Synchronized overproduction of neurotransmitter receptors in diverse regions of the primate cerebral cortex.

A remarkable diversity of neurotransmitter receptors develops concurrently in disparate areas of the primate cerebral cortex. The density of dopaminergic, adrenergic, serotonergic, cholinergic, and GABAergic receptors (where GABA is gamma-aminobutyric acid) in rhesus monkey reaches a maximum level between 2 and 4 months of age and then declines gradually to adult levels in all layers of sensory, motor, and association regions. The synchronized development of neurotransmitter receptors in diverse layers and regions of the neocortex occurs pari passu with synaptogenesis, demonstrating unusual coordination of biochemical and structural maturation and supporting the hypothesis that the entire cerebral cortex matures as an integrated network, rather than as a system-by-system cascade.     

Steroid receptors and action in the primate follicle.

Studies localizing steroid receptor (R) proteins and their mRNAs to the primate ovary are consistent with a local role for progesterone (P) and androgen (A) in modulating follicle growth and/or maturation via classic R-mediated pathways, but data in support of estrogen (E) action remain equivocal. Investigations of the hypothesis that steroids play a pivotal role in folliculogenesis or gametogenesis in macaques receiving inhibitors of steroid enzyme synthesis and in women with congenital steroid enzyme deficiencies reveal that (a) antral follicle growth, maturation, and luteinization, as well as oocyte meiosis, do not require high or increasing E levels; (b) elevated A to E ratios are detrimental to the gametogenic functions of the primate follicle, but do not alter growth and maturation of antral follicles; and (c) ovulation and luteinization of the mature follicle are dependent on local P actions. The genomic actions of steroids likely vary between compartments and with the developmental state of the follicle; however, potential nongenomic actions of steroids in follicles remain largely undefined. Future advances will rely on identification of discrete biochemical, morphological, and functional correlates of steroid hormone action in the somatic and gametogenic compartments of the primate follicle throughout its life cycle.     

Dopamine D2 receptors in the cerebral cortex: distribution and pharmacological characterization with [3H]raclopride.

An apparent involvement of dopamine in the regulation of cognitive functions and the recognition of a widespread dopaminergic innervation of the cortex have focused attention on the identity of cortical dopamine receptors. However, only the presence and distribution of dopamine D1 receptors in the cortex have been well documented. Comparable information on cortical D2 sites is lacking. We report here the results of binding studies in the cortex and neostriatum of rat and monkey using the D2 selective antagonist [3H]raclopride. In both structures [3H]raclopride bound in a sodium-dependent and saturable manner to a single population of sites with pharmacological profiles of dopamine D2 receptors. D2 sites were present in all regions of the cortex, although their density was much lower than in the neostriatum. The density of these sites in both monkey and, to a lesser extent, rat cortex displayed a rostral-caudal gradient with highest concentrations in the prefrontal and lowest concentrations in the occipital cortex, corresponding to dopamine levels in these areas. Thus, the present study establishes the presence and widespread distribution of dopamine D2 receptors in the cortex.     

D1A, D1B, and D1C dopamine receptors from Xenopus laevis.

Three distinct genes encoding members of the D1 dopamine receptor family were isolated from Xenopus laevis. Based on the deduced amino acid sequence, two of the receptors (Xen D1A and Xen D1B) appear to be homologues of mammalian D1/D1A and D5/D1B receptors. The third receptor, termed Xen D1C, displays equal overall amino acid and nucleotide sequence identity (approximately 55%) with mammalian D1A and D1B/D5 receptors. In agreement with their structural similarities, Xen D1A and D1B receptors, when expressed in COS-7 cells, displayed pharmacological profiles that paralleled those of their mammalian counterparts, with dopamine and 2-amino-6,7-dihydroxytetralin exhibiting 10-fold higher affinity for D1B than for D1A. The Xen D1C receptor displayed an overall rank order of potency and pharmacological profile clearly indicative of a D1-like receptor, with individual affinities for most agonists higher than those for either Xen or mammalian D1/D1A and D5/D1B receptors, whereas antagonist Ki values were intermediate to those for the D1/D1A and D5/D1B receptors. All three receptors stimulated adenylate cyclase activity in response to dopamine or SKF-82526. Xen D1A, D1B, and D1C receptor mRNAs were differentially distributed, with all three receptors expressed in brain and only D1B and D1C receptors expressed in kidney. The existence of a receptor which lacks appreciable overall sequence similarity to, but displays pharmacological homology with, mammalian D1-like receptors lends strong support to the contention that additional mammalian D1-like receptor gene products may exist to allow for the expression of the full spectrum of D1-like dopamine receptor-mediated events.     

