriatal pathway exerts a modulatory presynaptic action on corticostriatal glutamatergic transmission, counteracting increasing glutamatergic activity. In the case of chronic DA depletion induced by 6-OHDA, striatal adaptations may occur modifying the mechanisms acting at corticostriatal nerve terminal level.     

Ontogeny of Dopamine D1 Receptors in Rat Striatum

The development of dopamine D1 receptors in rat striatum during the early postnatal period is described, using [3H]piflutixol as ligand. Dopamine D1 receptors increase in number from day of birth until about 21 days of age, when they reach adult levels. This increase in number parallels the increase in several other dopamine markers in striatum during the same time period. The increase is reflected in an increase in Bmax of ligand binding to D1 receptors. All other properties of D1 receptors that were examined do not change throughout this developmental period and are essentially the same as those found in adult tissue. These include association and dissociation rates, affinity for piflutixol as determined by kinetic and saturation studies, and pharmacology. These studies provide a biochemical and pharmacological basis for further studies on the ontogeny of dopamine receptors and of striatum and on factors regulating development of this region.     

Electrophysiological Effects of Three Groups of Glutamate Metabotropic Receptors in Rat Piriform Cortex

1. The effects of three metabotropic glutamate receptor (mGluR) agonists were tested in two pathways of rat piriform cortex. The group I, II and III mGluR agonists used were RS-3,5-dihydroxyphenenylglycine (DHPG) (10–100 μM), (2S,1′S,2′S)-2-Carboxycyclopropyl (L-CCG) (20–100 μM) and L(+)-2-amino-4-phosphonobutyric acid (L-AP4) (5–500 μM), respectively.2. The effects of the three groups of agonists on synaptic transmission in the two piriform cortex pathways also were examined. All three agonists reduced the amplitude of the monosynaptic EPSPs generated by stimulation of the lateral olfactory tract (LOT) or of the association fiber pathway (ASSN). This was always accompanied by an increase in paired pulse facilitation.3. Group I and II mGluR agonists had similar synaptic effects on the two pathways, while the group III mGluR agonist suppressed the LOT pathway more than the association pathway.4. The group II and III mGluR agonists had no effect on passive membrane properties of pyramidal neurons. Group I agonists depolarized the pyramidal neuron membrane potential, and enhanced both membrane resistance and noise.5. Our data suggest that all three types of mGluRs modulate synaptic transmission in both of these pathways in piriform cortex. Only group I agonists alter post-synaptic membrane properties, while all three types of receptor regulate synaptic transmission. Groups I and II are equally potent in the LOT and association fiber pathways, while group III receptors are more potent in the LOT than the association fiber pathways.     

Endogenous Dopamine Increases Extracellular Concentrations of Glutamate and GABA in Striatum of the Freely Moving Rat: Involvement of D1 and D2 Dopamine Receptors

Interactions between endogenous dopamine, glutamate, GABA, and taurine were investigated in striatum of the freely moving rat by using microdialysis. Intrastriatal infusions of the selective dopamine uptake inhibitor nomifensine (NMF) were used to increase the endogenous extracellular dopamine. NMF produced a dose-related increase in extracellular dopamine and also increased extracellular concentrations of glutamate, GABA, and taurine. Extracellular increases of dopamine were significantly correlated with extracellular increases of glutamate and GABA, but not taurine. To investigate whether the increased extracelular dopamine produced by NMF was responsible for the concomitant increase of glutamate and GABA, D1, and D2 receptor antagonists were used. Dopamine receptor antagonists D1 (SCH23390) and D2 (sulpiride) significantly attenuated the increases of glutamate and GABA produced by NMF. These data suggest that endogenous dopamine, through both D1 and D2 dopamine receptors, plays a role in releasing glutamate and GABA in striatum of the freely moving rat.     

Metabotropic Glutamate Receptors Stimulate Phospholipase D via Different Pathways in the Adult and Neonate Rat Hippocampus

In order to characterize the ontogenetic profile of metabotropic glutamate (mGlu) receptors coupled to phospholipase D (PLD) we examined the effects of selected mGlu agents on PLD activity in immature and adult rat hippocampus. The group I mGlu receptor agonist 3,5-dihydroxyphenylglycine stimulated PLD in immature tissue, but reduced the PLD response evoked by the nonselective mGlu receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate [(1S,3R)-ACPD] in adult hippocampus. (2R,1S,2R,3S)-2-(2-Carboxy-3-phenylcyclopropyl)glycine (PCCG-13), a recently characterized selective antagonist of PLD-coupled mGlu receptors, displayed a much greater activity in reducing the PLD response to (1S,3R)-ACPD in adult than in neonate hippocampus. Our results lend support to the hypothesis that glutamatergic activation of PLD in the rat hippocampus is developmentally regulated.     

Distribution and Developmental Regulation of Metabotropic Glutamate Receptor 7a in Rat Brain

Abstract: To determine the regional and cellular distribution of the metabotropic glutamate receptor mGluR7a, we used rabbit anti-peptide polyclonal-targeted antibodies against the C-terminal domain of mGluR7a. Here we report that immunocytochemistry at the light-microscopic level revealed that mGluR7a is widely distributed throughout the adult rat brain, with a high level of expression in sensory areas, such as piriform cortex, superior colliculus, and dorsal cochlear nucleus. In most brain structures, mGluR7a immunoreactivity is characterized by staining of puncta and fibers. However, in some regions, including the locus ceruleus, cerebellum, and thalamic nuclei, both cell bodies and fibers are immunopositive. The changes in levels of mGluR7a during development were investigated with immunoblotting and immunocytochemical analysis. Immunoblot analysis revealed that the levels of mGluR7a are differentially regulated across brain regions during postnatal development. In cortical regions (hippocampus, neocortex, and olfactory cortex), mGluR7a levels were highest at postnatal day 7 (P7) and P14, then declined in older rats. In contrast, mGluR7a levels were highest at P7 in pons/medulla and cerebellum and decreased markedly between P7 and P14. In these regions, mGluR7a immunoreactivity was at similar low levels at P14 and P21 and in adults. Immunocytochemical analysis revealed that staining for mGluR7a was exceptionally high in fiber tracts in P7 animals relative to adults. Furthermore, the pattern of mGluR7a immunoreactivity in certain brain structures, including cerebellum, piriform cortex, and hippocampus, was significantly different in P7 and adult animals. In summary, these data suggest that mGluR7a is widely distributed throughout the rat brain and that this receptor undergoes a dynamic, regionally specific regulation during postnatal development.     

Turnover of Dopamine and Dopamine Metabolites in Rat Brain: Comparison Between Striatum and Substantia Nigra

Measurements of the turnover of dopamine (DA) and DA metabolites have been performed in the striatum and substantia nigra (SN) of the rat. Turnover rates of 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid have been assessed from the disappearance rates after blocking their formation by inhibition of monoamine oxidase by pargyline and of catechol-O-methyltransferase by tropolone. DA turnover has been measured as 3-methoxytyramine (3-MT) plus DA accumulation rate after MAO inhibition by pargyline and as accumulation rate of 3,4-dihydroxyphenylalanine (DOPA) after inhibition of aromatic amino acid decarboxylase by NSD 1015 or NSD 1034. These measures of DA turnover have been compared with alpha-methyl-p-tyrosine (alpha-MT)-induced DA disappearance rate. In SN all the different measures of DA turnover are in the same range (55-62 nmol/g protein/h) whereas in striatum DOPA accumulation rate after NSD 1015 and alpha-MT-induced DA disappearance rate (16-23 nmol/g/h) are much lower than DOPAC disappearance rate after pargyline, 3-MT plus DA accumulation rate after pargyline, and DOPA accumulation rate after NSD 1034 (39-46 nmol/g/h). The data confirm our previous findings indicating that the fractional turnover rate of DA is more rapid in SN than in striatum and that O-methylation of DA is relatively more important in SN. In striatum at least two pools of DA with different turnover rates appear to exist, whereas in SN, DA behaves as if located in a single compartment.     