Ontogenesis of insulin receptors in human cerebral cortex.

This study examines the ontogenesis of insulin receptors in human cerebral cortex. Synaptosomal membrane fraction was obtained after subcellular fractionation of human brain tissue. The 24 cases studied were classified according to the statistical differences found in: Group I, pre-term, up to 30 weeks of gestation; group II, full-term and newborns; group III from one year to adulthood. Scatchard plots of insulin binding to brain membranes were curvilinear and showed a decrease in insulin receptor number as a function of age with slight differences in affinity. Receptor number were 3.0 +/- 0.8 pmol/mg in Group I, 0.6 +/- 0.14 pmol/mg and 0.2 +/- 0.024 pmol/mg in Groups II and III respectively. Values of 5'nucleotidase and Na+ K+ ATPase activities, were similar in all groups, which indicates that the purity of the fraction used for binding was similar in each group. According to the ontogenic profile in insulin binding described in this work, it may be assumed that the higher concentration of insulin receptors in human brain during the fetal period can determine some insulin action in this early stage of maturation, even though the functionality of these receptors remains to be elucidated.     

Convergence and divergence of neurotransmitter action in human cerebral cortex.

The postsynaptic actions of acetylcholine, adenosine, gamma-aminobutyric acid, histamine, norepinephrine, and serotonin were analyzed in human cortical pyramidal cells maintained in vitro. The actions of these six putative neurotransmitters converged onto three distinct potassium currents. Application of acetylcholine, histamine, norepinephrine, or serotonin all increased spiking by reducing spike-frequency adaptation, in part by reducing the current that underlies the slow after hyperpolarization. In addition, application of muscarinic receptor agonists to all neurons or of serotonin to middle-layer cells substantially reduced or blocked the M-current (a K+ current that is voltage and time dependent). Inhibition of neuronal firing was elicited by adenosine, baclofen (a gamma-aminobutyric acid type B receptor agonist), or serotonin and appeared to be due to an increase in the same potassium current by all three agents. These data reveal that individual neuronal currents in the human cerebral cortex are under the control of several putative neurotransmitters and that each neurotransmitter may exhibit more than one postsynaptic action. The specific anatomical connections of these various neurotransmitter systems, as well as their heterogeneous distribution of postsynaptic receptors and responses, allows each to make a specific contribution to the modulation of cortical activity.     

D1 and D2 dopamine receptors in separate circuits cooperate to drive associative long-term potentiation in the prefrontal cortex

Dopamine release associated with motivational arousal is thought to drive goal-directed learning and consolidation of acquired memories. This dopamine hypothesis of learning and motivation directly suggests that dopamine is necessary for modifications of excitatory synapses in dopamine terminal fields, including the prefrontal cortex (PFC), to “stamp in” posttrial memory traces. It is unknown how such enabling occurs in native circuits tightly controlled by GABAergic inhibitory tone. Here we report that dopamine, via both D1-class receptors (D1Rs) and D2-class receptors (D2Rs), enables the induction of spike timing–dependent long-term potentiation (t-LTP) in layer V PFC pyramidal neurons over a “window” of more than 30 ms that is otherwise closed under intact inhibitory constraint. Dopamine acts at D2Rs in local GABAergic interneurons to suppress inhibitory transmission, gating the induction of t-LTP. Moreover, dopamine activates postsynaptic D1Rs in excitatory synapses to allow t-LTP induction at a substantially extended, normally ineffective, timing interval (+30 ms), thus increasing the associability of prepost coincident stimuli. Although the D2R-mediated disinhibition alone is sufficient to gate t-LTP at a normal timing (+10 ms), t-LTP at +30 ms requires concurrent activation of both D1Rs and D2Rs. Our results illustrate a previously unrecognized circuit-level mechanism by which dopamine receptors in separate microcircuits cooperate to drive Hebbian synaptic plasticity across a significant temporal window under intact inhibition. This mechanism should be important in functioning of interconnected PFC microcircuits, in which D1Rs and D2Rs are not colocalized but their coactivation is necessary.     