Evidence for Neuronal Origin and Metabotropic Receptor-Mediated Regulation of Extracellular Glutamate and Aspartate in Rat Striatum In Vivo Following Electrical Stimulation of the Prefrontal Cortex

Abstract: Extracellular levels of glutamate (Glu) and aspartate (Asp) were measured at 5-s intervals in the striatum of chloral hydrate-anesthetized rats by using microdialysis coupled to an automated assay system based on capillary electrophoresis with laser-induced fluorescence. Application of a single 10-s train of depolarizing pulses to the prefrontal cortex caused a rapid increase in Glu and Asp concentrations (200–300% of basal value), which returned to basal level within 60 s. The stimulated rise in Glu and Asp concentrations was blocked completely by 2 µ M tetrodotoxin or depletion of extracellular Ca²⁺, suggesting a neuronal origin of the Glu and Asp. Infusion of the Glu transport inhibitor l - trans -pyrrolidine-2,4-dicarboxylic acid (200 µ M ) increased resting Glu and Asp levels by 300–500% without altering electrically stimulated changes in Glu and Asp concentration. Stimulated Glu and Asp concentration changes were suppressed by 91 and 73%, respectively, by the metabotropic Glu receptor agonist (1 S ,3 R )-1-aminocyclopentane- trans -1,3-dicarboxylate (200 µ M ). This effect was blocked by the metabotropic Glu receptor antagonist ( RS )-α-methylcarboxyphenylglycine (MCPG; 200 µ M ). MCPG alone produced no effect on electrically stimulated changes in Glu and Asp levels; however, in the presence of l - trans -pyrrolidine-2,4-dicarboxylic acid, MCPG produced a five- to sixfold increase in stimulated overflow. Based on these results, it is concluded that release of Glu and Asp from corticostriatal neurons can be inhibited by activation of metabotropic Glu autoreceptors, which may be an important determinant of excitatory transmission at striatal synapses.     

Distribution and Localization of Estrogen-Sensitive Dopamine Receptors in the Rat Brain

Administration of estrogen to adult male rats increases the density of striatal dopamine receptors. The densities of the dopamine receptors in the nucleus accumbens and cortex are not altered, while the density of those in the hippocampus is decreased. In the pituitary the density, on a whole pituitary basis, is not changed. The increased density of striatal dopamine receptors normally observed after estrogen treatment is prevented by prior injection into the striatum of kainic acid, which destroys the intrinsic neurons in the striatum. In addition, the benzodiazepine receptors in the striatum, cortex, hippocampus, and cerebellum are not altered by estrogen treatment, showing the specificity of the estrogen treatment and suggesting that the effects of estrogen are not mediated through benzodiazepine receptors.     

Autoradiographic Localization of the Putative D4 Dopamine Receptor in Rat Brain

The putative dopamine D4 receptor protein in rat brain was labelled and quantified autoradiographically using two selective benzamides: [³H]YM-09151-2 which labels D2, D3 and D4 dopamine receptors and [³H]Raclopride which labels D2 and D3. The difference in densities of both ligands at saturable concentrations, show a regional distribution for the putative D4 receptor in the following rank order: hippocampus > caudate putamen > olfactory tubercle = substancia nigra > nucleus accumbens core > cerebral cortex > cerebellum. A calculated value of 0.34 pmol/mg protein was attributable to D4 receptor maximum capacity in caudate putamen and was obtained after subtracting the Bmax of the ligands. Our results show that the distribution of D4 receptor only partially overlaps with the D4 mRNA localization reported earlier and is not only associated to limbic structures but to motor areas as well.     