Thinning of capillary walls and declining numbers of endothelial mitochondria in the cerebral cortex of the aging primate, Macaca nemestrina.

Samples were obtained from the frontal and occipital cortex of Macaque monkeys at 4, 10 and 20 years of age. Electron microscopic studies revealed attenuation of capillary walls and declining numbers of endothelial mitochondria per capillary profile with increasing age. The basal lamina surrounding the capillary increased in thickness between 4 and 10 years of age; however, it did not undergo further change between 10 and 20 years. These results corroborate morphological and biochemical studies indicative of declining numbers of mitochondria, and decreasing mitochondrial ATP synthesis and ATPase activity in other tissues during aging.     

Localization of D1 and D2 dopamine receptors in brain with subtype-specific antibodies.

Five or more dopamine receptor genes are expressed in brain. However, the pharmacological similarities of the encoded D1-D5 receptors have hindered studies of the localization and functions of the subtypes. To better understand the roles of the individual receptors, antibodies were raised against recombinant D1 and D2 proteins and were shown to bind to the receptor subtypes specifically in Western blot and immunoprecipitation studies. Each antibody reacted selectively with the respective receptor protein expressed both in cells transfected with the cDNAs and in brain. By immunocytochemistry, D1 and D2 had similar regional distributions in rat, monkey, and human brain, with the most intense staining in striatum, olfactory bulb, and substantia nigra. Within each region, however, the precise distributions of each subtype were distinct and often complementary. D1 and D2 were differentially enriched in striatal patch and matrix compartments, in selective layers of the olfactory bulb, and in either substantia nigra pars compacta or reticulata. Electron microscopy demonstrated that D1 and D2 also had highly selective subcellular distributions. In the rat neostriatum, the majority of D1 and D2 immunoreactivity was localized in postsynaptic sites in subsets of spiny dendrites and spine heads in rat neostriatum. Presynaptic D1 and D2 receptors were also observed, indicating both subtypes may regulate neurotransmitter release. D1 was also present in axon terminals in the substantia nigra. These results provide a morphological substrate for understanding the pre- and postsynaptic functions of the genetically defined D1 and D2 receptors in discrete neuronal circuits in mammalian brain.     

A randomised trial of a pre-synaptic stimulator of DA2-dopaminergic and alpha2-adrenergic receptors on morbidity and mortality in patients with heart failure

BACKGROUND: By pre-synaptic stimulation of DA(2)-dopaminergic and alpha(2)-adrenergic receptors, nolomirole inhibits norepinephrine secretion from sympathetic nerve endings. We performed a clinical study with nolomirole in patients with heart failure (HF). METHODS: The study was designed as a multicentre, double blind, parallel group trial of 5 mg b.i.d. of nolomirole (n=501) versus placebo (n=499) in patients with severe left ventricular systolic dysfunction, recently in New York Heart Association (NYHA) class III/IV. The primary endpoint was time to all cause death or hospitalisation for HF, whichever came first. The study was event driven and required 420 primary events. The study was completed as scheduled. RESULTS: Mean age of patients was 70 years, and 73% were male. Heart rate and blood pressure were not different in the two treatment groups. There were no changes in blood pressure. There were 233 primary events in the nolomirole group versus 208 in the placebo group (p=0.1). There were 142/145 deaths and 369/374 all cause hospitalisations in the nolomirole/placebo groups. There were no differences in walking distance, quality of life or NYHA class. CONCLUSION: A dose of 5 mg b.i.d. of nolomirole was not beneficial (or harmful) in patients with heart failure.     