Local Activation of Metabotropic Glutamate Receptors Inhibits the Handling-Induced Increased Release of Dopamine in the Nucleus Accumbens but Not that of Dopamine or Noradrenaline in the Prefrontal Cortex: Comparison with Inhibition of Ionotropic Receptors

Abstract: On-line in vivo microdialysis was used to determine the effects of a 16-min handling period on release of dopamine (DA) in the nucleus accumbens and of DA and noradrenaline (NA) in the medial prefrontal cortex of awake, freely moving rats. DA and NA were determined in one HPLC run. Handling resulted in an immediate and strong increase of both catecholamines in the prefrontal cortex. Maximal values for DA were 295%, and for NA 225%, of controls. DA in the nucleus accumbens was also increased (to 135% of controls) but only after a short delay. Local inhibition of ionotropic glutamate receptors by continuous reversed dialysis of the drugs 6-cyano-7-nitroquinoxaline, d -2-amino-5-phosphonopentanoic acid, or dizocilpine did not significantly affect handling-induced increases in cortical DA and NA release. Neither did the agonist of metabotropic glutamate receptors, trans -(1 S ,3 R )-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), or the GABA-B agonist baclofen. Reversed dialysis of dizocilpine in the nucleus accumbens was equally ineffective, but ACPD inhibited the increase in DA release in this area. Stimulation of metabotropic glutamate receptors in the nucleus accumbens was previously reported to inhibit activation of DA release in that area after stimulation of glutamatergic or dopaminergic afferents. It is concluded that metabotropic receptors in the nucleus accumbens are important for the control of activation of DA release in the accumbens by physiological stimuli but that a similar mechanism is lacking in the prefrontal cortex.     

Depolarization Increases Chloride-Dependent Glutamate Sequestration in Synaptic Membranes of Rat Cerebral Cortex

To assess the functions of Cl- -dependent glutamate "binding" (Cl- -dependent glutamate uptake) in synaptic membranes, possible effects of depolarization on the uptake were examined. When rat cerebral cortical slices were preincubated with depolarizing agents such as veratrine (7 micrograms/ml), 10 microM aconitine, 56 mM K+, and 50 microM monensin, [3H]glutamate uptake by the crude synaptic membranes, which were subsequently prepared from the pretreated slices, was increased by 60-85%. Stimulation of the glutamate uptake by predepolarization was dependent on Na+ but not on Ca2+. The bindings of gamma-[3H]aminobutyric acid and 5-[3H]hydroxytryptamine were not significantly affected by the predepolarization. Veratrine pretreatment increased the maximal density of the glutamate uptake sites without affecting the affinity for glutamate. Several characteristics of the uptake sites increased by the veratrine pretreatment coincided with those of Cl- -dependent glutamate uptake sites. Na+-dependent glutamate binding (Na+-dependent glutamate uptake) to the membranes was not affected by pretreatment with veratrine. The content of endogenous glutamate and the noninulin space in the membrane fractions were not changed by the predepolarization. The increase in the glutamate uptake induced by pretreatment with high K+ was reversible: it returned to the control level after a second incubation of the slices in control medium. These results suggest that the Cl- -dependent glutamate sequestration system in synaptic membranes is regulated by the membrane potential.     

Phosphorylation of Mitogen-Activated Protein Kinase in Cultured Rat Cortical Glia by Stimulation of Metabotropic Glutamate Receptors

Abstract: Activation of metabotropic glutamate receptors (mGluRs) in glia results in significant physiological effects for both the glia and the neighboring neurons; but in many cases, the mGluR subtypes and signal transduction mechanisms mediating these effects have not been determined. In this study, we report that mGluR activation in primary cultures of rat cortical glia results in tyrosine phosphorylation of several proteins, including p44/p42 mitogen-activated protein kinases, also referred to as extracellular signal-regulated kinases (ERK1/2). Incubation of glial cultures with the general mGluR agonist 1-aminocyclopentane-1 S ,3 R -dicarboxylate and the mGluR group I-selective agonists ( RS )-3,5-dihydroxyphenylglycine (DHPG) and l -quisqualate resulted in increased tyrosine phosphorylation of ERK1/2. The group II-selective agonist (2 S ,2' R ,3' R )-2-(2',3'-dicarboxycyclopropyl)glycine and group III-selective agonist l (+)-2-amino-4-phosphonobutyric acid had no effect on tyrosine phosphorylation. DHPG-induced ERK1/2 phosphorylation could be inhibited by an antagonist that acts at group I or group II mGluRs but not by antagonists for group II and group III mGluRs. Protein kinase C (PKC) activators also induced ERK1/2 phosphorylation, but the PKC inhibitor bisindolylmaleimide I did not inhibit DHPG-induced ERK1/2 phosphorylation at a concentration that inhibited the response to phorbol 12,13-dibutyrate. These data suggest that mGluR activation of ERK1/2 in cultured glia is mediated by group I mGluRs and that this effect is independent of PKC activation. Furthermore, immunoblots with antibodies against various mGluR subtypes show expression of mGluR5, but no other mGluRs in our cultures. Taken together, these results suggest that mGluR5 stimulation results in tyrosine phosphorylation of ERK1/2 and other glial proteins.     

3,5-Dihydroxyphenylglycine Is a Highly Selective Agonist for Phosphoinositide-Linked Metabotropic Glutamate Receptors in the Rat Hippocampus

Abstract: Metabotropic glutamate receptors (mGluRs) are a heterogeneous family of G protein-coupled glutamate receptors that are linked to multiple second messenger systems in the CNS. In this study the selectivity of mGluR agonists for different mGluR second messenger effects was characterized in slices of the rat hippocampus. The mGluR agonists (1 S ,3 R )-1-aminocyclopentane-1,3-dicarboxylic acid and (2 S ,3 S ,4 S )α-(carboxycyclopropyl)glycine produced multiple effects on second messengers that included enhanced phosphoinositide hydrolysis in both adult and neonatal rat hippocampus, inhibition of forskolin-stimulated cyclic AMP (cAMP) formation in adult tissue, and increases in basal cAMP formation in the neonatal hippocampus. In contrast, 3,5-dihydroxyphenylglycine was potent and effective in increasing phosphoinositide hydrolysis in both adult and neonatal hippocampus but unlike the other mGluR agonists did not inhibit forskolin-stimulated cAMP formation (in the adult) or substantially enhance basal cAMP formation (in the neonate). Thus, in the rat hippocampus mGluR agonist-mediated increases or decreases in cAMP formation are not secondary to mGluR-mediated changes in phosphoinositide hydrolysis. Furthermore, 3,5-dihydroxyphenylglycine can be used to activate subpopulations of mGluRs coupled to phosphoinositide hydrolysis with minimal effects on cAMP-mGluR second messenger systems.     

Synaptic Membrane Antigens in Developing Rat Brain Cerebral Cortex and Cerebellum

Abstract: The contents of five synaptic membrane antigens (56K, 58K, 62K, 63K, and 64K) were determined in rat cerebral cortex and cerebellum at eight developmental time points: E9, E14, P < 1, P5, P14, P28, P60, and P180 (E, embryonic; P, postnatal). In cerebral cortex, the five antigens showed five different developmental patterns with respect both to specific content (i.e., quantity per unit of membrane) and total content (i.e., quantity per cortex). The 56K, 58K, and 62K polypeptides were first detected at E14, increased slightly to P5, then increased rapidly from P5 to P28 by 14-, 11-, and 18-fold, respectively. From P28 to PI80, the patterns of these antigens showed very large differences. The 63K and 64K antigens were first detected at P14 and P28, respectively. The specific content of 63K antigen continued to increase steadily throughout adult life; in contrast, the specific content of the 64K antigen did not change appreciably. In cerebellum only three antigens (56K, 58K, and 62K) were detected. These three antigens showed different developmental patterns. The 56K polypeptide was first detected at E14; its specific content increased very rapidly to a maximum at P < 1; it then decreased, first slowly, and then more rapidly, disappearing at P60. The 58K polypeptide also was detectable at E14 and increased very rapidly to a maximum at P < 1. It then decreased markedly to P5, followed by an increase, returning almost to its maximum level at P14. It then slowly decreased disappearing at P180. The 62K antigen was first detected at P14 and then it slowly decreased with disappearance at P60. The patterns with respect to total contents per cerebellum were similar for the three antigens, with a maximum at P28. We conclude that the highest increase in the contents of these antigens roughly corresponds to the period of maximal synaptogenesis (P9 to P28) in both regions. Differences among developmental patterns probably reflect changing molecular machinery required for development and functional differentiation of synapses in different brain regions. The fine structure of these patterns suggests that the quantitative measurement of synaptic membrane antigens will be useful for delineating complex processes occurring during synaptogenesis.     