Association of m1 and m2 muscarinic receptor proteins with asymmetric synapses in the primate cerebral cortex: morphological evidence for cholinergic modulation of excitatory neurotransmission.

Muscarinic m1 receptors traditionally are considered to be postsynaptic to cholinergic fibers, while m2 receptors are largely presynaptic receptors associated with axons. We have examined the distribution of these receptor proteins in the monkey cerebral cortex and obtained results that are at odds with this expectation. Using immunohistochemistry with specific antibodies to recombinant m1 and m2 muscarinic receptor proteins, we have demonstrated that both m1 and m2 receptors are prominently associated with noncholinergic asymmetric synapses as well as with the symmetric synapses that characterize the cholinergic pathways in the neocortex. At asymmetric synapses, both m1 and m2 receptor immunoreactivity is observed postsynaptically within spines and dendrites; the m2 receptor is also found in presynaptic axon terminals which, in the visual cortex, resemble the parvicellular geniculocortical pathway. In addition, m2 labeling was also found in a subset of nonpyramidal neurons. These findings establish that the m2 receptor is located postsynaptically as well as presynaptically. The association of m1 and m2 receptors with asymmetric synapses in central pathways, which use excitatory amino acids as neurotransmitters, provides a morphological basis for cholinergic modulation of excitatory neurotransmission.     

Regulation of the phosphorylation of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa in vivo by dopamine D1, dopamine D2, and adenosine A2A receptors

Dopamine D(1), dopamine D(2), and adenosine A(2A) receptors are highly expressed in striatal medium-sized spiny neurons. We have examined, in vivo, the influence of these receptors on the state of phosphorylation of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32). DARPP-32 is a potent endogenous inhibitor of protein phosphatase-1, which plays an obligatory role in dopaminergic transmission. A dose-dependent increase in the state of phosphorylation of DARPP-32 occurred in mouse striatum after systemic administration of the D(2) receptor antagonist eticlopride (0.1-2.0 mg/kg). This effect was abolished in mice in which the gene coding for the adenosine A(2A) receptor was disrupted by homologous recombination. A reduction was also observed in mice that had been pretreated with the selective A(2A) receptor antagonist SCH 58261 (10 mg/kg). The eticlopride-induced increase in DARPP-32 phosphorylation was also decreased by pretreatment with the D(1) receptor antagonist SCH 23390 (0.125 and 0.25 mg/kg) and completely reversed by combined pretreatment with SCH 23390 (0.25 mg/kg) plus SCH 58261 (10 mg/kg). SCH 23390, but not SCH 58261, abolished the increase in DARPP-32 caused by cocaine (15 mg/kg). The results indicate that, in vivo, the state of phosphorylation of DARPP-32 and, by implication, the activity of protein phosphatase-1 are regulated by tonic activation of D(1), D(2), and A(2A) receptors. The results also underscore the fact that the adenosine system plays a role in the generation of responses to dopamine D(2) antagonists in vivo.     

Androgen receptors in the cerebral cortex of fetal female rhesus monkeys

The present study characterizes putative androgen receptor activity in the cerebral cortex cytosol of the female fetal monkey (Macaca mulatta) on days 125-135 postconception. Binding activities were compared using tritiated 5 alpha-dihydrotestosterone (DHT) and methyltrienolone (R1881) as ligands. Receptor concentration and association constants (Ka) were estimated from bound/total vs. total ligand binding curves. For R1881 and DHT, Ka values were 4.3 X 10(9) and 7.0 X 10(9) M-1, respectively. Receptor concentrations using the two ligands were estimated to be 5.5 X 10(-15) mol/mg protein (R1881; n = 2) and 1.7 X 10(-15) mol/mg protein (DHT). Analysis of receptor binding on DEAE-cellulose columns (KCl gradient) revealed multiple binding peaks (0.05-0.3 M KCl); similar elution profiles were obtained for the two ligands. Competition with either R1881 or DHT significantly reduced [3H]DHT binding throughout the KCl gradient, while triamcinolone acetonide had no effect. In contrast, [3H]R1881 binding was reduced by all three competitors. Cytosol from fetal male seminal vesicles had [3H]ligand-binding activity that was chromatographically similar to that of cerebral cortex. In contrast, binding in fetal male serum was negligible. Competition of cerebral cortex binding with a variety of hormones, including SCH-16423, progesterone, and cortisol, and analysis of the results on DEAE-cellulose suggested that there may be additional species of ligand-binding molecules. In summary, the findings verify the existence of a specific androgen receptor(s) in the cerebral cortex of fetal female rhesus monkeys. Its presence may be important for understanding both the influence of androgens on central nervous system development and the potential for teratogenic agents to disrupt normal patterns of central nervous system development.     

Potentiation of NMDA receptor currents by dopamine D1 receptors in prefrontal cortex

Interactions between dopamine and N-methyl-D-aspartate receptors (NMDARs) in prefrontal cortex (PFC) and other brain regions are believed to play an important role in normal mental function and neuropsychiatric disorders. In this study, we examined the regulation of NMDAR currents by the dopamine D1 receptor in PFC pyramidal neurons. Application of the D1 receptor agonist SKF81297 caused a prominent increase of the steady-state NMDA-evoked current in acutely isolated PFC pyramidal neurons. The D1 effect on NMDARs was independent of protein kinase A or protein phosphatase 1, but was abolished by incubation of neurons in Ca2+-free medium. Intracellular application of the Ca2+ chelator, calmodulin, or calmodulin inhibitors largely prevented the D1 modulation of NMDAR currents. Moreover, inhibiting PKC activity or disrupting PKC association with its anchoring protein also significantly reduced the D1 effect on NMDAR currents. This upregulation of NMDAR activity by dopamine D1 receptors and the previous finding on up-regulation of dopamine D1 receptors by NMDAR activation provide a cellular mechanism for the reciprocal interactions between D1 and NMDARs. These interactions may play an important role in modulating synaptic plasticity and thus in cognitive and emotional processes.     

Effects of clozapine and haloperidol on body weight,neuropeptide Y and leptin in patients with schizophrenia

Objective To investigate the changes of neuropeptide Y(NPY) ,leptin,body weight and their relationship in schizophrenics with clozapine and haloperidol treatment. Methods Thirty schizophrenic patients treated     

Stimulation of high-affinity adenosine A2 receptors decreases the affinity of dopamine D2 receptors in rat striatal membranes.

Since high-affinity adenosine A2 receptors (A2a) are localized exclusively in dopamine-rich regions in the central nervous system and mediate inhibition of locomotor activity, we have examined the effect of A2a receptor activation on D1 and D2 receptor binding in membrane preparations of the rat striatum. The A2a agonist 2-[p-(2-carboxyethyl)phenethylamino]-5'- N-ethylcarboxamidoadenosine (CGS 21680) increased the Kd of the dopamine D2 agonist L-(-)-N-[3H]propylnorapomorphine without affecting the Bmax. The increase in Kd was maximal (40%) at 30 nM CGS 21680. CGS 21680 (30 nM) decreased the dopamine-induced inhibition of [3H]raclopride (a D2 antagonist) binding due to an increase (about 3-fold) in KH and KL, the dissociation constants of high- and low-affinity binding sites. The effects of CGS 21680 were antagonized by the adenosine antagonist 8-phenyltheophylline (10 microM). (-)-N6-(2-Phenylisopropyl)adenosine produced an effect similar to that of CGS 21680, provided the concentration used was high enough to stimulate A2a receptors (300 nM). GTP (50 microM) also decreased the dopamine-induced inhibition of [3H]raclopride binding but, in contrast to CGS 21680, GTP decreased the proportion of D2 receptors in the high-affinity state. CGS 21680 (30 nM) did not affect the Kd or Bmax of [3H]raclopride and failed to affect ligand binding to D1 receptors. Thus, stimulation of A2a receptors potently reduces the affinity of D2 agonist binding sites within the plasma membrane of striatal neurons. This A2a-D2 interaction may underlie the neuroleptic-like actions of adenosine agonists and the enhancing effects of adenosine antagonists, such as caffeine, on locomotor activity.     