Enhanced Sensitivity of "Metabotropic" Glutamate Receptors After Induction of Long-Term Potentiation in Rat Hippocampus

Stimulation of [3H]inositol monophosphate ([3H]InsP) formation by ibotenate or trans-1-aminocyclopentyl-1,3-dicarboxylic acid (t-ACPD) in rat hippocampal slices was enhanced after tetanic stimulation of the Schaffer collaterals projecting to the CA1 region (in vitro) or the perforant pathway projecting to the dentate gyrus (in freely moving animals). This effect was observed 5 h (but not 2 h) after long-term potentiation (LTP) induction and was abolished if tetanic stimulation was performed in the presence of specific antagonists of N-methyl-D-aspartate receptors. The delayed increase in excitatory amino acid-induced polyphosphoinositide (PPI) hydrolysis was accompanied by an enhanced responsiveness to norepinephrine, whereas the basal and carbamylcholine-stimulated [3H]InsP formation were unchanged. These results suggest that an increased activity of "metabotropic" glutamate receptors may contribute to the synaptic mechanisms enabling the late expression and or maintenance of LTP. Accordingly, LTP decayed more rapidly (within 5 h) in rats repeatedly injected with LiCl (60-120 mg/kg, i.p., for 10 days), a treatment that led to a reduced efficacy of ibotenate and norepinephrine in stimulating PPI hydrolysis in hippocampal slices.     

大鼠大脑皮层中钙调神经磷酸酶活力的时空变化

以ＰＮＰＰ为底物测定了超离心制备的大鼠出生后早期和成年大脑皮层亚细胞各组分中钙调神经磷酸酶的活力。实验结果表明：（ｌ）钙调神经磷酸酶活力广泛地存在于胞液和突触部分,并且各亚细胞组分有明显差异。成年大鼠大脑皮层中ＣａＮ活力相对最高水平是在突触体,突触质,胞液,重的和轻的突触膜部分。（２）大鼠大脑皮层突触体中ＣａＮ活力在出生后第２周和第３周出现高峰的平台期,这与突触发生的高峰期是一致的。在胞液和重的突触膜中ＣａＮ活力最高水平是在出生后的第７ｄ,而在突触质和轻的突触膜中是在第２０ｄ。总之,这些发现证实,在脑发育期间,ＣａＮ活力是依照区域和时间性控制的,提示ＣａＮ可能参与了突触功能作用。     

GABA Autoreceptors Are Not Coupled to Benzodiazepine Receptors in Rat Cerebral Cortex

Abstract: Depolarization-induced release of [³H] γ -aminobutyric acid ([³H]-GABA) from preloaded slices of rat cerebral cortex was inhibited by muscimol and THIP in a dose-dependent fashion. This inhibition of release was prevented by the GABA antagonists bicuculline and picrotoxin. These results confirm previous reports postulating the existence of GABA autoreceptors on GABAergic terminals. Since benzodiazapines are known to facilitate postsynaptic GABA actions, the effect of flunitrazepam on the inhibition of GABA release mediated through the autoreceptors has been examined. At a concentration of 1 μ m or 10 μ m , flunitrazepam had no effect on the IC₅₀ values for muscimol or THIP in inhibiting stimulated GABA release. It thus seems that GABA autoreceptors are not functionally coupled to benzodiazepine receptors in rat cerebral cortex.