Presence of hormonally-sensitive adenylate cyclase receptors in capillary-enriched fractions from rat cerebral cortex.

The 10 000 g particulate fraction from capillary-enriched fractions isolated from rat cerebral cortex was shown to possess an adenylate cyclase highly sensitive to activation by sodium fluoride, norepinephrine, epinephrine, isoproterenol and dopamine. To a lesser extent histamine and three dopamine agonists, namely M-7 (5,6-dihydroxy-2-dimethylamino tetralin), ET-495 (methane sulfonate of pyribedil), and S-584 (metabolite of pyribedil) stimulated the enzyme preparation. The action of norepinephrine was blocked by propanolol while phenotolamine and haloperidol were relatively ineffective except at highest concentrations. Phentolamine and propanolol at only highest concentrations (10(-4) M) antagonized the action of dopamine. Haloperidol was seen to be a potent inhibitor of either dopamine- or dopamine agonist-sensitive adenylate cyclase. No effects on the enzyme were observed with methoxamine, octopamine or serotonin. These preliminary data suggest the presence of a mixed population of receptors for adenylate cyclase in rat brain capillaries.     

Affinity labeling of muscarinic receptors in rat cerebral cortex with a photolabile antagonist.

Highly potent photoaffinity probes for muscarinic binding sites were prepared by the incorporation of an azido group into the benzilic acid moiety in two compound, 3-quinuclidinyl benzilate (3QNB) and N-methyl-4-piperidyl benzilate (4NMPB). Inactivation of muscarinic sites in rat cortex depends on the formation of a reversible complex with the azides prior to their photolytic conversion to the highly reactive nitrenes. During photolysis, radiolabeled azido-4NMPB interacted specifically and with high affinity (Kd = 1.06 nM) with the muscarinic receptors, and the ligand could be covalently incorporated into a macromolecule of about 86,000 Mr, presumably the muscarinic receptor. The incorporation was almost stoichiometric when compared to determination of receptor density by reversible ligands. Atropine (10 microM) afforded specific protection (greater than 83%) of the receptor against inactivation by azido-[3H]4NMPB. This compound and the other ligands described here (i.e., amino-4NMPB, amino-3QNB, and azido-3QNB) represent powerful potential probes for the biochemical isolation and characterization of muscarinic receptors.     

Colocalization of integrin receptors and reelin in dendritic spine postsynaptic densities of adult nonhuman primate cortex

The expression of telencephalic reelin (Reln) and glutamic acid decarboxylase mRNAs and their respective cognate proteins is down-regulated in postmortem brains of schizophrenia and bipolar disorder patients. To interpret the pathophysiological significance of this finding, immunoelectron microscopic experiments are required, but these cannot be carried out in postmortem human brains. As an alternative, we carried out such experiments in the cortex of rats and nonhuman primates. We found that Reln is expressed predominantly in layer I of both cortices and is localized to bitufted (double-bouquet), horizontal, and multipolar gamma-aminobutyric acid-ergic interneurons, which secrete Reln into extracellular matrix. Reln secretion is mediated by a constitutive mechanism that depends on the expression of a specific signal peptide present in the Reln carboxy-terminal domain. Extracellular matrix Reln is found to aggregate in proximity of postsynaptic densities expressed in apical dendrite spines, which include also the alpha(3) subunit of integrin receptors. Most pyramidal neurons of various cortical layers express the mouse-disabled 1 (Dab1) protein, which, after phosphorylation by a soluble tyrosine kinase, functions as an adapter protein, probably mediating a modulation of cytoskeleton protein expression. We hypothesize that the decrease of neuropil and dendritic spine density reported to exist in the neocortex of psychiatric patients may be related to a down-regulation of Reln-integrin interactions and the consequent decrease of cytoskeleton protein turnover.